The synergistic inhibitory effect of PRKAA1 activation on ECM mechanical stress-induced skin fibrosis

The synergistic inhibitory effect of PRKAA1 activation on ECM mechanical stress-induced skin fibrosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The synergistic inhibitory effect of PRKAA1 activation on ECM mechanical stress-induced skin fibrosis shihui zhu, Yang Xiang, Yuanyuan Chen, Jin Zhang, Bohan Pan, Jianyu Lu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8422435/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Modulating mechanically mediated fibrosis to alleviate scar formation represents a critical frontier in current research. In previous fibrosis studies, metabolic reprogramming was largely viewed as a passive, secondary process. By combining biological experiments with multiomics analyses, our study reveals that pharmacological activation of AMP-activated catalytic subunit alpha1 ( PRKAA1/AMPKα1 ) affects mechanical signal transduction pathways and initiates regenerative metabolic rewiring. In dermal fibroblasts, elevated mechanical stress triggers a vicious cycle: insufficient PRKAA1 activation fails to supply adequate energy for YAP1 -driven fibrotic progression and increases the production of lactate and proinflammatory molecules. In this context, pharmacologically enhancing PRKAA1 activity results in a ​synergistic antifibrotic effect​ through two key mechanisms: First, PRKAA1 directly phosphorylates YAP1 to suppress hyperactive mechanical signaling, reducing the expression of fibrotic effector factors and thereby limiting their nuclear localization and transcriptional activity. Second, it reprograms cellular metabolism to fulfil the energy requirements for adapting to high stress, increasing the production of anti-inflammatory molecules and accelerating the synthesis of the extracellular matrix (ECM). This dual antifibrotic action rooted in PRKAA1 activity not only deepens our understanding of the regulatory role of metabolism in fibrosis but also offers a valuable translational framework for clinical scar management. Biological sciences/Cell biology/Cell adhesion/Extracellular matrix Biological sciences/Cell biology/Proteolysis/Ubiquitylation skin fibrosis fibroblast mechanical response metabolism PRKAA1 YAP1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Skin fibrosis is a pathological outcome of wound healing and is characterized by excessive extracellular matrix (ECM) deposition and myofibroblast activation 1 , 2 . Researchers have extensively investigated the mechanisms by which mechanical stress-generated by skin stretching and ECM stiffening-regulates fibrosis 3 . Mascharak et al. proposed that blocking YAP1 -mediated mechanical transduction signaling promotes regeneration, establishing YAP1 as a central mediator of mechanotransduction 4 , 5 . In addition to the classical Hippo / LATS pathway, other pathways and molecules (e.g., RhoA / ROCK , Integrinβ1 / FAK / Src , and Piezo1 ) modulate YAP1 activity by controlling cytoskeletal dynamics and kinase activity, thereby inhibiting fibrosis progression 6 – 10 . These findings have further clarified the regulatory network through which external mechanical stress induces fibrosis via cellular mechanotransduction. However, processes—particularly energy metabolism—synergize with mechanotransduction to regulate fibrosis remains poorly understood. Previous studies have demonstrated that mechanical stimulation can induce ​metabolic reprogramming​ in cells 11 , 12 . As a critical energy-sensing kinase, AMPK plays a pivotal role in cellular metabolic regulation. The cAMP/AMPK signaling axis, a key regulator of energy metabolism, inherently has the potential to establish regulatory communication with signal transduction processes activated by mechanical stimuli 13 . Rather than merely mediating basic energy storage and supply conversion, cAMP/AMPK may have essential regulatory functions in ​mechanoresponses​ and fibrosis 14 . Existing evidence reveals that under high mechanical modulus stimulation, cells enter an ​energy stress state, which is​ characterized by activated AMPK 15 , 16 . AMPK activation through pharmacological means reduces collagen release and inflammation by influencing TGF-β, mTOR, NF-κB , and mitochondrial activity, thus preventing fibrosis 17 – 19 . The paradox of AMPK lies in its ​disconnectedness between metabolic and mechanical cues: while it senses energy status, it lacks clear links to mechanical signals. Phosphorylation serves as a key post-translational modification (PTM) that acts as a functional switch for YAP1 ; as an energy sensor, AMPK may influence YAP1 activity through direct or indirect phosphorylation pathways. Therefore, investigating whether the kinase AMPK directly phosphorylates YAP1 and regulates its upstream/downstream signaling will help elucidate the signaling network of cells under mechanical stress and metabolic reprogramming, providing novel insights and strategies for scar treatment. Here, we integrated molecular biology experiments with multiomics techniques to demonstrate that ​pharmacological activation of PRKAA1 directly phosphorylates YAP1 at Ser127 and Thr156 under high mechanical stress. This modification promotes YAP1 degradation via the proteasome pathway and inhibits its nuclear translocation, which in turn reduces YAP1-TEAD1 binding and suppresses downstream profibrotic factor expression. Additionally, metabolic reprogramming induced by PRKAA1 activation enhances energy supply and alters "regenerative" metabolic intermediates. The ​synergistic interplay of PRKAA1 -mediated phosphorylation and metabolic reprogramming​ ultimately results in fibrosis inhibition. Our study’s key breakthrough lies in revealing that ​phosphorylation regulation and metabolic reprogramming are not isolated events—they act synergistically to form the core mechanism through which PRKAA1 inhibits fibrosis. This work substantially expands our understanding of the complex crosstalk between mechanical signal transduction and cellular metabolism, and elucidates in detail how mechanical stress precisely regulates YAP1 activity via the critical node PRKAA1 . It provides new molecular insights into mechanobiology and offers promising therapeutic targets for fibrotic diseases such as pathological scars in clinical practice. RESULTS High mechanical stress induces the differentiation of human dermal fibroblasts (HDFs) into myofibroblasts (MFs) and energy stress Mechanical stress not only affects cell morphology and function but also promotes cell differentiation towards specific lineages by regulating gene expression and signaling pathways. To investigate the critical role of mechanical cues in modulating cell behavior and fibrosis, we ​seeded HDFs onto hydrogels of varying stiffness​ (Extended Data Fig. 1 a-b) to simulate static mechanical stress. While observing HDF differentiation and energy stress, we explored the correlation between YAP1 and PRKAA1 activity. Flow cytometry revealed that as mechanical stress increased, the proportion of ​Vimentin ⁺ and ACTA2 ⁺ myofibroblasts (MFs)​​ increased significantly (Fig. 1 a, Extended Data Fig. 1 c). Concurrently, the expression of ACTA2 , the COL I/COL III ratio, and levels of other profibrotic molecules were upregulated (Fig. 1 b-e, Extended Data Fig. 1 d), indicating enhanced HDF-to-MF differentiation. Moreover, ADP levels increased with higher mechanical stress, suggesting that cellular energy demand increased under stress (Fig. 1 f). The detection of key metabolic enzymes revealed ​enhanced glycolysis and oxidative phosphorylation (OXPHOS)​​ (Fig. 1 g-i), reflecting increased energy supply. Further analysis using ​the extracellular acidification rate (ECAR; Fig. 1 j-k; Extended Data Fig. 1 e)​​ (a proxy for glycolysis) and the ​oxygen consumption rate (OCR; Fig. 1 l-m, Extended Data Fig. 1 f)​​ (a proxy for OXPHOS) confirmed significantly elevated glycolytic and OXPHOS activity. Concurrently, increasing lactate levels (Fig. 1 n) indicated that glycolysis dominated cellular metabolism. The mitochondrial activity and fission capacity were also enhanced (Fig. 1 o-p, Extended Data Fig. 1 g-h). These findings collectively indicate that HDFs slowly transform into MFs when subjected to high mechanical stress, a process that is mainly fueled by increased glycolysis leading to energy stress. Next, we assessed YAP1 and PRKAA1 activity. Consistent with expectations, both PRKAA1 expression and phosphorylation levels increased significantly under energy stress (Fig. 1 r-s), suggesting that metabolic reprogramming was initiated to boost the ATP supply (Fig. 1 q). YAP1 expression and activity (reflected by its unphosphorylated form) also increased (Fig. 1 t), alongside increased YAP1 nuclear translocation (Fig. 1 u-v); this change aligned with the upregulation of the MF marker ACTA2 (Fig. 1 w, Extended Data Fig. 1 i). Collectively, these results indicate that YAP1 activation is correlated with enhanced MF differentiation and fibrotic factor expression, indicating that YAP1 is a critical mediator of mechanical stress-induced fibrotic phenotypes. However, a notable finding emerged: ​ PRKAA1 phosphorylation levels exhibited an inverse trend to YAP1 phosphorylation. These observations contradict the established role of PRKAA1 as a YAP1 kinase, suggesting that the regulatory relationship between PRKAA1 and YAP1 may be more complex under these specific mechanical conditions. Pharmacological activation of PRKAA1 attenuates mechanical stress-induced fibrosis progression by inhibiting YAP1 nuclear translocation. To investigate whether the regulatory effect of PRKAA1 on YAP1 is constrained under ​basal conditions, we pharmacologically modulated PRKAA1 activity. Activation of PRKAA1 with the AMPK agonist GSK621 led to a significant decrease in the proportion of Vimentin ⁺ and ACTA2 ⁺ cells among HDFs (Fig. 2 a, Extended Data Fig. 2 a), indicating ​marked inhibition of myofibroblast (MF) differentiation. Consistently, both YAP1 nuclear translocation (Fig. 2 b-c, Extended Data Fig. 2 b-d) and activity (which inversely correlated with its phosphorylation level; Fig. 2 e-f) were suppressed. Notably, regarding extracellular matrix (ECM) components, the expression of the MF marker ACTA2 and the COL I / COL III ratio were decreased following PRKAA1 activation (Fig. 2 d, Fig. 2 g-h, Extended Data Fig. 2 b-d). Conversely, ​ COL III expression increased​ upon PRKAA1 activation (Extended Data Fig. 2 e-g). In contrast, inhibiting PRKAA1 with the AMPK inhibitor Dorsomorphin induced ​opposite phenotypic changes. Together, these results confirm that PRKAA1 activity ​negatively regulates YAP1 activity and function. More importantly, the upregulation of YAP1 activity upon PRKAA1 inhibition suggests that under physiological mechanical stress, endogenous PRKAA1 activation may be insufficient to fully phosphorylate and inhibit the active form of YAP1 , which accumulates in response to stress. In contrast, pharmacological activation potentiates this function, effectively reversing YAP1 -driven fibrotic phenotypes. To explore the impact of mechanical stress on fibrosis during ​in vivo wound healing, we applied controlled mechanical stress to full-thickness skin defects on the backs of C57BL/6 mice using hydrogels. We then locally injected drugs around and in the wound bed to modulate PRKAA1 activity and assess fibrosis progression. Masson's trichrome staining revealed that the activation of PRKAA1 in conditions of low mechanical modulus led to distinct collagen fiber alignment, akin to that in the control group and normal skin. However, PRKAA1 inhibition led to disorganized collagen fiber deposition (Fig. 2 i, Extended Data Fig. 2 h). Sirius Red staining revealed that PRKAA1 activation significantly reduced the COL I / COL III ratio (Fig. 2 j-k, Extended Data Fig. 2 i-j). Additionally, activating PRKAA1 with GSK621 effectively reversed the mechanical stress-induced upregulation of ACTA2 in the dermis—an outcome consistent with our in vitro findings (Fig. 2 l-m, Extended Data Fig. 2 k). Further Western blotting and immunofluorescence analyses revealed that PRKAA1 activation significantly downregulated YAP1 activity and the expression of ACTA2 , while also reducing the COL I / COL III ratio (Fig. 2 n-q), indicating the inhibition of skin fibrosis progression. Deeper analysis revealed that this reduced ratio was primarily due to ​increased COL III secretion​ (Extended Data Fig. 2 l-m), which is consistent with the findings of previous studies suggesting that elevated COL III attenuates tissue fibrosis 20 . In the late healing phase, ACTA2 -high cells were enriched primarily in the superficial dermis—a spatial distribution consistent with the current findings 21 . However, PRKAA1 inhibition with Dorsomorphin reversed these phenomena, further validating the efficacy of PRKAA1 activation in inhibiting fibrosis. Together, these ​in vivo data​ confirm that PRKAA1 activation effectively inhibits mechanical stress-induced skin wound fibrosis by regulating YAP1 activity and collagen expression. PRKAA1 -dependent YAP1 regulation is key to inhibiting fibrosis To investigate whether there is a ​direct upstream/downstream regulatory relationship​ between PRKAA1 and YAP1 , we performed in-depth validation using genetic editing tools. Even under ​ LATS1 knockdown ( LATS1 KD )​, activation of PRKAA1 with GSK621 still effectively induced YAP1 phosphorylation and significantly inhibited the expression of the myofibroblast marker ACTA2 (Fig. 3 a-c, Extended Data Fig. 3 a). This key finding suggests that the phosphorylation of YAP1 by PRKAA1 is ​independent of the classical Hippo pathway core kinase LATS1 , initially establishing PRKAA1 as an ​upstream regulator​ of YAP1 . To further confirm the role of PRKAA1 in YAP1 activity, we generated cell lines with: PRKAA1 overexpression ( PRKAA1 OE ), PRKAA1 knockdown ( PRKAA1 KD ), YAP1 overexpression ( YAP1 OE ), YAP1 knockdown ( YAP1 KD ), and double manipulations ( PRKAA1 OE YAP1 OE and PRKAA1 KD YAP1 OE ). When YAP1 OE cells were treated with GSK621, their ACTA2 expression was significantly higher than that in the ​vector-transfected group. In contrast, YAP1 KD cells presented reduced YAP1 nuclear translocation, and compared with the vector control, GSK621 treatment had no significant effect on ACTA2 expression, although both of these parameters remained lower (Fig. 3 d-f, Extended Data Fig. 3 b). Flow cytometry results also demonstrated that YAP1 overexpression significantly increased the ​proportion of myofibroblast differentiation, whereas YAP1 knockdown had the opposite effect (Extended Data Fig. 3 c). These findings indicate that ACTA2 and myofibroblast differentiation are regulated by YAP1 expression and activity, further supporting the role of YAP1 as a ​core molecule in mechanotransduction​ and providing a theoretical basis for subsequent validation. Compared with those in vector group, both ​ YAP1 activity​ (Fig. 3 g) and ​nuclear translocation​ (Fig. 3 e) were significantly decreased in GSK621-treated PRKAA1 OE cells (Extended Data Fig. 3 d). Additionally, the proportion of myofibroblast differentiation and ACTA2 expression were markedly reduced (Fig. 3 h, Extended Data Fig. 3 e). Conversely, in PRKAA1 KD cells, even with GSK621 treatment, YAP1 activity was significantly higher than in the vector + GSK621 group. These findings from both overexpression and knockdown experiments confirm that PRKAA1 is a ​critical upstream negative regulator​ of YAP1 . Taken together, these results suggest that under ​basal physiological conditions, the activation level of endogenous PRKAA1 is likely a key factor limiting its ability to fully phosphorylate and inhibit YAP1 . This inference was validated in subsequent pharmacological experiments: no statistically significant difference in YAP1 activity was observed between vector and PRKAA1 OE cells, regardless of GSK621 treatment (Fig. 3 g). This phenomenon suggests that YAP1 activity may have a physiological ​​“floor” threshold—we hypothesize that this is related to the basic functions required for cells adhere to and grow on culture surfaces. Furthermore, even under GSK621-activated PRKAA1 conditions, both YAP1 activity and the fibrotic phenotype driven by YAP1 were significantly higher in PRKAA1 OE YAP1 OE cells than in vector-transfected control cells (Fig. 3 i-j, Extended Data Fig. 3 f-h). These results further confirm that YAP1 is a ​key driver of fibrosis progression, and that the anti-fibrotic effect of PRKAA1 activation primarily acts by ​enhancing the phosphorylation-mediated inhibition of YAP1 . To validate the critical role of PRKAA1 expression/activity in fibrosis regulation, we used a ​ PRKAA1 +/− heterozygous mouse model. In contrast to the results in wild-type (WT) mice, Masson staining of high-mechanical-stress-induced full-thickness back wounds in PRKAA1 +/− mice revealed ​significantly denser collagen fibers​ in the PRKAA1 knockdown group than in the WT group (Extended Data Fig. 3 i). Even with drug-induced PRKAA1 activation, no significant decrease in the ​ COL I / COL III ratio​ was observed via Sirius Red staining (Fig. 3 k-l), and the expression of the myofibroblast marker ACTA2 remained high in the wound tissue during the late healing stage (Fig. 3 m). Western blotting further revealed that with decreasing PRKAA1 phosphorylation levels, ​ YAP1 activity​ (Fig. 3 n-p), ​ ACTA2 expression, and the ​ COL I / COL III ratio​ all increased significantly (Fig. 3 q-r, Extended Data Fig. 3 j), indicating that intact PRKAA1 gene function is essential for normal pathway activity. Together, these in vitro and in vivo results confirm that: PRKAA1 ​gene expression level​ is a prerequisite for its anti-fibrotic function. Pharmacological activation of PRKAA1 inhibits fibrosis by promoting YAP1 phosphorylation. Insufficient PRKAA1 activation under physiological conditions is the main factor limiting the suppression of fibrosis. PRKAA1 regulates YAP1 protein stability via LATS1 -Independent direct interaction To clarify whether there is a ​direct molecular interaction​ between PRKAA1 and YAP1 , we conducted systematic validation by combining ​computational predictions​ with multiple experimental approaches. The computational results from Rosetta software revealed a binding free energy of -24.997 kcal/mol between PRKAA1 and YAP1 (Fig. 4 a, Extended Data Fig. 3 k), indicating a strong propensity for spontaneous binding. This finding was further supported by AlphaFold 3 protein structure prediction, which also predicted binding potential between the two proteins (Extended Data Fig. 3 l). Co-immunoprecipitation (Co-IP) analysis confirmed that PRKAA1 interacts with YAP1 in cells ​regardless of exogenous drug treatment​ (Fig. 4 b). However, given that PRKAA1 is known to promote YAP1 phosphorylation via the Hippo pathway kinase LATS1 , the interaction captured by Co-IP might arise from the PRKAA1 / LATS1 / YAP1 ​ternary complex​ rather than from direct binding between PRKAA1 and YAP1 . To rule out this possibility, we used a ​ GST fusion protein pull-down assay​ to test for direct in vitro interactions: purified GST - PRKAA1 fusion protein was co-incubated with Flag -tagged YAP1 recombinant protein. The results clearly revealed that GST - PRKAA1 directly pulled down Flag - YAP1 (Fig. 4 c), whereas the GST tag protein control did not exhibit such binding. This in vitro evidence effectively eliminated interference from other bridging proteins (e.g., LATS1 ) in cells, ​confirming a direct molecular interaction between PRKAA1 and YAP1 . To elucidate whether PRKAA1 regulates YAP1 stability through a mechanism independent of the classical Hippo pathway core kinase LATS1 , we further constructed PRKAA1 -overexpressing ( PRKAA1 OE LATS1 KD ) and PRKAA1 -knockdown ( PRKAA1 KD LATS1 KD ) cell lines with a LATS1 -knockdown ( LATS1 KD ) genetic background. Under controlled proteasome pathway activation conditions, we evaluated YAP1 ubiquitination levels and its proteasomal degradation after PRKAA1 activation. The experimental results demonstrated that in the absence of LATS1 , PRKAA1 activation effectively increased YAP1 ubiquitination even when the proteasome pathway was unopened. After proteasome pathway activation, YAP1 degradation was significantly enhanced (Fig. 4 d-g). These findings strongly suggest the existence of a LATS1 -independent pathway for PRKAA1 -mediated regulation of YAP1 stability. Further analysis revealed that in PRKAA1 KD LATS1 KD cells, YAP1 degradation was reduced accompanied by decreased ubiquitination. Conversely, PRKAA1 activation in PRKAA1 OE LATS1 KD cells accelerated YAP1 degradation with increased ubiquitination. These findings confirm that PRKAA1 activation effectively promotes YAP1 degradation. PRKAA1 inhibits the transcriptional co-activation function of YAP1 by directly phosphorylating it at the Ser127 and Thr156 residues Our results revealed that PRKAA1 likely regulates the ubiquitin-proteasome degradation pathway of YAP1 by directly interacting with it and modulating its phosphorylation status. To precisely identify the key PRKAA1 -mediated phosphorylation sites on YAP1 , we used phosphorylation mass spectrometry to systematically analyze changes in the phosphorylation levels of various YAP1 peptides under three treatment conditions: ​PRKAA1 activation, ​no intervention, and ​ PRKAA1 inhibition. The analysis revealed that only the phosphorylation level of the peptide spanning the N125–C161 region significantly decreased after PRKAA1 activation (Table 1 ), suggesting that this region is a potential key target of PRKAA1 . This peptide contains 10 potential phosphorylation sites: Ser127 , Ser128 , Ser131 , Ser138 , Thr141 , Thr143 , Thr145 , Ser149 , Thr154 , and Thr156 . To screen for the most likely PRKAA1 -targeted sites, we combined predictions from two professional phosphorylation site prediction platforms—PhosphoSitePlus​ and ​GPS 6.0. The PhosphoSitePlus results (Table 2 ) indicated that Ser127 and Thr156 were more likely to be phosphorylated by PRKAA1 , with Ser127 showing greater kinase specificity. GPS 6.0 (Table 3 ) further confirmed the importance of Ser127 , as its prediction score was significantly higher than that of all the other sites. Additionally, GPS 6.0 identified other sites with scores > 0.001, ranked by score as follows: Ser128 > Thr156 > Ser131 > Ser138 > Thr143 . Combining data from both platforms revealed that Ser127 and Thr156 were consistently highlighted as potential targets, whereas Ser128 had a relatively high score on GPS 6.0. Thus, we selected ​ Ser127 , Ser128 , and Thr156 ​ as key candidates for subsequent functional validation. To clarify the specific molecular sites and functional hierarchy of the direct phosphorylation of YAP1 , we constructed HDF models with YAP1 mutations at Ser127 , Ser128 , or Thr156 using site-directed mutagenesis. After pharmacological PRKAA1 activation, phosphorylation analysis revealed that ​only the Ser127 and Thr156 mutants failed to undergo significant phosphorylation upregulation—whereas the Ser128 mutant remained phosphorylatable, with no statistical difference from the vector + GSK621 group (Fig. 4 g-h). These findings confirm that PRKAA1 specifically phosphorylates YAP1 at Ser127 and Thr156 , but that Ser128 is not a key target. To explore the relative functional importance of Ser127 and Thr156 , we assessed the binding of the YAP1 mutant to TEAD1 under GSK621 treatment. The mutation at ​ Ser127 greatly enhanced the binding of YAP1 to TEAD1 , with a greater fold-change than that induced by Thr156 mutation, suggesting that phosphorylation at Ser127 more effectively inhibits the YAP1 - TEAD1 interaction. Consistent with these functional observations, the transcriptional response of ​its target genes ( CTGF and CYR61 ), in which TEAD1 binds to the promoter regions, exhibited trends aligned with those of the aforementioned binding assays: the Ser127 mutation exerted the most pronounced inhibitory effect on downstream transcriptional activity (Fig. 4 k-l). Combining all the evidence, we ​precisely identified Ser127 and Thr156 as the key PRKAA1 phosphorylation sites on YAP1 ​ and uncovered their functional hierarchy: PRKAA1 primarily inhibits YAP1 - TEAD1 complex formation and downstream profibrotic transcription by phosphorylating ​ Ser127 , while Thr156 plays a secondary, auxiliary regulatory role. Metabolomics reveals the unique role of PRKAA1 -induced metabolic reprogramming in inhibiting fibrosis Another key mechanism through which PRKAA1 activation inhibits fibrosis progression lies in its ability to induce induced ​metabolic reprogramming​. This effect first manifests as an overall improvement in cellular energy supply levels (Fig. 5 a-b). More importantly, PRKAA1 activation prompts a ​fundamental shift in the cell’s metabolic pattern—from being primarily dominated by anaerobic glycolysis to being dominated by oxidative phosphorylation (Fig. 5 c-f)—which directly leads to a significant decrease in extracellular lactate levels (Fig. 5 h). The WB results further support changes in the expression of key proteins related to glycolysis and oxidative phosphorylation, strengthening the conclusion of this metabolic switch (Fig. 5 g, i-j). Additionally, mitochondrial dynamic fission and functional levels are enhanced due to PRKAA1 activation (Fig. 5 g, k-m). Many studies have shown that elevated energy levels and reduced lactate content are key factors in inhibiting fibrosis and promoting tissue regeneration. To systematically dissect the regulatory differences in cellular metabolism between PRKAA1 activation (HA) and simple high mechanical modulus intervention (HN) under high mechanical stress, this study first screened for differentially expressed metabolites using ​untargeted metabolomics​ and constructed a heatmap to visualize metabolic phenotypes (Fig. 5 n). The cluster analysis results indicated that samples from the HA and HN groups formed two separate clusters, demonstrating significant metabolic differences. The heatmap displays 20 differentially expressed metabolites across four categories: amino acids, carbohydrates, lipids, and nucleotides. Specifically, 11 metabolites were upregulated in HA, including N-acetyl-L-aspartate (amino acids), palmitic acid (lipids), and methylated uridine (nucleotides); and 7 metabolites were downregulated, exemplified by glucosamine-6-phosphate (a key glycolytic intermediate). Notably, upregulated metabolites were associated with energy storage ( palmitic acid ), redox homeostasis ( N-acetyl-L-aspartate in glutathione synthesis), and nucleotide metabolism ( methylated uridine , which regulates RNA stability). The downregulated metabolites were concentrated in the late glycolysis stage, suggesting that PRKAA1 activation may antagonize the profibrotic metabolic phenotypes induced by high mechanical stress by reprogramming energy metabolism and oxidative stress pathways. To further explore the interactions between differentially expressed metabolites and gene expression, we integrated transcriptomic data to construct a transcriptome–metabolome network​ for the HA vs. HN groups (Fig. 5 o). Central hubs with high connectivity to surrounding nodes were formed by key pathways such as insulin secretion, fatty acid biosynthesis, and arginine biosynthesis, suggesting that PRKAA1 reshapes metabolic networks through the synergy of multiple pathways. The arginine biosynthesis pathway was significantly upregulated in HA: three key metabolites ( ornithine , citrulline , and arginine ) were elevated, and positively correlated with N-acetylglutamate (a urea cycle node). Moreover, the downstream metabolite N-acetyl-L-aspartate (upregulated in HA heatmaps) formed a positive regulatory cluster with the purine metabolism genes GMPS (GMP synthase) and XMPK (XMP kinase), suggesting that the ​arginine purine axis​ supports anti-fibrotic ECM remodeling by promoting nucleotide synthesis. Coordinated activation of the pyruvate metabolism-fatty acid biosynthesis axis was another hallmark: PKM2 (key pyruvate kinase) expression increased in HA, driving the conversion of pyruvate to acetyl-CoA (upregulated) and activating fatty acid biosynthesis (upregulation of ACACA / FASN ). This finding was consistent with the increased levels of palmitic acid (a lipid metabolite) in HA, suggesting that the activity of PRKAA1 shifts from profibrotic glycolysis to the synthesis of new fatty acids, thereby decreasing lipotoxic fibroblast activation. Notably, the TCA cycle and HIF1α signaling exhibited ​pathological coupling​ in HN: citrate (a downregulated TCA metabolite) formed a negative cluster with HIF1α and its target LDHA ( lactate dehydrogenase A ). These findings suggest that high mechanical stress inhibits the TCA cycle, causing pyruvate accumulation, activating HIF1α -mediated glycolytic reprogramming, and promoting the formation of a lactate buildup/fibrotic microenvironment. In HA, restored citrate levels and downregulated HIF1α indicated PRKAA1 blocks this cascade by restoring TCA cycle function. To validate the results of the combined metabolomics and transcriptomics analysis, we selected three metabolites— N-acetyl-L-aspartate , ​ palmitic acid , and ​ methylated uridine . Under high mechanical modulus conditions, we observed that ​GSK621 , ​ N-acetyl-L-aspartate , and ​palmitic acid​ effectively reduced ​ ACTA2 protein expression in HDFs​ (Fig. 5 p-q) and ​immunofluorescence intensity​ (Fig. 5 r-s). However, ​palmitic acid exacerbated fibrotic phenotypes​ induced by high mechanical modulus. These findings corroborate the key insights from the integrative metabolomic-transcriptomic analysis. Taken together, these data demonstrate that PRKAA1 is a central integrator of metabolic and mechanical signals, thereby establishing a direct link between metabolism and fibrosis regulation (Fig. 5 t) DISCUSSION For decades, the interplay between mechanical stress and cellular metabolism in fibrosis pathogenesis has intrigued researchers 22 , 23 . However, how energy metabolism pathways coordinately regulate fibrotic signaling and mechanotransduction remains poorly defined 24 – 26 . Our study elucidates a ​dual regulatory axis​ in which pharmacological activation of PRKAA1 ( AMPKα1 ) not only directly phosphorylates YAP1 at the Ser127 and Thr156 residues to induce its proteasomal degradation but also coordinates a metabolic reprogramming switch from glycolysis to oxidative phosphorylation—jointly inhibiting mechanical stress-induced fibrosis. This synergistic mechanism challenges the traditional view that YAP1 is solely regulated via the Hippo pathway and highlights potential crosstalk between energy homeostasis and mechanotransduction signaling in fibrotic diseases 5 , 27 , 28 . AMPK is traditionally viewed as a metabolic sensor, but our study uncovers its ​dual role as a modulator of mechanotransduction. While previous studies have suggested that LATS1 -mediated phosphorylation is the primary mechanism underlying YAP1 inactivation, our data demonstrate that PRKAA1 bypasses this canonical pathway via direct interaction with YAP1 . Co-IP and GST pull-down assays revealed a high-affinity interaction, indicating that PRKAA1 is a regulator of YAP1 activity under energy stress. These findings demonstrate that PRKAA1 acts as a ​parallel regulatory pathway alongside the Hippo cascade. This independence is functionally relevant: even when LATS1 is inhibited during fibrosis development, PRKAA1 activation still induces YAP1 degradation, indicating that PRKAA1 can bypass canonical Hippo signaling to induce YAP1 inactivation. Notably, this phosphorylation event relies on promoting YAP1 degradation and reducing its nuclear localization to inhibit YAP1 - TEAD1 complex formation: YAP1 - Ser127 plays a dominant role, while Thr156 plays a minor role. This hierarchical phosphorylation mechanism aligns with recent findings on YAP1 structural plasticity but expands its regulatory scope to include metabolic sensors 5 , 27 . Our experimental results emphasize the ​physiological relevance of this axis. In both in vivo and in vitro experiments, we observed no significant changes in YAP1 activity in the context of PRKAA1 deficiency or the absence of GSK621 activation, indicating that PRKAA1 expression and activity determine the activation threshold of YAP1 . These findings suggest the following important mechanism: partial AMPK activation preserves the tissue-repair activity of YAP1 , whereas full activation inhibits pathological remodeling. Exogenous PRKAA1 activation failed to further inhibit YAP1 activity in PRKAA1 OE cells, indicating a ​saturation effect​ of this axis. This observation implies that endogenous YAP1 activity sets a baseline constraint—a concept previously unappreciated in fibrosis biology 29 – 31 . Furthermore, we observed metabolic changes characterized by reduced lactate production and increased oxidative phosphorylation, which increase the complexity of the regulation of fibrosis. By integrating metabolomic and transcriptomic analyses, we identified a metabolic network centered on arginine biosynthesis–fatty acid oxidation– pyruvate –TCA cycle– HIF1α . Specifically, under high mechanical stress, PRKAA1 activation restores TCA cycle function–thereby inhibiting the hypoxic signaling driven by HIF1α , which activates YAP1 . This metabolic shift also produces anti-fibrotic metabolite, such as N-acetyl-L-aspartate , a known modulator of TGF-β signaling 32 , 33 . The downregulation of fibrotic pathways, including HIF1α , under PRKAA1 activation further supports the idea that metabolic reprogramming disrupts the ​hypoxia-mimicking microenvironment​ that perpetuates fibrosis 34 . Notably, this metabolic shift mirrors the “Warburg effect reversal” reported in regenerative environments, where enhanced oxidative metabolism promotes tissue repair 35 , 36 . This homeostatic mechanism is consistent with recent discoveries about mitochondrial retrograde signaling in fibrosis, highlighting PRKAA1 as a key integrator of metabolic and mechanical signals, thereby directly connecting metabolism with the regulation of fibrosis. Our findings hold ​transformative potential for fibrosis therapy. First, targeting the PRKAA1-YAP1 interaction circumvents the limitations of current anti-fibrotic strategies that broadly inhibit TGF-β or collagen deposition 37 , 38 . Small-molecule AMPK activators such as GSK621 show promise in preclinical models but require optimization for clinical translation to avoid off-target effects on glucose metabolism 39 , 40 . Second, the metabolic reprogramming patterns identified in this study, marked by increased oxidative phosphorylation and arginine production, serve as new biomarkers for assessing fibrosis stages and tracking treatment effectiveness. 41–43 . For example, upregulated N-acetyl-L-aspartate can serve as a non-invasive indicator of PRKAA1 activity in fibrotic tissues. However, challenges remain. The ​spatial heterogeneity of YAP1 activation​ within fibrotic lesions suggests that PRKAA1 -based therapies may need to be administered locally to avoid systemic metabolic disruption. Additionally, the fibroblast heterogeneity observed in human fibrosis requires cell type-specific targeting strategies to maintain tissue-specific homeostasis. Furthermore, the temporal kinetics of PRKAA1 - YAP1 phosphorylation during fibrosis progression remain unclear. Does sustained mechanical stress lead to PRKAA1 depletion or adaptive resistance? In addition, it is necessary to investigate how other metabolic nodes, such as the pentose phosphate pathway or lipid droplet dynamics, influence YAP1 activity. Addressing these questions requires advanced spatiotemporal imaging and organoid models to recapitulate tissue-level mechanical heterogeneity. In summary, our work bridges a critical gap in fibrosis biology by elucidating how energy stress regulates mechanical transduction signaling. By demonstrating that PRKAA1 acts as a ​metabolic checkpoint that inhibits YAP1 overactivation, this study not only enhances our understanding of fibrosis pathogenesis but also opens new avenues for precision therapies targeting the intersection of metabolism and mechanobiology. MATERIALS AND METHODS Experimental reagents GSK621 (cat. # HY-100548), Dorsomorphin (cat. #HY-13418A) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, a photoinitiator, cat. #HY-44076) were purchased from MedChem Express (NJ, USA). Gelatin methacryloyl (GelMA, cat. #EFL-GM-90) was purchased from Engforlife Tech (Suzhou, China). Cell lines and cell culture ​ Human dermal fibroblasts (HDFs) were obtained from Pricella (CM-H103, Wuhan, China). The cells were maintained in Dulbecco’s Modified Eagle Medium (11885084, ThermoScientific, US) supplemented with 10% fetal bovine serum (A5256701, ThermoScientific, US) and 1% penicillin-streptomycin (C100C5, NCM Biotech, China) at 37°C in a humidified incubator with 5% CO₂. GelMA hydrogel preparation ​ GelMA was dissolved in sterile phosphate-buffered saline (PBS) containing 0.25% LAP, under dark conditions at 60°C until fully dissolved. The solutions were then adjusted to concentrations of 5%, 10%, or 15% (w/v) and sterilized by filtration. For hydrogel formation, 50–100 µL of the GelMA precursor was added to each well of a 6-well plate or 10 cm culture dish, exposed to 405 nm light for 1 minute for crosslinking, and allowed to hydrate in complete medium for 2 hours at 37°C with 5% CO₂ before cell seeding. Cell seeding and drug treatment ​ HDFs were seeded onto GelMA hydrogels at a density of 1×10⁵ cells per well (6-well plate) or 1×10⁶ cells per dish (10 cm plate). After 24 hours of adhesion, cells were treated with fresh medium containing vehicle (DMSO) or test compounds. Drug solutions were prepared as 10 mM stock solutions in DMSO and diluted to working concentrations in PBS or DMEM before use. The final concentrations were: 30 µM GSK621 (a PRKAA1 activator) and 10 µM Dorsomorphin (a PRKAA1 inhibitor). Plasmid/siRNA transfection Plasmids and siRNAs were designed and validated by GeneChem (Shanghai, China). For transfection, HDFs were seeded in 6-well plates and transfected with Lipofectamine Lipo6000 (C0526; Beyotime, China) according to the manufacturer’s protocol. GFP plasmids were used for transfection efficiency validation in pilot experiments. Lentiviral infection and stable cell line construction Lentiviruses encoding shRNAs or overexpression constructs were generated by GeneChem (Shanghai, China). For infection, HDFs were seeded at 50% confluence and treated with lentivirus (MOI = 20) plus 8 µg/mL polybrene. Stable cells were selected with puromycin (2 µg/mL) for 7 days. Site-directed mutagenesis Ser127 , Ser128 , and Thr156 mutants of YAP1 were generated with primers designed by GeneChem (Shanghai, China). Mutant plasmids were validated by Sanger sequencing. Animals ​ Male C57BL/6 mice (6 weeks old, ~ 25 g) were purchased from Gempharmatech (Jiangsu, China). Heterozygous PRKAA1 knockout mice ( PRKAA1⁺/⁻ ) were generated and genotyped by Beijing Jieyalife Biotechnology Co., Ltd. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Yishang Biotechnology (approval NO. IACUC-2024-Mi-059) and conducted in accordance with the 3Rs principles. The mice were housed in specific pathogen-free (SPF) facilities with controlled temperature (22–25°C), humidity (40–60%), and a 12-hour light-dark cycle, with free access to standard chow and water. Full-thickness wound model ​ The mice were anesthetized via isoflurane inhalation. The back was removed using electric clippers followed by depilatory cream (Veet®). Two 8 mm diameter full-thickness skin wounds were created on the back using a biopsy punch. Wounds were secured with 12 mm polyvinyl chloride (PVC) rings and dressed with petroleum gauze, moist chlorhexidine gauze, and iodine film. Dressings were changed on days 3, 7, 10, 14, and 17, with hydrogel application (5% or 15% w/v GelMA) and wound photography. The contraction rings were removed on day 10. Six wound tissue samples were collected on days 10 and 20. For mechanotransduction studies, drugs (100 µL) were injected into the wound bed and surrounding area at day 0 and every 2 days (same concentrations as in vitro). Mechanical property testing GelMA hydrogels (5%, 10%, and 15% w/w) were cast into cylindrical molds (diameter: 26.5 mm, height: 5 mm). The compressive modulus at 40% strain was measured using a computer-controlled universal testing machine (HD-B602, Haida, China) after 2 hours and 50 hours of immersion in culture medium. Young’s modulus was calculated from the linear elastic region of the stress-strain curve. Cell viability assay (CCK-8) HDFs were seeded in 96-well plates at 3,000 cells per well and cultured for 12 hours. After being washed with PBS, the cells were treated with gradient concentrations of GelMA (0.1%, 0.5%, 1%, and 5%) dissolved in DMEM containing 2% FBS. A blank control (no cells) was included. At 0, 12 and 24 hour post-treatment, 10 µL of CCK-8 reagent (CK04, Dojindo, Japan) was added to each well, followed by incubation at 37°C with 5% CO₂ for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Relative cell viability was normalized to that of the blank control. Quantitative Real-time PCR (qPCR) Total RNA was extracted using TRIzol reagent (RC112-01, Vazyme, China) and reverse-transcribed into cDNA with a PrimeScript™ RT Reagent Kit (RR036A, Takara, Japan). qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using TB Green Premix Ex Taq II (RR820A, Takara, Japan). The cycling program was as follows: 95°C for 30 seconds; 40 cycles of 95°C for 5 seconds, 60°C for 30 seconds; and a melting curve analysis. Gene expression levels were normalized to TUBULIN using the 2 ⁻ΔΔCt method. The primer sequences are listed in Table S1 . Flow cytometry ​ Immunophenotyping ​ For surface marker analysis, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Antibodies against Vimentin (ab8069, Abcam, UK) and α-SMA (#19245, CST, US) were added and the samples were incubated for 1 hour at room temperature. Fluorescent secondary antibodies (12-4739-81, 48-4015-82, ThermoScientific, US) were added and the samples were incubated for 1 hour at room temperature. Data were acquired on a Beckman CytoFlex and analyzed using Cytexpert2.5. Western blotting (WB) Cells were lysed in RIPA buffer (WB3100, NCM Biotech, China) containing protlytic protease and phosphatase inhibitor (P002, NCM Biotech, China) on ice for 1 hour. The lysates were subsequently were centrifuged at 12,000 × g for 20 minutes at 4°C, and protein concentrations were determined using a NanoDrop (Oxford, US). Equal amounts of protein (10–20 µg) were separated by SDS-PAGE (8–12% gels) and transferred to PVDF membranes (Millipore, US). Membranes were blocked with 5% BSA in TBST for 2 hours and probed with primary antibodies overnight at 4°C. After being washed, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. The protein bands were visualized using ECL Plus (P2100, NCM Biotech, China) and quantified with ImageJ. Co-iImmunoprecipitation (Co-IP) HDFs (1×10⁶) were seeded in 10 cm plates, treated with GSK621 (30 µM) or vehicle for 48 hours, and lysed in IP lysis buffer (P0013, Beyotime, China) containing protease/phosphatase inhibitors. Lysates were incubated with anti-PRKAA1 or IgG antibodies (5 µg/mL) overnight at 4°C, followed by incubation with Protein A/G magnetic beads (P2179S, Beyotime, China) for 2 hours. The beads were subsequently were washed, and bound proteins were eluted with elution buffer. Input and IP samples were analyzed by WB. GST pull-down assay GST, GST- PRKAA1 , and Flag- YAP1 plasmids were transformed into Rosetta competent cells (D1065S, Beyotime, China). Bacterial cultures were induced with 1 mM IPTG (ST098, Beyotime, China) at 37°C for 6 hours. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing lysozyme and protease inhibitors. Cleared lysates were incubated with Anti-Flag or anti-GST magnetic beads (P2115, P2138, Beyotime, China) overnight at 4°C. The beads were subsequently washed, after which the bound proteins were eluted. For kinase assays, GST- PRKAA1 and Flag- YAP1 were co-incubated in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) supplemented with 200 µM ATP at 37°C for 30 minutes. Interactions were validated by WB using anti-GST, anti-Flag, anti- PRKAA1 , and anti- YAP1 antibodies. Chromatin immunoprecipitation (ChIP) HDFs were crosslinked with 1% formaldehyde for 10 minutes at room temperature, quenched with 0.125 M glycine, and lysed in ChIP lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 10 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100). Nuclei were isolated and resuspended in sonication buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA). Chromatin was sheared using a Covaris S220 sonicator (175 W, 10% duty factor, 200 cycles, 10 minutes) to generate 200–500 bp fragments. Immunoprecipitation was performed with anti- YAP1 or IgG antibodies, followed by protein A/G bead incubation. DNA was purified and quantified by qPCR. Prediction of Protein-Protein interaction PRKAA1 - YAP1 interactions were predicted using the RosettaDock and AlphaFold 3. Structural models of PRKAA1 (Q13131-1) and YAP1 (P46937-1) were downloaded from UniProt and visualized with PyMOL. Prediction of in silico phosphorylation site ​ Potential phosphorylation sites on YAP1 targeted by PRKAA1 were predicted using PhosphoSitePlus ( https://www.phosphosite.org/ ) and GPS ( https://gps.biocuckoo.cn/ ). Public phosphoproteomics data were mined to validate candidate sites. Immunofluorescence Staining ​ In vitro ​ Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.3% Triton X-100 for 10 minutes, and blocked with 5% BSA. Primary antibodies (e.g., anti-YAP1, anti-PRKAA1) were added and the samples were incubated overnight at 4°C. Fluorescent secondary antibodies were added and incubated for 1 hour at room temperature. The nuclei were counterstained with DAPI. Images were captured using a Leica confocal microscope. ​ In vivo ​ Paraffin-embedded tissues were sectioned (4–6 µm), deparaffinized, and rehydrated. Antigen retrieval was performed using citrate buffer (pH 6.0) via microwave heating. Subsequent steps followed the in vitro protocol. Histological staining ​ Masson’s trichrome staining ​ Sections were stained with Weigert’s iron hematoxylin (nuclei), acid fuchsin (cytoplasm/muscle), and aniline blue (collagen). Images were analyzed for collagen content. ​ Sirius Red Staining ​ The sections were incubated with 0.1% Sirius Red (in saturated picric acid) for 1 hour, rinsed with acidic ethanol, and dehydrated. Collagen fibers were visualized under polarized light. Statistical Analysis The data are presented as mean ± standard deviation (SD) and were analyzed using GraphPad Prism 9.0. Normality was assessed using the Shapiro-Wilk test. Differences between groups were evaluated by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. P-value < 0.05 was considered to indicate statistical significance ( P < 0.05, ​P < 0.01, P < 0.001, ​ ​ P < 0.0001; ns, not significant). Declarations ACKNOWLEDGEMENTS We appreciate the data from UniProt, PhosphoSitePlus (https://www.phosphosite.org/) and GPS (https://gps.biocuckoo.cn/). We express our gratitude to Cosmos Wisdom (Hangzhou, China) for its support in bioinformatics sequencing and analysis. AUTHOR CONTRIBUTIONS YX: Conceptualization, Formal analysis, Investigation, Validation, Visualization, Writing-original draft. YyC: Data curation, Formal analysis, Methodology, Validation, Writing-original draft. JZ: Conceptualization, Methodology, Resources, Validation, Writing-original draft. BhP: Data curation, Investigation, Methodology, Writing- review & editing. JyL: Investigation, Methodology, Validation, Writing-review & editing. CrW Investigation, Writing- review & editing. JqC: Investigation, Writing- review & editing. CL: Investigation, Writing-review & editing. YyY: Funding acquisition, Investigation, Supervision, review & editing. SzJ: Funding acquisition, Supervision, Writing-review & editing. ShZ: Funding acquisition, Project administration, Supervision, Writing – review & editing. FUNDING The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Key Research Project (2019YSL010), National Key R&D Program of China (2024YFA1108405), National Natural Science Foundation of China (No.82473580 and No. 82574032), and the Academic General project (No.2023MS006). We thank Biorender (https://biorender.com) for the production of the figures in this article. DATA AVAILABILITY The transcriptome sequencing and metabolomics data used in the study have been uploaded to the National Genomics Data Center (https://www.cncb.ac.cn/; BioProject Number: PRJCA051113). The data are expected to be released in November 2027. Further inquiries can be directed to the corresponding authors. ETHICS APPROVAL All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Yishang Biotechnology (approval NO. IACUC-2024-Mi-059) and conducted in accordance with the 3Rs principles. COMPETING INTERESTS The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflicts of interest. CONSENT TO PARTICIPATE Not applicable. References Peña, O. A. & Martin, P. Cellular and molecular mechanisms of skin wound healing. Nat Rev Mol Cell Biol 25, 599–616 (2024). Talbott, H. E., Mascharak, S., Griffin, M., Wan, D. C. & Longaker, M. T. Wound healing, fibroblast heterogeneity, and fibrosis. Cell Stem Cell 29, 1161–1180 (2022). Li, Y. Y., Ji, S. F., Fu, X. B., Jiang, Y. F. & Sun, X. Y. Biomaterial-based mechanical regulation facilitates scarless wound healing with functional skin appendage regeneration. Mil Med Res 11, 13 (2024). 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Tables Table 1 Mass spectrometry results of protein phosphorylation modification PRKAA1 function Phosphorylatable sites within the sequence Confidence Sequence Xcorr score Activated K315-AC;UB High LQQLQMEK 1.33 Inhibited K315-AC;UB High LQQLQMEK 1.3 Activated K315-AC;UB High LQQLQMEK 1.04 untreated K315-AC;UB Low LQQLQMEK 1 untreated K497-ME;UB;S499-P High LDKESFLTWL 2.66 Activated K497-ME;UB;S499-P Medium LDKESFLTWL 2.34 Inhibited K497-ME;UB;S499-P High LDKESFLTWL 2.05 Inhibited S109-P;GL/T110-P/T114-P/T119-P High QASTDAGTAGALTPQHVR 3.51 untreated S109-P;GL/T110-P/T114-P/T119-P High QASTDAGTAGALTPQHVR 3.17 untreated S109-P;GL/T110-P/T114-P/T119-P High SQLPTLEQDGGTQNPVSSPGMSQELR 2.86 Activated S109-P;GL/T110-P/T114-P/T119-P High QASTDAGTAGALTPQHVR 1.86 Activated S127-P/S128-P/S131-P/S138-P/T141-P/T143-P/T145-P/S149-P/T154-P/T156-P High AHSSPASLQLGAVSPGTLTPTGVVSGPAATPTAQHLR 5.92 untreated S127-P/S128-P/S131-P/S138-P/T141-P/T143-P/T145-P/S149-P/T154-P/T156-P High AHSSPASLQLGAVSPGTLTPTGVVSGPAATPTAQHLR 4.96 Inhibited S127-P/S128-P/S131-P/S138-P/T141-P/T143-P/T145-P/S149-P/T154-P/T156-P High AHSSPASLQLGAVSPGTLTPTGVVSGPAATPTAQHLR 2.29 Inhibited S340-P/K342-ME;UB High NINPSTANSPK 2.62 Activated S340-P/K342-ME;UB High NINPSTANSPK 1.63 Inhibited S61-P/T63-P/K76-UB High GDSETDLEALFNAVMNPK 3.67 untreated S61-P/T63-P/K76-UB High GDSETDLEALFNAVMNPK 3.62 Activated S61-P/T63-P/K76-UB High GDSETDLEALFNAVMNPK 3.61 untreated S94-P/K97-AC;UB;SM/K102-UB High KLPDSFFKPPEPK 2.27 Inhibited S94-P/K97-AC;UB;SM/K102-UB High KLPDSFFKPPEPK 1.62 Activated S94-P/K97-AC;UB;SM/K102-UB High KLPDSFFKPPEPK 1.56 untreated S94-P/K97-AC;UB;SM/K102-UB Medium LPDSFFKPPEPK 1.13 Activated S94-P/K97-AC;UB;SM/K102-UB Medium LPDSFFKPPEPK 1.05 Inhibited T354-P/T361-P/S366-P/S367-P/S371-P High SQLPTLEQDGGTQNPVSSPGMSQELR 2.72 Activated T354-P/T361-P/S366-P/S367-P/S371-P High SQLPTLEQDGGTQNPVSSPGMSQELR 1.52 Inhibited T354-P/T361-P/S366-P/S367-P/S371-P High SQLPTLEQDGGTQNPVSSPGMSQELR 1.43 untreated T398-P/S200-P/S403-P/S405-P/Y407-P High DESTDSGLSMSSYSVPR 2.74 Activated T398-P/S200-P/S403-P/S405-P/Y407-P High DESTDSGLSMSSYSVPR 1.85 Inhibited T398-P/S200-P/S403-P/S405-P/Y407-P High DESTDSGLSMSSYSVPR 1.45 Inhibited T412-P/T419-P/S436-P High TPDDFLNSVDEMDTGDTINQSTLPSQQNR 3.71 Activated T412-P/T419-P/S436-P High TPDDFLNSVDEMDTGDTINQSTLPSQQNR 3.24 Inhibited T77-P/T83-P High TANVPQTVPMR 2.51 untreated T77-P/T83-P High TANVPQTVPMR 2.46 untreated T77-P/T83-P High TANVPQTVPMR 1.85 Activated T77-P/T83-P High TANVPQTVPMR 1.84 Activated T77-P/T83-P High TANVPQTVPMR 1.77 Inhibited T77-P/T83-P High TANVPQTVPMR 1.62 untreated Y188-P High YFLNHIDQTTTWQDPR 1.95 Inhibited Y188-P High YFLNHIDQTTTWQDPR 1.79 Activated Y188-P High YFLNHIDQTTTWQDPR 1.3 Table 2 Predictions from the PhosphoSitePlus Platform site site sequence log2(score)* site percentile* S127 AHs*sPAsLQLGAVsPGtLtPtGVVsGPAAtPtAQHLR 0.07 81.03% S128 AHss*PAsLQLGAVsPGtLtPtGVVsGPAAtPtAQHLR -0.223 77.88% S131 AHssPAs*LQLGAVsPGtLtPtGVVsGPAAtPtAQHLR -2.696 38.61% S138 AHssPAsLQLGAVs*PGtLtPtGVVsGPAAtPtAQHLR -2.654 39.43% T141 AHssPAsLQLGAVsPGt*LtPtGVVsGPAAtPtAQHLR -4.099 17.13% T143 AHssPAsLQLGAVsPGtLt*PtGVVsGPAAtPtAQHLR -4.245 15.38% T145 AHssPAsLQLGAVsPGtLtPt*GVVsGPAAtPtAQHLR -2.954 34.26% S149 AHssPAsLQLGAVsPGtLtPtGVVs*GPAAtPtAQHLR -1.891 52.89% T154 AHssPAsLQLGAVsPGtLtPtGVVsGPAAt*PtAQHLR -3.407 26.79% T156 AHssPAsLQLGAVsPGtLtPtGVVsGPAAtPt*AQHLR 1.103 89.64% *in site sequence, phosphorylation site. *log2(score)=0, neural; >0, favorable; <0, unfavorable. *site percentile: percentage of sites in the phosphoproteome with lower scores, kinase specific. Table 3 Predictions from the GPS 6.0 Platform Position Code Kinase Peptide sequence Score 127 Ser CAMK/CAMKL/AMPK/AMPKα1 PQHVRAHSSPASLQL 0.0075 128 Ser CAMK/CAMKL/AMPK/AMPKα1 QHVRAHSSPASLQLG 0.0025 131 Ser CAMK/CAMKL/AMPK/AMPKα1 RAHSSPASLQLGAVS 0.0014 138 Ser CAMK/CAMKL/AMPK/AMPKα1 SLQLGAVSPGThrLThrPThr 0.0011 141 Thr CAMK/CAMKL/AMPK/AMPKα1 LGAVSPGThrLThrPThrGVV 0.0009 143 Thr CAMK/CAMKL/AMPK/AMPKα1 AVSPGThrLThrPThrGVVSG 0.0011 145 Thr CAMK/CAMKL/AMPK/AMPKα1 SPGThrLThrPThrGVVSGPA 0.0009 149 Ser CAMK/CAMKL/AMPK/AMPKα1 LThrPThrGVVSGPAAThrPThr 0.0007 154 Thr CAMK/CAMKL/AMPK/AMPKα1 VVSGPAAThrPThrAQHLR 0.0009 156 Thr CAMK/CAMKL/AMPK/AMPKα1 SGPAAThrPThrAQHLRQS 0.0015 Additional Declarations There is a duality of interest Supplementary Files ExtendingFigure1.jpg Extended Data Figure 1 ExtendingFigure2.jpg Extended Data Figure 2 ExtendingFigure3.jpg Extended Data Figure 3 SupplementaryInformationCDD.docx Supplementary Information Fulllengthuncroppedoriginalwesternblots.pdf Full length uncropped original western blots Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8422435","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":569465346,"identity":"cfb73e54-4a4f-42b5-a451-7c9fba06bab7","order_by":0,"name":"shihui 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Flow cytometry characterization of changes in the proportion of \u003cem\u003eVIM\u003c/em\u003e⁺\u003cem\u003eACTA2\u003c/em\u003e⁺ HDFs under 1, 15, and 25 kPa mechanical stimulation (n=3).\u003c/p\u003e\n\u003cp\u003eb. Western blotting images of \u003cem\u003eCOL I\u003c/em\u003e, \u003cem\u003eCOL III\u003c/em\u003e, and \u003cem\u003eACTA2\u003c/em\u003e protein levels under 1, 15, and 25 kPa mechanical stimulation.\u003c/p\u003e\n\u003cp\u003ec-e. Relative expression levels of \u003cem\u003eCOL I\u003c/em\u003e, \u003cem\u003eCOL III\u003c/em\u003e, and \u003cem\u003eACTA2\u003c/em\u003e compared to the low mechanical stimulation group, quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003ef. Changes in intracellular ADP content of HDFs under 1, 15, and 25 kPa mechanical stimulation (n=6).\u003c/p\u003e\n\u003cp\u003eg. Western blotting images of key glycolytic enzymes (\u003cem\u003eHK2\u003c/em\u003e, \u003cem\u003eLDHA\u003c/em\u003e) and oxidative phosphorylation (OXPHOS) enzymes (\u003cem\u003eMTCO2\u003c/em\u003e, \u003cem\u003eATP5A1\u003c/em\u003e) under 1, 15, and 25 kPa mechanical stimulation.\u003c/p\u003e\n\u003cp\u003eh. Relative expression levels of glycolytic enzymes (\u003cem\u003eHK2\u003c/em\u003e, \u003cem\u003eLDHA\u003c/em\u003e) compared to the low mechanical stimulation group, quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003ei. Relative expression levels of OXPHOS enzymes (\u003cem\u003eMTCO2\u003c/em\u003e, \u003cem\u003eATP5A1\u003c/em\u003e) compared to the low mechanical stimulation group, quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003ej. Extracellular acidification rate (ECAR).\u003c/p\u003e\n\u003cp\u003ek. Glycolysis capacity of ECAR (n=6).\u003c/p\u003e\n\u003cp\u003el. Oxygen consumption rate (OCR).\u003c/p\u003e\n\u003cp\u003em. ATP turnover capacity of OCR (n=6).\u003c/p\u003e\n\u003cp\u003en. Changes in intracellular lactate content of HDFs under 1, 15, and 25 kPa mechanical stimulation.\u003c/p\u003e\n\u003cp\u003eo. Relative mitochondrial immunofluorescence intensity of HDFs under different mechanical stresses (n=5).\u003c/p\u003e\n\u003cp\u003ep. MitoTracker Red CMXRos-labeled mitochondrial immunofluorescence images.\u003c/p\u003e\n\u003cp\u003eq. Changes in the intracellular ATP content of HDFs under 1, 15, and 25 kPa mechanical stimulation (n=6).\u003c/p\u003e\n\u003cp\u003er. Western blotting images of \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eYAP1\u003c/em\u003e, and their phosphorylated forms (\u003cem\u003ep-PRKAA1 \u003c/em\u003eand \u003cem\u003ep-YAP1\u003c/em\u003e) under 1, 15, and 25 kPa mechanical stimulation.\u003c/p\u003e\n\u003cp\u003es-t. Relative expression and phosphorylation levels of \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eYAP1\u003c/em\u003e, and their phosphorylated forms, quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003eu. Immunofluorescence images of \u003cem\u003eYAP1\u003c/em\u003e nuclear localization (FITC green channel) and merged DAPI staining under differential mechanical stress conditions.\u003c/p\u003e\n\u003cp\u003ev. Relative immunofluorescence intensity of \u003cem\u003eYAP1\u003c/em\u003e nuclear localization under differential mechanical stress (n=4).\u003c/p\u003e\n\u003cp\u003ew. Relative immunofluorescence intensity of \u003cem\u003eACTA2\u003c/em\u003e under different mechanical stresses (n=4). ​\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e: HDF, human dermal fibroblast; \u003cem\u003eVIM\u003c/em\u003e, vimentin; \u003cem\u003eACTA2\u003c/em\u003e, Actin Alpha 2, Smooth Muscle; \u003cem\u003eCOL I\u003c/em\u003e, collagen type I;\u003cem\u003e COL III\u003c/em\u003e, collagen type III; \u003cem\u003eHK2\u003c/em\u003e, Hexokinase 2; \u003cem\u003eLDHA\u003c/em\u003e, Lactate Dehydrogenase A; \u003cem\u003eMTCO2\u003c/em\u003e, Mitochondrially Encoded Cytochrome C Oxidase II; \u003cem\u003eATP5A1\u003c/em\u003e, ATP Synthase F1 Subunit Alpha; ADP, adenosine diphosphate; ATP, adenosine triphosphate; \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eAMPKα1\u003c/em\u003e, Protein Kinase AMP-Activated Catalytic Subunit Alpha 1; \u003cem\u003eYAP1\u003c/em\u003e, Yes1 Associated Transcriptional Regulator; \u003cem\u003eβ-TUB\u003c/em\u003e, β-Tubulin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rote/AA, rotenone/antimycin; 2-DG, 2-deoxyglucose. \u003cstrong\u003eStatistical notation\u003c/strong\u003e: Horizontal lines indicate comparisons between two groups; ns, nonsignificant; *，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/bc824c9a7ddd52ef6fb96458.jpg"},{"id":101401941,"identity":"19096caa-c6e5-4297-b99f-2f54a7acec3e","added_by":"auto","created_at":"2026-01-29 10:04:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1400643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological activation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKAA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with GSK621 effectively inhibits mechanical modulus-induced \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eYAP1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e activation and fibrosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Flow cytometry characterization of changes in the proportion of \u003cem\u003eVIM\u003c/em\u003e⁺\u003cem\u003eACTA2\u003c/em\u003e⁺ HDFs after GSK621 or Dorsomorphin intervention concurrent with mechanical stimulation (n=3).\u003c/p\u003e\n\u003cp\u003eb. Immunofluorescence images of \u003cem\u003eYAP1\u003c/em\u003e nuclear localization (FITC green channel) and merged DAPI staining under mechanical stimulation and drug intervention.\u003c/p\u003e\n\u003cp\u003ec. Relative immunofluorescence intensity of \u003cem\u003eYAP1\u003c/em\u003e nuclear localization (n=4).\u003c/p\u003e\n\u003cp\u003ed. Relative immunofluorescence intensity of \u003cem\u003eACTA2\u003c/em\u003e under different mechanical stresses (n=4).\u003c/p\u003e\n\u003cp\u003ee. Western blotting images of \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eYAP1\u003c/em\u003e, and their phosphorylated forms (p-\u003cem\u003ePRKAA1 \u003c/em\u003eand \u003cem\u003ep-YAP1\u003c/em\u003e), and of \u003cem\u003eACTA2\u003c/em\u003e, \u003cem\u003eCOL I\u003c/em\u003e, and \u003cem\u003eCOL III\u003c/em\u003eunder combined mechanical stimulation and drug treatment.\u003c/p\u003e\n\u003cp\u003ef-h. Relative expression and phosphorylation levels of \u003cem\u003eYAP1\u003c/em\u003e, \u003cem\u003ep-YAP1\u003c/em\u003e, \u003cem\u003eACTA2\u003c/em\u003e, and \u003cem\u003eCOL I\u003c/em\u003e/\u003cem\u003eCOL III\u003c/em\u003e ratio compared to the simple mechanical stimulation group, and quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003ei. Masson staining images of mouse dorsal full-thickness defect wounds under 25 kPa mechanical stress on day 20.\u003c/p\u003e\n\u003cp\u003ej. Sirius Red-stained images of mouse dorsal full-thickness defect wounds under 25 kPa mechanical stress on day 20.\u003c/p\u003e\n\u003cp\u003ek. Differences in the \u003cem\u003eCOL I\u003c/em\u003e/\u003cem\u003eCOL III\u003c/em\u003e ratio among different mechanical stimuli on day 20, as quantified by Sirius Red staining (n=3).\u003c/p\u003e\n\u003cp\u003el. Immunofluorescence staining images of \u003cem\u003eACTA2\u003c/em\u003e in wounds under 25 kPa mechanical stimulation and drug intervention on day 10.\u003c/p\u003e\n\u003cp\u003em. Relative immunofluorescence intensity of \u003cem\u003eACTA2\u003c/em\u003e in wounds under differential mechanical stimulation and drug intervention on day 10 (n=5).\u003c/p\u003e\n\u003cp\u003en-p. Relative phosphorylation level of \u003cem\u003eYAP1\u003c/em\u003e, relative expression level of \u003cem\u003eACTA2\u003c/em\u003e, and relative \u003cem\u003eCOL I\u003c/em\u003e/\u003cem\u003eCOL III\u003c/em\u003e ratio in wound tissue on day 20 under differential mechanical stimulation and drug intervention, quantified by Western blotting (n=5).\u003c/p\u003e\n\u003cp\u003eq. Western blotting images of \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eYAP1\u003c/em\u003e and its phosphorylated forms, \u003cem\u003eACTA2\u003c/em\u003e, \u003cem\u003eCOL I\u003c/em\u003e, and \u003cem\u003eCOL III\u003c/em\u003e in wound tissue on day 20 under differential mechanical stimulation and drug intervention. ​\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e: HDF, human dermal fibroblast; \u003cem\u003eVIM\u003c/em\u003e, vimentin; \u003cem\u003eACTA2\u003c/em\u003e, Actin Alpha 2, Smooth Muscle; \u003cem\u003eCOL I\u003c/em\u003e, collagen type I; \u003cem\u003eCOL III\u003c/em\u003e, collagen type III; \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eAMPKα1\u003c/em\u003e, Protein Kinase AMP-Activated Catalytic Subunit Alpha 1; \u003cem\u003eYAP1\u003c/em\u003e, Yes1 Associated Transcriptional Regulator; \u003cem\u003eβ-TUB\u003c/em\u003e, β-Tubulin. ​\u003cstrong\u003eStatistical notation\u003c/strong\u003e: Horizontal lines indicate comparisons between two groups; ns, non-significant; *，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/bb89f597d9c8e05d602353ee.jpg"},{"id":101401955,"identity":"486a8c36-d9a5-4312-a7be-c30d852e0f3e","added_by":"auto","created_at":"2026-01-29 10:04:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":936743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\n\u003cstrong\u003eAbbreviations\u003c/strong\u003e: HDF, human dermal fibroblast; OE, over-expression; KD, knockdown; \u003cem\u003eACTA2\u003c/em\u003e, Actin Alpha 2, Smooth Muscle; \u003cem\u003eCOL I\u003c/em\u003e, collagen type I; \u003cem\u003eCOL III\u003c/em\u003e, collagen type III; \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eAMPKα1\u003c/em\u003e, Protein Kinase AMP-Activated Catalytic Subunit Alpha 1; \u003cem\u003eYAP1\u003c/em\u003e, Yes1 Associated Transcriptional Regulator; \u003cem\u003eβ-TUB\u003c/em\u003e, β-Tubulin.\u003cbr\u003e\n\u003cstrong\u003eStatistical notation\u003c/strong\u003e: Horizontal lines indicate comparisons between two groups; ns, non-significant;\u003cem\u003e P \u0026lt; 0.05;\u003c/em\u003e *，\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05; **，\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.01; ***，\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.001; ****，\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePRKAA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e activation inhibits mechanical modulus-induced fibrosis by suppressing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eYAP1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Western blotting images of intracellular \u003cem\u003eYAP1\u003c/em\u003e phosphorylation levels and \u003cem\u003eACTA2\u003c/em\u003e expression in cells treated with GSK621 under 25 kPa mechanical stimulation and \u003cem\u003eLATS1\u003c/em\u003e knockdown (LATS1\u003csup\u003eKD\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003eb-c. Relative phosphorylation level of \u003cem\u003eYAP1\u003c/em\u003e and relative expression level of \u003cem\u003eACTA2\u003c/em\u003e, quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003ed. Relative \u003cem\u003eACTA2\u003c/em\u003e expression levels in \u003cem\u003eYAP1\u003c/em\u003e-overexpressing (\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e) or \u003cem\u003eYAP1\u003c/em\u003e knockdown (\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e) cells after GSK621 treatment, assessed by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003ee. Immunofluorescence images of \u003cem\u003eYAP1\u003c/em\u003e nuclear localization in WT+GSK621, \u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e+GSK621, \u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e+GSK621, \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e+GSK621, \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e+GSK621, and \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e+GSK621 cells under 25 kPa mechanical stimulation.\u003c/p\u003e\n\u003cp\u003ef. Western blotting images of \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eYAP1\u003c/em\u003e, their phosphorylated forms, and \u003cem\u003eACTA2\u003c/em\u003e in cells with \u003cem\u003ePRKAA1\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e overexpression or knockdown.\u003c/p\u003e\n\u003cp\u003eg-h. Relative \u003cem\u003eYAP1\u003c/em\u003e phosphorylation level and relative \u003cem\u003eACTA2\u003c/em\u003e expression level in \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e cells after GSK621 intervention, quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003ei-j. Relative \u003cem\u003eYAP1\u003c/em\u003e phosphorylation level and relative \u003cem\u003eACTA2\u003c/em\u003e expression level in \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e cells after GSK621 intervention, quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003ek-l. Sirius Red staining images and \u003cem\u003eCOL I\u003c/em\u003e/\u003cem\u003eCOL III\u003c/em\u003e ratio comparison of mouse dorsal full-thickness defect wounds on day 20 under 25 kPa mechanical stimulation, with or without GSK621 intervention (n=3).\u003c/p\u003e\n\u003cp\u003em. Immunofluorescence staining images of \u003cem\u003eACTA2\u003c/em\u003e in wound tissue on day 20.\u003c/p\u003e\n\u003cp\u003en. Western blotting images of \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eYAP1\u003c/em\u003e, its phosphorylated forms, \u003cem\u003eACTA2\u003c/em\u003e, \u003cem\u003eCOL I\u003c/em\u003e, and \u003cem\u003eCOL III\u003c/em\u003e in wound tissue on day 20.\u003c/p\u003e\n\u003cp\u003eo-r. Relative phosphorylation level of \u003cem\u003ePRKAA1\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e, relative expression level of \u003cem\u003eACTA2\u003c/em\u003e, and relative \u003cem\u003eCOL I\u003c/em\u003e/\u003cem\u003eCOL III\u003c/em\u003e ratio in wound tissue, quantified by Western blotting (n=5).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/fd7f3e077ea519bdcab62659.jpg"},{"id":101401952,"identity":"d9954c54-b476-4734-ae38-860d531f6b7f","added_by":"auto","created_at":"2026-01-29 10:04:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1009039,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\n\u003cstrong\u003eAbbreviations\u003c/strong\u003e: HDF, human dermal fibroblast; OE, overexpression; KD, knockdown; \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eAMPKα1\u003c/em\u003e, Protein Kinase AMP-Activated Catalytic Subunit Alpha 1; \u003cem\u003eYAP1\u003c/em\u003e, Yes1 Associated Transcriptional Regulator; \u003cem\u003eβ-TUB\u003c/em\u003e, β-Tubulin; \u003cem\u003eTEAD1\u003c/em\u003e, TEA domain transcription factor 1; \u003cem\u003eCTGF\u003c/em\u003e, connective tissue growth factor; \u003cem\u003eCYR61\u003c/em\u003e, cysteine-rich angiogenic inducer 61; CHX, cycloheximide.\u003cbr\u003e\n\u003cstrong\u003eStatistical notation\u003c/strong\u003e: Horizontal lines indicate comparisons between two groups; ns, non-significant; *，\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05; **，\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.01; ***，\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.001; ****，\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePRKAA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e regulates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eYAP1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protein stability via direct \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLATS1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-independent interactions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Computational prediction of the \u003cem\u003ePRKAA1\u003c/em\u003e-\u003cem\u003eYAP1\u003c/em\u003e protein interaction using\u003cbr\u003e\n\u003cstrong\u003eRosetta\u003c/strong\u003e\u003cbr\u003e\nsoftware, with 3D visualization via PyMOL.\u003c/p\u003e\n\u003cp\u003eb. Co-immunoprecipitation (Co-IP) Western blotting images of \u003cem\u003ePRKAA1\u003c/em\u003e and \u003cem\u003eYAP1\u003c/em\u003e in HDFs with or without GSK621 treatment.\u003c/p\u003e\n\u003cp\u003ec. In vitro GST pull-down Western blotting images of recombinant\u003cem\u003e PRKAA1\u003c/em\u003e-GST and \u003cem\u003eYAP1\u003c/em\u003e-Flag proteins.\u003c/p\u003e\n\u003cp\u003ed. Western blotting images of \u003cem\u003eYAP1 and its \u003c/em\u003eubiquitination in vector, \u003cem\u003ePRKAA1\u003c/em\u003e-overexpressing (\u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e), and \u003cem\u003ePRKAA1\u003c/em\u003e-knockdown (\u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e) HDFs treated with GSK621 and CHX (cycloheximide), with the proteasome pathway manipulated by MG132.\u003c/p\u003e\n\u003cp\u003ee. Relative \u003cem\u003eYAP1\u003c/em\u003e expression levels in vector, \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e HDFs (inversely correlated with the degradation rate, n=3).\u003c/p\u003e\n\u003cp\u003ef. Relative \u003cem\u003eYAP1\u003c/em\u003e ubiquitination levels in vector, \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e HDFs (inversely correlated with degradation rate, n=3).\u003c/p\u003e\n\u003cp\u003eg. Western blotting images of \u003cem\u003eYAP1\u003c/em\u003e and its phosphorylated forms in HDFs with \u003cem\u003eYAP1-Ser127\u003c/em\u003e, \u003cem\u003e-Ser128\u003c/em\u003e, or \u003cem\u003e-Thr156\u003c/em\u003e mutations after GSK621 treatment.\u003c/p\u003e\n\u003cp\u003eh. Changes in the relative phosphorylation level of \u003cem\u003eYAP1\u003c/em\u003e after mutations of \u003cem\u003eYAP1 \u003c/em\u003eat\u003cem\u003e Ser127\u003c/em\u003e, \u003cem\u003eSer128\u003c/em\u003e, or \u003cem\u003eThr156\u003c/em\u003e (n=3).\u003c/p\u003e\n\u003cp\u003ei. Co-IP Western blotting images of the \u003cem\u003eYAP1\u003c/em\u003e-\u003cem\u003eTEAD1\u003c/em\u003e interaction in \u003cem\u003eYAP1\u003c/em\u003e-\u003cem\u003eSer127\u003c/em\u003e or -\u003cem\u003eThr156\u003c/em\u003e HDFs treated with GSK621.\u003c/p\u003e\n\u003cp\u003ej. Relative binding levels of \u003cem\u003eYAP1\u003c/em\u003e-\u003cem\u003eTEAD1\u003c/em\u003e in \u003cem\u003eYAP1\u003c/em\u003e-\u003cem\u003eSer127\u003c/em\u003e or -\u003cem\u003eThr156\u003c/em\u003e HDFs (n=3).\u003c/p\u003e\n\u003cp\u003ek. Relative \u003cem\u003eTEAD1\u003c/em\u003e binding to \u003cem\u003eCTGF\u003c/em\u003e or \u003cem\u003eCYR61\u003c/em\u003e gene promoter regions in \u003cem\u003eYAP1\u003c/em\u003e-\u003cem\u003eSer127\u003c/em\u003e, -\u003cem\u003eSer128\u003c/em\u003e, or -\u003cem\u003eThr156\u003c/em\u003e mutated HDFs treated with GSK621.\u003c/p\u003e\n\u003cp\u003el. Relative mRNA expression levels of \u003cem\u003eCTGF\u003c/em\u003e and \u003cem\u003eCYR61\u003c/em\u003e in \u003cem\u003eYAP1\u003c/em\u003e-\u003cem\u003eSer127\u003c/em\u003e, -\u003cem\u003eSer128\u003c/em\u003e, or -\u003cem\u003eThr156\u003c/em\u003e mutated HDFs treated with GSK621 (n=6).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/b5b5515fae7c6ed72f78469f.jpg"},{"id":101401956,"identity":"19032545-f533-46cf-a295-b098984187ef","added_by":"auto","created_at":"2026-01-29 10:04:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1651117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePRKAA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eactivation initiates metabolic reprogramming to ameliorate fibrosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Changes in intracellular ATP content in HDFs under 1 kPa or 25 kPa mechanical stimulation with GSK621 or Dorsomorphin intervention (n=6).\u003c/p\u003e\n\u003cp\u003eb. Changes in intracellular ADP content under the same conditions (n=6).\u003c/p\u003e\n\u003cp\u003ec. Extracellular acidification rate (ECAR).\u003c/p\u003e\n\u003cp\u003ed. Oxygen consumption rate (OCR).\u003c/p\u003e\n\u003cp\u003ee. Glycolysis capacity of ECAR (n=6).\u003c/p\u003e\n\u003cp\u003ef. ATP turnover capacity of OCR (n=6).\u003c/p\u003e\n\u003cp\u003eg. Western blotting images of key glycolytic enzymes (\u003cem\u003eHK2\u003c/em\u003e, \u003cem\u003eLDHA\u003c/em\u003e), oxidative phosphorylation (OXPHOS) enzymes (\u003cem\u003eMTCO2\u003c/em\u003e, \u003cem\u003eATP5A1\u003c/em\u003e), and mitochondrial fission proteins (\u003cem\u003eDRP1\u003c/em\u003e, \u003cem\u003eFIS1\u003c/em\u003e) under 1/25 kPa mechanical stimulation and drug intervention.\u003c/p\u003e\n\u003cp\u003eh. Relative expression levels of glycolytic enzymes (\u003cem\u003eHK2\u003c/em\u003e, \u003cem\u003eLDHA\u003c/em\u003e) quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003ei. Relative expression levels of OXPHOS enzymes (\u003cem\u003eMTCO2\u003c/em\u003e, \u003cem\u003eATP5A1\u003c/em\u003e) quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003ej. Relative intracellular lactate content under 1/25 kPa mechanical stimulation and drug intervention (n=6).\u003c/p\u003e\n\u003cp\u003ek. Relative expression levels of mitochondrial fission proteins (\u003cem\u003eDRP1 \u003c/em\u003eand \u003cem\u003eFIS1\u003c/em\u003e) quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003el. Relative mitochondrial immunofluorescence intensity of HDFs under differential mechanical stress and drug intervention conditions (n=5).\u003c/p\u003e\n\u003cp\u003em. MitoTracker Red CMXRos-labeled mitochondrial immunofluorescence images.\u003c/p\u003e\n\u003cp\u003en. Heatmap of differential metabolites in HDFs under 25 kPa mechanical stimulation: \u003cem\u003ePRKAA1\u003c/em\u003eactivation (HA) vs. control (HN) (n=6).\u003c/p\u003e\n\u003cp\u003eo. Integrated metabolomic-transcriptomic network analysis comparing the HA and HN groups.\u003c/p\u003e\n\u003cp\u003ep. Immunoblotting images of \u003cem\u003eACTA2\u003c/em\u003e protein in HDFs (human dermal fibroblasts) under high mechanical modulus conditions and after treatment with different drugs.\u003c/p\u003e\n\u003cp\u003eq. Relative expression levels of \u003cem\u003eACTA2\u003c/em\u003e quantified by Western blotting (n=3).\u003c/p\u003e\n\u003cp\u003er. Immunofluorescence images of \u003cem\u003eACTA2\u003c/em\u003e (red channel) and merged DAPI staining under mechanical stimulation and drug intervention.\u003c/p\u003e\n\u003cp\u003es. Relative immunofluorescence intensity of \u003cem\u003eACTA2\u003c/em\u003e (n=4).​\u003c/p\u003e\n\u003cp\u003et. The schematic work model of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e: HDF, human dermal fibroblast; \u003cem\u003eHK2\u003c/em\u003e, Hexokinase 2; \u003cem\u003eLDHA\u003c/em\u003e, Lactate Dehydrogenase A; \u003cem\u003eMTCO2\u003c/em\u003e, Mitochondrially Encoded Cytochrome C Oxidase II; \u003cem\u003eATP5A1\u003c/em\u003e, ATP Synthase F1 Subunit Alpha; ADP, adenosine diphosphate; ATP, adenosine triphosphate; \u003cem\u003ePRKAA1\u003c/em\u003e, \u003cem\u003eAMPKα1\u003c/em\u003e, Protein Kinase AMP-Activated Catalytic Subunit Alpha 1; \u003cem\u003eDRP1\u003c/em\u003e, Dynamin-Related Protein 1; \u003cem\u003eFIS1\u003c/em\u003e, Mitochondrial Fission 1; \u003cem\u003eβ-TUB\u003c/em\u003e, β-Tubulin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rote/AA, rotenone/antimycin; 2-DG, 2-deoxyglucose; HA, \u003cem\u003ePRKAA1\u003c/em\u003e activation group; HN, high mechanical modulus control group; \u003cem\u003eACTA2\u003c/em\u003e, Actin Alpha 2, Smooth Muscle. ​\u003cstrong\u003eStatistical notation\u003c/strong\u003e: Horizontal lines indicate comparisons between two groups; ns, non-significant; *，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****，\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/7a8981804efe1d5f38f33459.jpg"},{"id":101751642,"identity":"52774241-053b-44e2-8d23-ea083a28d11d","added_by":"auto","created_at":"2026-02-03 10:21:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8382606,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/c8f12b87-189c-4e62-a975-2cc19cb11399.pdf"},{"id":101401938,"identity":"f0e90c4b-2330-462d-a300-43ce3972731d","added_by":"auto","created_at":"2026-01-29 10:04:07","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":877118,"visible":true,"origin":"","legend":"Extended Data Figure 1","description":"","filename":"ExtendingFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/2558f9372b0891792430ffeb.jpg"},{"id":101401936,"identity":"7bdd1d15-8cf6-4dc1-93b4-a8490e2ff314","added_by":"auto","created_at":"2026-01-29 10:04:07","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2053802,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Figure 2\u003c/p\u003e","description":"","filename":"ExtendingFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/e98454d60e969bd8ade48c3c.jpg"},{"id":101401954,"identity":"66bdfef6-a864-49ce-8e1d-b4550c2b82f5","added_by":"auto","created_at":"2026-01-29 10:04:11","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1589644,"visible":true,"origin":"","legend":"Extended Data Figure 3","description":"","filename":"ExtendingFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/4a3c388770bff8be811fd3b0.jpg"},{"id":101401957,"identity":"698b9f7d-eb39-424c-bf00-cd660dd5e384","added_by":"auto","created_at":"2026-01-29 10:04:12","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1969985,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformationCDD.docx","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/9f301fd7b88b65ee1f6a215d.docx"},{"id":101401953,"identity":"3c29c952-69db-4e26-889d-7ab4a66166cb","added_by":"auto","created_at":"2026-01-29 10:04:10","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12152006,"visible":true,"origin":"","legend":"\u003cp\u003eFull length uncropped original western blots\u003c/p\u003e","description":"","filename":"Fulllengthuncroppedoriginalwesternblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8422435/v1/9a7caf42449b621c0f1ec67a.pdf"}],"financialInterests":"There is a duality of interest","formattedTitle":"The synergistic inhibitory effect of PRKAA1 activation on ECM mechanical stress-induced skin fibrosis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSkin fibrosis is a pathological outcome of wound healing and is characterized by excessive extracellular matrix (ECM) deposition and myofibroblast activation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Researchers have extensively investigated the mechanisms by which mechanical stress-generated by skin stretching and ECM stiffening-regulates fibrosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Mascharak et al. proposed that blocking \u003cem\u003eYAP1\u003c/em\u003e-mediated mechanical transduction signaling promotes regeneration, establishing \u003cem\u003eYAP1\u003c/em\u003e as a central mediator of mechanotransduction\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In addition to the classical \u003cem\u003eHippo\u003c/em\u003e/\u003cem\u003eLATS\u003c/em\u003e pathway, other pathways and molecules (e.g., \u003cem\u003eRhoA\u003c/em\u003e/\u003cem\u003eROCK\u003c/em\u003e, \u003cem\u003eIntegrinβ1\u003c/em\u003e/\u003cem\u003eFAK\u003c/em\u003e/\u003cem\u003eSrc\u003c/em\u003e, and \u003cem\u003ePiezo1\u003c/em\u003e) modulate \u003cem\u003eYAP1\u003c/em\u003e activity by controlling cytoskeletal dynamics and kinase activity, thereby inhibiting fibrosis progression\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These findings have further clarified the regulatory network through which external mechanical stress induces fibrosis via cellular mechanotransduction. However, processes\u0026mdash;particularly energy metabolism\u0026mdash;synergize with mechanotransduction to regulate fibrosis remains poorly understood.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that mechanical stimulation can induce ​metabolic reprogramming​ in cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. As a critical energy-sensing kinase, \u003cem\u003eAMPK\u003c/em\u003e plays a pivotal role in cellular metabolic regulation. The \u003cem\u003ecAMP/AMPK\u003c/em\u003e signaling axis, a key regulator of energy metabolism, inherently has the potential to establish regulatory communication with signal transduction processes activated by mechanical stimuli\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Rather than merely mediating basic energy storage and supply conversion, \u003cem\u003ecAMP/AMPK\u003c/em\u003e may have essential regulatory functions in ​mechanoresponses​ and fibrosis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Existing evidence reveals that under high mechanical modulus stimulation, cells enter an ​energy stress state, which is​ characterized by activated \u003cem\u003eAMPK\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eAMPK\u003c/em\u003e activation through pharmacological means reduces collagen release and inflammation by influencing \u003cem\u003eTGF-β, mTOR, NF-κB\u003c/em\u003e, and mitochondrial activity, thus preventing fibrosis\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The paradox of \u003cem\u003eAMPK\u003c/em\u003e lies in its ​disconnectedness between metabolic and mechanical cues: while it senses energy status, it lacks clear links to mechanical signals. Phosphorylation serves as a key post-translational modification (PTM) that acts as a functional switch for \u003cem\u003eYAP1\u003c/em\u003e; as an energy sensor, \u003cem\u003eAMPK\u003c/em\u003e may influence \u003cem\u003eYAP1\u003c/em\u003e activity through direct or indirect phosphorylation pathways. Therefore, investigating whether the kinase \u003cem\u003eAMPK\u003c/em\u003e directly phosphorylates \u003cem\u003eYAP1\u003c/em\u003e and regulates its upstream/downstream signaling will help elucidate the signaling network of cells under mechanical stress and metabolic reprogramming, providing novel insights and strategies for scar treatment.\u003c/p\u003e \u003cp\u003eHere, we integrated molecular biology experiments with multiomics techniques to demonstrate that ​pharmacological activation of \u003cem\u003ePRKAA1\u003c/em\u003e directly phosphorylates \u003cem\u003eYAP1\u003c/em\u003e at \u003cem\u003eSer127\u003c/em\u003e and \u003cem\u003eThr156\u003c/em\u003e under high mechanical stress. This modification promotes \u003cem\u003eYAP1\u003c/em\u003e degradation via the proteasome pathway and inhibits its nuclear translocation, which in turn reduces \u003cem\u003eYAP1-TEAD1\u003c/em\u003e binding and suppresses downstream profibrotic factor expression. Additionally, metabolic reprogramming induced by \u003cem\u003ePRKAA1\u003c/em\u003e activation enhances energy supply and alters \"regenerative\" metabolic intermediates. The ​synergistic interplay of \u003cem\u003ePRKAA1\u003c/em\u003e-mediated phosphorylation and metabolic reprogramming​ ultimately results in fibrosis inhibition. Our study\u0026rsquo;s key breakthrough lies in revealing that ​phosphorylation regulation and metabolic reprogramming are not isolated events\u0026mdash;they act synergistically to form the core mechanism through which \u003cem\u003ePRKAA1\u003c/em\u003e inhibits fibrosis. This work substantially expands our understanding of the complex crosstalk between mechanical signal transduction and cellular metabolism, and elucidates in detail how mechanical stress precisely regulates \u003cem\u003eYAP1\u003c/em\u003e activity via the critical node \u003cem\u003ePRKAA1\u003c/em\u003e. It provides new molecular insights into mechanobiology and offers promising therapeutic targets for fibrotic diseases such as pathological scars in clinical practice.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eHigh mechanical stress induces the differentiation of human dermal fibroblasts (HDFs) into myofibroblasts (MFs) and energy stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMechanical stress not only affects cell morphology and function but also promotes cell differentiation towards specific lineages by regulating gene expression and signaling pathways. To investigate the critical role of mechanical cues in modulating cell behavior and fibrosis, we ​seeded HDFs onto hydrogels of varying stiffness​ (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b) to simulate static mechanical stress. While observing HDF differentiation and energy stress, we explored the correlation between \u003cem\u003eYAP1\u003c/em\u003e and \u003cem\u003ePRKAA1\u003c/em\u003e activity. Flow cytometry revealed that as mechanical stress increased, the proportion of \u003cem\u003e​Vimentin\u003c/em\u003e⁺ and \u003cem\u003eACTA2\u003c/em\u003e⁺ myofibroblasts (MFs)​​ increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Concurrently, the expression of \u003cem\u003eACTA2\u003c/em\u003e, the \u003cem\u003eCOL I/COL III\u003c/em\u003e ratio, and levels of other profibrotic molecules were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-e, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), indicating enhanced HDF-to-MF differentiation. Moreover, ADP levels increased with higher mechanical stress, suggesting that cellular energy demand increased under stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). The detection of key metabolic enzymes revealed ​enhanced glycolysis and oxidative phosphorylation (OXPHOS)​​ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-i), reflecting increased energy supply. Further analysis using ​the extracellular acidification rate (ECAR; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej-k; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee)​​ (a proxy for glycolysis) and the ​oxygen consumption rate (OCR; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el-m, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef)​​ (a proxy for OXPHOS) confirmed significantly elevated glycolytic and OXPHOS activity. Concurrently, increasing lactate levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003en) indicated that glycolysis dominated cellular metabolism. The mitochondrial activity and fission capacity were also enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eo-p, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-h). These findings collectively indicate that HDFs slowly transform into MFs when subjected to high mechanical stress, a process that is mainly fueled by increased glycolysis leading to energy stress. Next, we assessed \u003cem\u003eYAP1\u003c/em\u003e and \u003cem\u003ePRKAA1\u003c/em\u003e activity. Consistent with expectations, both \u003cem\u003ePRKAA1\u003c/em\u003e expression and phosphorylation levels increased significantly under energy stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003er-s), suggesting that metabolic reprogramming was initiated to boost the ATP supply (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eq). \u003cem\u003eYAP1\u003c/em\u003e expression and activity (reflected by its unphosphorylated form) also increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003et), alongside increased \u003cem\u003eYAP1\u003c/em\u003e nuclear translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eu-v); this change aligned with the upregulation of the MF marker \u003cem\u003eACTA2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ew, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). Collectively, these results indicate that \u003cem\u003eYAP1\u003c/em\u003e activation is correlated with enhanced MF differentiation and fibrotic factor expression, indicating that \u003cem\u003eYAP1\u003c/em\u003e is a critical mediator of mechanical stress-induced fibrotic phenotypes. However, a notable finding emerged: ​\u003cem\u003ePRKAA1\u003c/em\u003e phosphorylation levels exhibited an inverse trend to \u003cem\u003eYAP1\u003c/em\u003e phosphorylation. These observations contradict the established role of \u003cem\u003ePRKAA1\u003c/em\u003e as a \u003cem\u003eYAP1\u003c/em\u003e kinase, suggesting that the regulatory relationship between \u003cem\u003ePRKAA1\u003c/em\u003e and \u003cem\u003eYAP1\u003c/em\u003e may be more complex under these specific mechanical conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePharmacological activation of\u003c/b\u003e \u003cb\u003ePRKAA1\u003c/b\u003e \u003cb\u003eattenuates mechanical stress-induced fibrosis progression by inhibiting\u003c/b\u003e \u003cb\u003eYAP1\u003c/b\u003e \u003cb\u003enuclear translocation.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate whether the regulatory effect of \u003cem\u003ePRKAA1\u003c/em\u003e on \u003cem\u003eYAP1\u003c/em\u003e is constrained under ​basal conditions, we pharmacologically modulated \u003cem\u003ePRKAA1\u003c/em\u003e activity. Activation of \u003cem\u003ePRKAA1\u003c/em\u003e with the \u003cem\u003eAMPK\u003c/em\u003e agonist GSK621 led to a significant decrease in the proportion of \u003cem\u003eVimentin\u003c/em\u003e⁺ and \u003cem\u003eACTA2\u003c/em\u003e⁺ cells among HDFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), indicating ​marked inhibition of myofibroblast (MF) differentiation. Consistently, both \u003cem\u003eYAP1\u003c/em\u003e nuclear translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d) and activity (which inversely correlated with its phosphorylation level; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-f) were suppressed. Notably, regarding extracellular matrix (ECM) components, the expression of the MF marker \u003cem\u003eACTA2\u003c/em\u003e and the \u003cem\u003eCOL I\u003c/em\u003e/\u003cem\u003eCOL III\u003c/em\u003e ratio were decreased following \u003cem\u003ePRKAA1\u003c/em\u003e activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-h, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). Conversely, ​\u003cem\u003eCOL III\u003c/em\u003e expression increased​ upon \u003cem\u003ePRKAA1\u003c/em\u003e activation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-g). In contrast, inhibiting \u003cem\u003ePRKAA1\u003c/em\u003e with the \u003cem\u003eAMPK\u003c/em\u003e inhibitor Dorsomorphin induced ​opposite phenotypic changes. Together, these results confirm that \u003cem\u003ePRKAA1\u003c/em\u003e activity ​negatively regulates \u003cem\u003eYAP1\u003c/em\u003e activity and function. More importantly, the upregulation of \u003cem\u003eYAP1\u003c/em\u003e activity upon \u003cem\u003ePRKAA1\u003c/em\u003e inhibition suggests that under physiological mechanical stress, endogenous \u003cem\u003ePRKAA1\u003c/em\u003e activation may be insufficient to fully phosphorylate and inhibit the active form of \u003cem\u003eYAP1\u003c/em\u003e, which accumulates in response to stress. In contrast, pharmacological activation potentiates this function, effectively reversing \u003cem\u003eYAP1\u003c/em\u003e-driven fibrotic phenotypes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the impact of mechanical stress on fibrosis during ​in vivo wound healing, we applied controlled mechanical stress to full-thickness skin defects on the backs of C57BL/6 mice using hydrogels. We then locally injected drugs around and in the wound bed to modulate \u003cem\u003ePRKAA1\u003c/em\u003e activity and assess fibrosis progression. Masson's trichrome staining revealed that the activation of \u003cem\u003ePRKAA1\u003c/em\u003e in conditions of low mechanical modulus led to distinct collagen fiber alignment, akin to that in the control group and normal skin. However, \u003cem\u003ePRKAA1\u003c/em\u003e inhibition led to disorganized collagen fiber deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Sirius Red staining revealed that \u003cem\u003ePRKAA1\u003c/em\u003e activation significantly reduced the \u003cem\u003eCOL I\u003c/em\u003e/\u003cem\u003eCOL III\u003c/em\u003e ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej-k, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei-j). Additionally, activating \u003cem\u003ePRKAA1\u003c/em\u003e with GSK621 effectively reversed the mechanical stress-induced upregulation of \u003cem\u003eACTA2\u003c/em\u003e in the dermis\u0026mdash;an outcome consistent with our in vitro findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el-m, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). Further Western blotting and immunofluorescence analyses revealed that \u003cem\u003ePRKAA1\u003c/em\u003e activation significantly downregulated \u003cem\u003eYAP1\u003c/em\u003e activity and the expression of \u003cem\u003eACTA2\u003c/em\u003e, while also reducing the \u003cem\u003eCOL I\u003c/em\u003e/\u003cem\u003eCOL III\u003c/em\u003e ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003en-q), indicating the inhibition of skin fibrosis progression. Deeper analysis revealed that this reduced ratio was primarily due to ​increased \u003cem\u003eCOL III\u003c/em\u003e secretion​ (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el-m), which is consistent with the findings of previous studies suggesting that elevated \u003cem\u003eCOL III\u003c/em\u003e attenuates tissue fibrosis\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In the late healing phase, \u003cem\u003eACTA2\u003c/em\u003e-high cells were enriched primarily in the superficial dermis\u0026mdash;a spatial distribution consistent with the current findings\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, \u003cem\u003ePRKAA1\u003c/em\u003e inhibition with Dorsomorphin reversed these phenomena, further validating the efficacy of \u003cem\u003ePRKAA1\u003c/em\u003e activation in inhibiting fibrosis. Together, these ​in vivo data​ confirm that \u003cem\u003ePRKAA1\u003c/em\u003e activation effectively inhibits mechanical stress-induced skin wound fibrosis by regulating \u003cem\u003eYAP1\u003c/em\u003e activity and collagen expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePRKAA1\u003c/b\u003e \u003cb\u003e-dependent\u003c/b\u003e \u003cb\u003eYAP1\u003c/b\u003e \u003cb\u003eregulation is key to inhibiting fibrosis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate whether there is a ​direct upstream/downstream regulatory relationship​ between \u003cem\u003ePRKAA1\u003c/em\u003e and \u003cem\u003eYAP1\u003c/em\u003e, we performed in-depth validation using genetic editing tools. Even under ​\u003cem\u003eLATS1\u003c/em\u003e knockdown (\u003cem\u003eLATS1\u003c/em\u003e\u003csup\u003eKD\u003c/sup\u003e)​, activation of \u003cem\u003ePRKAA1\u003c/em\u003e with GSK621 still effectively induced \u003cem\u003eYAP1\u003c/em\u003e phosphorylation and significantly inhibited the expression of the myofibroblast marker \u003cem\u003eACTA2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This key finding suggests that the phosphorylation of \u003cem\u003eYAP1\u003c/em\u003e by \u003cem\u003ePRKAA1\u003c/em\u003e is ​independent of the classical Hippo pathway core kinase \u003cem\u003eLATS1\u003c/em\u003e, initially establishing \u003cem\u003ePRKAA1\u003c/em\u003e as an ​upstream regulator​ of \u003cem\u003eYAP1\u003c/em\u003e. To further confirm the role of \u003cem\u003ePRKAA1\u003c/em\u003e in \u003cem\u003eYAP1\u003c/em\u003e activity, we generated cell lines with: \u003cem\u003ePRKAA1\u003c/em\u003e overexpression (\u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e), \u003cem\u003ePRKAA1\u003c/em\u003e knockdown (\u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003eKD\u003c/sup\u003e), \u003cem\u003eYAP1\u003c/em\u003e overexpression (\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e), \u003cem\u003eYAP1\u003c/em\u003e knockdown (\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003eKD\u003c/sup\u003e), and double manipulations (\u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e and \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003eKD\u003c/sup\u003e\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e). When \u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e cells were treated with GSK621, their \u003cem\u003eACTA2\u003c/em\u003e expression was significantly higher than that in the ​vector-transfected group. In contrast, \u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003eKD\u003c/sup\u003e cells presented reduced \u003cem\u003eYAP1\u003c/em\u003e nuclear translocation, and compared with the vector control, GSK621 treatment had no significant effect on \u003cem\u003eACTA2\u003c/em\u003e expression, although both of these parameters remained lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Flow cytometry results also demonstrated that \u003cem\u003eYAP1\u003c/em\u003e overexpression significantly increased the ​proportion of myofibroblast differentiation, whereas \u003cem\u003eYAP1\u003c/em\u003e knockdown had the opposite effect (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These findings indicate that \u003cem\u003eACTA2\u003c/em\u003e and myofibroblast differentiation are regulated by \u003cem\u003eYAP1\u003c/em\u003e expression and activity, further supporting the role of \u003cem\u003eYAP1\u003c/em\u003e as a ​core molecule in mechanotransduction​ and providing a theoretical basis for subsequent validation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with those in vector group, both ​\u003cem\u003eYAP1\u003c/em\u003e activity​ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg) and ​nuclear translocation​ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) were significantly decreased in GSK621-treated \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Additionally, the proportion of myofibroblast differentiation and \u003cem\u003eACTA2\u003c/em\u003e expression were markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Conversely, in \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003eKD\u003c/sup\u003e cells, even with GSK621 treatment, \u003cem\u003eYAP1\u003c/em\u003e activity was significantly higher than in the vector\u0026thinsp;+\u0026thinsp;GSK621 group. These findings from both overexpression and knockdown experiments confirm that \u003cem\u003ePRKAA1\u003c/em\u003e is a ​critical upstream negative regulator​ of \u003cem\u003eYAP1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTaken together, these results suggest that under ​basal physiological conditions, the activation level of endogenous \u003cem\u003ePRKAA1\u003c/em\u003e is likely a key factor limiting its ability to fully phosphorylate and inhibit \u003cem\u003eYAP1\u003c/em\u003e. This inference was validated in subsequent pharmacological experiments: no statistically significant difference in \u003cem\u003eYAP1\u003c/em\u003e activity was observed between vector and \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e cells, regardless of GSK621 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). This phenomenon suggests that \u003cem\u003eYAP1\u003c/em\u003e activity may have a physiological ​​\u0026ldquo;floor\u0026rdquo; threshold\u0026mdash;we hypothesize that this is related to the basic functions required for cells adhere to and grow on culture surfaces. Furthermore, even under GSK621-activated \u003cem\u003ePRKAA1\u003c/em\u003e conditions, both \u003cem\u003eYAP1\u003c/em\u003e activity and the fibrotic phenotype driven by \u003cem\u003eYAP1\u003c/em\u003e were significantly higher in \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e\u003cem\u003eYAP1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e cells than in vector-transfected control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei-j, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-h). These results further confirm that \u003cem\u003eYAP1\u003c/em\u003e is a ​key driver of fibrosis progression, and that the anti-fibrotic effect of \u003cem\u003ePRKAA1\u003c/em\u003e activation primarily acts by ​enhancing the phosphorylation-mediated inhibition of \u003cem\u003eYAP1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo validate the critical role of \u003cem\u003ePRKAA1\u003c/em\u003e expression/activity in fibrosis regulation, we used a ​\u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e heterozygous mouse model. In contrast to the results in wild-type (WT) mice, Masson staining of high-mechanical-stress-induced full-thickness back wounds in \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e mice revealed ​significantly denser collagen fibers​ in the \u003cem\u003ePRKAA1\u003c/em\u003e knockdown group than in the WT group (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). Even with drug-induced \u003cem\u003ePRKAA1\u003c/em\u003e activation, no significant decrease in the ​\u003cem\u003eCOL I\u003c/em\u003e/\u003cem\u003eCOL III\u003c/em\u003e ratio​ was observed via Sirius Red staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek-l), and the expression of the myofibroblast marker \u003cem\u003eACTA2\u003c/em\u003e remained high in the wound tissue during the late healing stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em). Western blotting further revealed that with decreasing \u003cem\u003ePRKAA1\u003c/em\u003e phosphorylation levels, ​\u003cem\u003eYAP1\u003c/em\u003e activity​ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003en-p), ​\u003cem\u003eACTA2\u003c/em\u003e expression, and the ​\u003cem\u003eCOL I\u003c/em\u003e/\u003cem\u003eCOL III\u003c/em\u003e ratio​ all increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eq-r, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej), indicating that intact \u003cem\u003ePRKAA1\u003c/em\u003e gene function is essential for normal pathway activity.\u003c/p\u003e \u003cp\u003eTogether, these in vitro and in vivo results confirm that: \u003cem\u003ePRKAA1\u003c/em\u003e ​gene expression level​ is a prerequisite for its anti-fibrotic function. Pharmacological activation of \u003cem\u003ePRKAA1\u003c/em\u003e inhibits fibrosis by promoting \u003cem\u003eYAP1\u003c/em\u003e phosphorylation. Insufficient \u003cem\u003ePRKAA1\u003c/em\u003e activation under physiological conditions is the main factor limiting the suppression of fibrosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePRKAA1\u003c/b\u003e \u003cb\u003eregulates\u003c/b\u003e \u003cb\u003eYAP1\u003c/b\u003e \u003cb\u003eprotein stability via\u003c/b\u003e \u003cb\u003eLATS1\u003c/b\u003e\u003cb\u003e-Independent direct interaction\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo clarify whether there is a ​direct molecular interaction​ between \u003cem\u003ePRKAA1\u003c/em\u003e and \u003cem\u003eYAP1\u003c/em\u003e, we conducted systematic validation by combining ​computational predictions​ with multiple experimental approaches. The computational results from Rosetta software revealed a binding free energy of -24.997 kcal/mol between \u003cem\u003ePRKAA1\u003c/em\u003e and \u003cem\u003eYAP1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek), indicating a strong propensity for spontaneous binding. This finding was further supported by AlphaFold 3 protein structure prediction, which also predicted binding potential between the two proteins (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el). Co-immunoprecipitation (Co-IP) analysis confirmed that \u003cem\u003ePRKAA1\u003c/em\u003e interacts with \u003cem\u003eYAP1\u003c/em\u003e in cells ​regardless of exogenous drug treatment​ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). However, given that \u003cem\u003ePRKAA1\u003c/em\u003e is known to promote \u003cem\u003eYAP1\u003c/em\u003e phosphorylation via the Hippo pathway kinase \u003cem\u003eLATS1\u003c/em\u003e, the interaction captured by Co-IP might arise from the \u003cem\u003ePRKAA1\u003c/em\u003e/\u003cem\u003eLATS1\u003c/em\u003e/\u003cem\u003eYAP1\u003c/em\u003e ​ternary complex​ rather than from direct binding between \u003cem\u003ePRKAA1\u003c/em\u003e and \u003cem\u003eYAP1\u003c/em\u003e. To rule out this possibility, we used a ​\u003cem\u003eGST\u003c/em\u003e fusion protein pull-down assay​ to test for direct in vitro interactions: purified \u003cem\u003eGST\u003c/em\u003e-\u003cem\u003ePRKAA1\u003c/em\u003e fusion protein was co-incubated with \u003cem\u003eFlag\u003c/em\u003e-tagged \u003cem\u003eYAP1\u003c/em\u003e recombinant protein. The results clearly revealed that \u003cem\u003eGST\u003c/em\u003e-\u003cem\u003ePRKAA1\u003c/em\u003e directly pulled down \u003cem\u003eFlag\u003c/em\u003e-\u003cem\u003eYAP1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), whereas the \u003cem\u003eGST\u003c/em\u003e tag protein control did not exhibit such binding. This in vitro evidence effectively eliminated interference from other bridging proteins (e.g., \u003cem\u003eLATS1\u003c/em\u003e) in cells, ​confirming a direct molecular interaction between \u003cem\u003ePRKAA1\u003c/em\u003e and \u003cem\u003eYAP1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo elucidate whether \u003cem\u003ePRKAA1\u003c/em\u003e regulates \u003cem\u003eYAP1\u003c/em\u003e stability through a mechanism independent of the classical Hippo pathway core kinase \u003cem\u003eLATS1\u003c/em\u003e, we further constructed \u003cem\u003ePRKAA1\u003c/em\u003e-overexpressing (\u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eLATS1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e) and \u003cem\u003ePRKAA1\u003c/em\u003e-knockdown (\u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eLATS1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e) cell lines with a \u003cem\u003eLATS1\u003c/em\u003e-knockdown (\u003cem\u003eLATS1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e) genetic background. Under controlled proteasome pathway activation conditions, we evaluated \u003cem\u003eYAP1\u003c/em\u003e ubiquitination levels and its proteasomal degradation after \u003cem\u003ePRKAA1\u003c/em\u003e activation. The experimental results demonstrated that in the absence of \u003cem\u003eLATS1\u003c/em\u003e, \u003cem\u003ePRKAA1\u003c/em\u003e activation effectively increased \u003cem\u003eYAP1\u003c/em\u003e ubiquitination even when the proteasome pathway was unopened. After proteasome pathway activation, \u003cem\u003eYAP1\u003c/em\u003e degradation was significantly enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-g). These findings strongly suggest the existence of a \u003cem\u003eLATS1\u003c/em\u003e-independent pathway for \u003cem\u003ePRKAA1\u003c/em\u003e-mediated regulation of \u003cem\u003eYAP1\u003c/em\u003e stability. Further analysis revealed that in \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eLATS1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e cells, \u003cem\u003eYAP1\u003c/em\u003e degradation was reduced accompanied by decreased ubiquitination. Conversely, \u003cem\u003ePRKAA1\u003c/em\u003e activation in \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eOE\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eLATS1\u003c/em\u003e\u003csup\u003e\u003cem\u003eKD\u003c/em\u003e\u003c/sup\u003e cells accelerated \u003cem\u003eYAP1\u003c/em\u003e degradation with increased ubiquitination. These findings confirm that \u003cem\u003ePRKAA1\u003c/em\u003e activation effectively promotes \u003cem\u003eYAP1\u003c/em\u003e degradation.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePRKAA1\u003c/b\u003e \u003cb\u003einhibits the transcriptional co-activation function of\u003c/b\u003e \u003cb\u003eYAP1\u003c/b\u003e \u003cb\u003eby directly phosphorylating it at the\u003c/b\u003e \u003cb\u003eSer127\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eThr156\u003c/b\u003e \u003cb\u003eresidues\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur results revealed that \u003cem\u003ePRKAA1\u003c/em\u003e likely regulates the ubiquitin-proteasome degradation pathway of \u003cem\u003eYAP1\u003c/em\u003e by directly interacting with it and modulating its phosphorylation status. To precisely identify the key \u003cem\u003ePRKAA1\u003c/em\u003e-mediated phosphorylation sites on \u003cem\u003eYAP1\u003c/em\u003e, we used phosphorylation mass spectrometry to systematically analyze changes in the phosphorylation levels of various YAP1 peptides under three treatment conditions: \u003cem\u003e​PRKAA1\u003c/em\u003e activation, ​no intervention, and ​\u003cem\u003ePRKAA1\u003c/em\u003e inhibition. The analysis revealed that only the phosphorylation level of the peptide spanning the \u003cem\u003eN125\u0026ndash;C161\u003c/em\u003e region significantly decreased after \u003cem\u003ePRKAA1\u003c/em\u003e activation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting that this region is a potential key target of \u003cem\u003ePRKAA1\u003c/em\u003e. This peptide contains 10 potential phosphorylation sites: \u003cem\u003eSer127\u003c/em\u003e, \u003cem\u003eSer128\u003c/em\u003e, \u003cem\u003eSer131\u003c/em\u003e, \u003cem\u003eSer138\u003c/em\u003e, \u003cem\u003eThr141\u003c/em\u003e, \u003cem\u003eThr143\u003c/em\u003e, \u003cem\u003eThr145\u003c/em\u003e, \u003cem\u003eSer149\u003c/em\u003e, \u003cem\u003eThr154\u003c/em\u003e, and \u003cem\u003eThr156\u003c/em\u003e. To screen for the most likely \u003cem\u003ePRKAA1\u003c/em\u003e-targeted sites, we combined predictions from two professional phosphorylation site prediction platforms\u0026mdash;PhosphoSitePlus​ and ​GPS 6.0. The PhosphoSitePlus results (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) indicated that \u003cem\u003eSer127\u003c/em\u003e and \u003cem\u003eThr156\u003c/em\u003e were more likely to be phosphorylated by \u003cem\u003ePRKAA1\u003c/em\u003e, with \u003cem\u003eSer127\u003c/em\u003e showing greater kinase specificity. GPS 6.0 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) further confirmed the importance of \u003cem\u003eSer127\u003c/em\u003e, as its prediction score was significantly higher than that of all the other sites. Additionally, GPS 6.0 identified other sites with scores\u0026thinsp;\u0026gt;\u0026thinsp;0.001, ranked by score as follows: \u003cem\u003eSer128\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eThr156\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eSer131\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eSer138\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eThr143\u003c/em\u003e. Combining data from both platforms revealed that \u003cem\u003eSer127\u003c/em\u003e and \u003cem\u003eThr156\u003c/em\u003e were consistently highlighted as potential targets, whereas \u003cem\u003eSer128\u003c/em\u003e had a relatively high score on GPS 6.0. Thus, we selected ​\u003cem\u003eSer127\u003c/em\u003e, \u003cem\u003eSer128\u003c/em\u003e, and \u003cem\u003eThr156\u003c/em\u003e​ as key candidates for subsequent functional validation.\u003c/p\u003e \n \u003cp\u003eTo clarify the specific molecular sites and functional hierarchy of the direct phosphorylation of \u003cem\u003eYAP1\u003c/em\u003e, we constructed HDF models with \u003cem\u003eYAP1\u003c/em\u003e mutations at \u003cem\u003eSer127\u003c/em\u003e, \u003cem\u003eSer128\u003c/em\u003e, or \u003cem\u003eThr156\u003c/em\u003e using site-directed mutagenesis. After pharmacological \u003cem\u003ePRKAA1\u003c/em\u003e activation, phosphorylation analysis revealed that ​only the \u003cem\u003eSer127\u003c/em\u003e and \u003cem\u003eThr156\u003c/em\u003e mutants failed to undergo significant phosphorylation upregulation\u0026mdash;whereas the \u003cem\u003eSer128\u003c/em\u003e mutant remained phosphorylatable, with no statistical difference from the vector\u0026thinsp;+\u0026thinsp;GSK621 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-h). These findings confirm that \u003cem\u003ePRKAA1\u003c/em\u003e specifically phosphorylates \u003cem\u003eYAP1\u003c/em\u003e at \u003cem\u003eSer127\u003c/em\u003e and \u003cem\u003eThr156\u003c/em\u003e, but that \u003cem\u003eSer128\u003c/em\u003e is not a key target. To explore the relative functional importance of \u003cem\u003eSer127\u003c/em\u003e and \u003cem\u003eThr156\u003c/em\u003e, we assessed the binding of the \u003cem\u003eYAP1\u003c/em\u003e mutant to \u003cem\u003eTEAD1\u003c/em\u003e under GSK621 treatment. The mutation at ​\u003cem\u003eSer127\u003c/em\u003e greatly enhanced the binding of \u003cem\u003eYAP1\u003c/em\u003e to \u003cem\u003eTEAD1\u003c/em\u003e, with a greater fold-change than that induced by \u003cem\u003eThr156\u003c/em\u003e mutation, suggesting that phosphorylation at \u003cem\u003eSer127\u003c/em\u003e more effectively inhibits the \u003cem\u003eYAP1\u003c/em\u003e-\u003cem\u003eTEAD1\u003c/em\u003e interaction. Consistent with these functional observations, the transcriptional response of ​its target genes (\u003cem\u003eCTGF\u003c/em\u003e and \u003cem\u003eCYR61\u003c/em\u003e), in which \u003cem\u003eTEAD1\u003c/em\u003e binds to the promoter regions, exhibited trends aligned with those of the aforementioned binding assays: the \u003cem\u003eSer127\u003c/em\u003e mutation exerted the most pronounced inhibitory effect on downstream transcriptional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek-l). Combining all the evidence, we ​precisely identified \u003cem\u003eSer127\u003c/em\u003e and \u003cem\u003eThr156\u003c/em\u003e as the key \u003cem\u003ePRKAA1\u003c/em\u003e phosphorylation sites on \u003cem\u003eYAP1\u003c/em\u003e​ and uncovered their functional hierarchy: \u003cem\u003ePRKAA1\u003c/em\u003e primarily inhibits \u003cem\u003eYAP1\u003c/em\u003e-\u003cem\u003eTEAD1\u003c/em\u003e complex formation and downstream profibrotic transcription by phosphorylating ​\u003cem\u003eSer127\u003c/em\u003e, while \u003cem\u003eThr156\u003c/em\u003e plays a secondary, auxiliary regulatory role.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetabolomics reveals the unique role of\u003c/b\u003e \u003cb\u003ePRKAA1\u003c/b\u003e\u003cb\u003e-induced metabolic reprogramming in inhibiting fibrosis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnother key mechanism through which \u003cem\u003ePRKAA1\u003c/em\u003e activation inhibits fibrosis progression lies in its ability to induce induced ​metabolic reprogramming​. This effect first manifests as an overall improvement in cellular energy supply levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). More importantly, \u003cem\u003ePRKAA1\u003c/em\u003e activation prompts a ​fundamental shift in the cell\u0026rsquo;s metabolic pattern\u0026mdash;from being primarily dominated by anaerobic glycolysis to being dominated by oxidative phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-f)\u0026mdash;which directly leads to a significant decrease in extracellular lactate levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). The WB results further support changes in the expression of key proteins related to glycolysis and oxidative phosphorylation, strengthening the conclusion of this metabolic switch (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, i-j). Additionally, mitochondrial dynamic fission and functional levels are enhanced due to \u003cem\u003ePRKAA1\u003c/em\u003e activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, k-m). Many studies have shown that elevated energy levels and reduced lactate content are key factors in inhibiting fibrosis and promoting tissue regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo systematically dissect the regulatory differences in cellular metabolism between \u003cem\u003ePRKAA1\u003c/em\u003e activation (HA) and simple high mechanical modulus intervention (HN) under high mechanical stress, this study first screened for differentially expressed metabolites using ​untargeted metabolomics​ and constructed a heatmap to visualize metabolic phenotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003en). The cluster analysis results indicated that samples from the HA and HN groups formed two separate clusters, demonstrating significant metabolic differences. The heatmap displays 20 differentially expressed metabolites across four categories: amino acids, carbohydrates, lipids, and nucleotides. Specifically, 11 metabolites were upregulated in HA, including \u003cem\u003eN-acetyl-L-aspartate\u003c/em\u003e (amino acids), \u003cem\u003epalmitic acid\u003c/em\u003e (lipids), and \u003cem\u003emethylated uridine\u003c/em\u003e (nucleotides); and 7 metabolites were downregulated, exemplified by \u003cem\u003eglucosamine-6-phosphate\u003c/em\u003e (a key glycolytic intermediate). Notably, upregulated metabolites were associated with energy storage (\u003cem\u003epalmitic acid\u003c/em\u003e), redox homeostasis (\u003cem\u003eN-acetyl-L-aspartate\u003c/em\u003e in glutathione synthesis), and nucleotide metabolism (\u003cem\u003emethylated uridine\u003c/em\u003e, which regulates RNA stability). The downregulated metabolites were concentrated in the late glycolysis stage, suggesting that \u003cem\u003ePRKAA1\u003c/em\u003e activation may antagonize the profibrotic metabolic phenotypes induced by high mechanical stress by reprogramming energy metabolism and oxidative stress pathways.\u003c/p\u003e \u003cp\u003eTo further explore the interactions between differentially expressed metabolites and gene expression, we integrated transcriptomic data to construct a transcriptome\u0026ndash;metabolome network​ for the HA vs. HN groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eo). Central hubs with high connectivity to surrounding nodes were formed by key pathways such as insulin secretion, fatty acid biosynthesis, and arginine biosynthesis, suggesting that \u003cem\u003ePRKAA1\u003c/em\u003e reshapes metabolic networks through the synergy of multiple pathways.\u003c/p\u003e \u003cp\u003eThe arginine biosynthesis pathway was significantly upregulated in HA: three key metabolites (\u003cem\u003eornithine\u003c/em\u003e, \u003cem\u003ecitrulline\u003c/em\u003e, and \u003cem\u003earginine\u003c/em\u003e) were elevated, and positively correlated with \u003cem\u003eN-acetylglutamate\u003c/em\u003e (a urea cycle node). Moreover, the downstream metabolite \u003cem\u003eN-acetyl-L-aspartate\u003c/em\u003e (upregulated in HA heatmaps) formed a positive regulatory cluster with the purine metabolism genes \u003cem\u003eGMPS\u003c/em\u003e (GMP synthase) and \u003cem\u003eXMPK\u003c/em\u003e (XMP kinase), suggesting that the ​arginine purine axis​ supports anti-fibrotic ECM remodeling by promoting nucleotide synthesis.\u003c/p\u003e \u003cp\u003eCoordinated activation of the pyruvate metabolism-fatty acid biosynthesis axis was another hallmark: \u003cem\u003ePKM2\u003c/em\u003e (key pyruvate kinase) expression increased in HA, driving the conversion of pyruvate to \u003cem\u003eacetyl-CoA\u003c/em\u003e (upregulated) and activating fatty acid biosynthesis (upregulation of \u003cem\u003eACACA\u003c/em\u003e/\u003cem\u003eFASN\u003c/em\u003e). This finding was consistent with the increased levels of palmitic acid (a lipid metabolite) in HA, suggesting that the activity of \u003cem\u003ePRKAA1\u003c/em\u003e shifts from profibrotic glycolysis to the synthesis of new fatty acids, thereby decreasing lipotoxic fibroblast activation.\u003c/p\u003e \u003cp\u003eNotably, the TCA cycle and \u003cem\u003eHIF1α\u003c/em\u003e signaling exhibited ​pathological coupling​ in HN: \u003cem\u003ecitrate\u003c/em\u003e (a downregulated TCA metabolite) formed a negative cluster with \u003cem\u003eHIF1α\u003c/em\u003e and its target \u003cem\u003eLDHA\u003c/em\u003e (\u003cem\u003elactate dehydrogenase A\u003c/em\u003e). These findings suggest that high mechanical stress inhibits the TCA cycle, causing \u003cem\u003epyruvate\u003c/em\u003e accumulation, activating \u003cem\u003eHIF1α\u003c/em\u003e-mediated glycolytic reprogramming, and promoting the formation of a lactate buildup/fibrotic microenvironment. In HA, restored citrate levels and downregulated \u003cem\u003eHIF1α\u003c/em\u003e indicated \u003cem\u003ePRKAA1\u003c/em\u003e blocks this cascade by restoring TCA cycle function.\u003c/p\u003e \u003cp\u003eTo validate the results of the combined metabolomics and transcriptomics analysis, we selected three metabolites\u0026mdash;\u003cem\u003eN-acetyl-L-aspartate\u003c/em\u003e, ​\u003cem\u003epalmitic acid\u003c/em\u003e, and ​\u003cem\u003emethylated uridine\u003c/em\u003e. Under high mechanical modulus conditions, we observed that \u003cem\u003e​GSK621\u003c/em\u003e, ​\u003cem\u003eN-acetyl-L-aspartate\u003c/em\u003e, and \u003cem\u003e​palmitic acid​\u003c/em\u003e effectively reduced ​\u003cem\u003eACTA2\u003c/em\u003e protein expression in HDFs​ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ep-q) and ​immunofluorescence intensity​ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003er-s). However, \u003cem\u003e​palmitic acid\u003c/em\u003e exacerbated fibrotic phenotypes​ induced by high mechanical modulus. These findings corroborate the key insights from the integrative metabolomic-transcriptomic analysis. Taken together, these data demonstrate that \u003cem\u003ePRKAA1\u003c/em\u003e is a central integrator of metabolic and mechanical signals, thereby establishing a direct link between metabolism and fibrosis regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003et)\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eFor decades, the interplay between mechanical stress and cellular metabolism in fibrosis pathogenesis has intrigued researchers\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, how energy metabolism pathways coordinately regulate fibrotic signaling and mechanotransduction remains poorly defined\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Our study elucidates a ​dual regulatory axis​ in which pharmacological activation of \u003cem\u003ePRKAA1\u003c/em\u003e (\u003cem\u003eAMPKα1\u003c/em\u003e) not only directly phosphorylates \u003cem\u003eYAP1\u003c/em\u003e at the \u003cem\u003eSer127\u003c/em\u003e and \u003cem\u003eThr156\u003c/em\u003e residues to induce its proteasomal degradation but also coordinates a metabolic reprogramming switch from glycolysis to oxidative phosphorylation\u0026mdash;jointly inhibiting mechanical stress-induced fibrosis. This synergistic mechanism challenges the traditional view that \u003cem\u003eYAP1\u003c/em\u003e is solely regulated via the Hippo pathway and highlights potential crosstalk between energy homeostasis and mechanotransduction signaling in fibrotic diseases\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAMPK\u003c/em\u003e is traditionally viewed as a metabolic sensor, but our study uncovers its ​dual role as a modulator of mechanotransduction. While previous studies have suggested that \u003cem\u003eLATS1\u003c/em\u003e-mediated phosphorylation is the primary mechanism underlying \u003cem\u003eYAP1\u003c/em\u003e inactivation, our data demonstrate that \u003cem\u003ePRKAA1\u003c/em\u003e bypasses this canonical pathway via direct interaction with \u003cem\u003eYAP1\u003c/em\u003e. Co-IP and GST pull-down assays revealed a high-affinity interaction, indicating that \u003cem\u003ePRKAA1\u003c/em\u003e is a regulator of \u003cem\u003eYAP1\u003c/em\u003e activity under energy stress. These findings demonstrate that \u003cem\u003ePRKAA1\u003c/em\u003e acts as a ​parallel regulatory pathway alongside the \u003cem\u003eHippo\u003c/em\u003e cascade. This independence is functionally relevant: even when \u003cem\u003eLATS1\u003c/em\u003e is inhibited during fibrosis development, \u003cem\u003ePRKAA1\u003c/em\u003e activation still induces \u003cem\u003eYAP1\u003c/em\u003e degradation, indicating that \u003cem\u003ePRKAA1\u003c/em\u003e can bypass canonical Hippo signaling to induce \u003cem\u003eYAP1\u003c/em\u003e inactivation. Notably, this phosphorylation event relies on promoting \u003cem\u003eYAP1\u003c/em\u003e degradation and reducing its nuclear localization to inhibit \u003cem\u003eYAP1\u003c/em\u003e-\u003cem\u003eTEAD1\u003c/em\u003e complex formation: \u003cem\u003eYAP1\u003c/em\u003e-\u003cem\u003eSer127\u003c/em\u003e plays a dominant role, while \u003cem\u003eThr156\u003c/em\u003e plays a minor role. This hierarchical phosphorylation mechanism aligns with recent findings on \u003cem\u003eYAP1\u003c/em\u003e structural plasticity but expands its regulatory scope to include metabolic sensors\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur experimental results emphasize the ​physiological relevance of this axis. In both in vivo and in vitro experiments, we observed no significant changes in \u003cem\u003eYAP1\u003c/em\u003e activity in the context of \u003cem\u003ePRKAA1\u003c/em\u003e deficiency or the absence of GSK621 activation, indicating that \u003cem\u003ePRKAA1\u003c/em\u003e expression and activity determine the activation threshold of \u003cem\u003eYAP1\u003c/em\u003e. These findings suggest the following important mechanism: partial \u003cem\u003eAMPK\u003c/em\u003e activation preserves the tissue-repair activity of \u003cem\u003eYAP1\u003c/em\u003e, whereas full activation inhibits pathological remodeling. Exogenous \u003cem\u003ePRKAA1\u003c/em\u003e activation failed to further inhibit \u003cem\u003eYAP1\u003c/em\u003e activity in \u003cem\u003ePRKAA1\u003c/em\u003e\u003csup\u003eOE\u003c/sup\u003e cells, indicating a ​saturation effect​ of this axis. This observation implies that endogenous \u003cem\u003eYAP1\u003c/em\u003e activity sets a baseline constraint\u0026mdash;a concept previously unappreciated in fibrosis biology\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, we observed metabolic changes characterized by reduced lactate production and increased oxidative phosphorylation, which increase the complexity of the regulation of fibrosis. By integrating metabolomic and transcriptomic analyses, we identified a metabolic network centered on \u003cem\u003earginine\u003c/em\u003e biosynthesis\u0026ndash;fatty acid oxidation\u0026ndash;\u003cem\u003epyruvate\u003c/em\u003e\u0026ndash;TCA cycle\u0026ndash;\u003cem\u003eHIF1α\u003c/em\u003e. Specifically, under high mechanical stress, \u003cem\u003ePRKAA1\u003c/em\u003e activation restores TCA cycle function\u0026ndash;thereby inhibiting the hypoxic signaling driven by \u003cem\u003eHIF1α\u003c/em\u003e, which activates \u003cem\u003eYAP1\u003c/em\u003e. This metabolic shift also produces anti-fibrotic metabolite, such as \u003cem\u003eN-acetyl-L-aspartate\u003c/em\u003e, a known modulator of \u003cem\u003eTGF-β\u003c/em\u003e signaling\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The downregulation of fibrotic pathways, including \u003cem\u003eHIF1α\u003c/em\u003e, under \u003cem\u003ePRKAA1\u003c/em\u003e activation further supports the idea that metabolic reprogramming disrupts the ​hypoxia-mimicking microenvironment​ that perpetuates fibrosis\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Notably, this metabolic shift mirrors the \u0026ldquo;Warburg effect reversal\u0026rdquo; reported in regenerative environments, where enhanced oxidative metabolism promotes tissue repair\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This homeostatic mechanism is consistent with recent discoveries about mitochondrial retrograde signaling in fibrosis, highlighting PRKAA1 as a key integrator of metabolic and mechanical signals, thereby directly connecting metabolism with the regulation of fibrosis.\u003c/p\u003e \u003cp\u003eOur findings hold ​transformative potential for fibrosis therapy. First, targeting the PRKAA1-YAP1 interaction circumvents the limitations of current anti-fibrotic strategies that broadly inhibit \u003cem\u003eTGF-β\u003c/em\u003e or collagen deposition\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Small-molecule \u003cem\u003eAMPK\u003c/em\u003e activators such as GSK621 show promise in preclinical models but require optimization for clinical translation to avoid off-target effects on glucose metabolism\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Second, the metabolic reprogramming patterns identified in this study, marked by increased oxidative phosphorylation and arginine production, serve as new biomarkers for assessing fibrosis stages and tracking treatment effectiveness. \u003csup\u003e41\u0026ndash;43\u003c/sup\u003e. For example, upregulated N-acetyl-L-aspartate can serve as a non-invasive indicator of \u003cem\u003ePRKAA1\u003c/em\u003e activity in fibrotic tissues.\u003c/p\u003e \u003cp\u003eHowever, challenges remain. The ​spatial heterogeneity of \u003cem\u003eYAP1\u003c/em\u003e activation​ within fibrotic lesions suggests that \u003cem\u003ePRKAA1\u003c/em\u003e-based therapies may need to be administered locally to avoid systemic metabolic disruption. Additionally, the fibroblast heterogeneity observed in human fibrosis requires cell type-specific targeting strategies to maintain tissue-specific homeostasis. Furthermore, the temporal kinetics of \u003cem\u003ePRKAA1\u003c/em\u003e-\u003cem\u003eYAP1\u003c/em\u003e phosphorylation during fibrosis progression remain unclear. Does sustained mechanical stress lead to \u003cem\u003ePRKAA1\u003c/em\u003e depletion or adaptive resistance? In addition, it is necessary to investigate how other metabolic nodes, such as the pentose phosphate pathway or lipid droplet dynamics, influence YAP1 activity. Addressing these questions requires advanced spatiotemporal imaging and organoid models to recapitulate tissue-level mechanical heterogeneity.\u003c/p\u003e \u003cp\u003eIn summary, our work bridges a critical gap in fibrosis biology by elucidating how energy stress regulates mechanical transduction signaling. By demonstrating that \u003cem\u003ePRKAA1\u003c/em\u003e acts as a ​metabolic checkpoint that inhibits \u003cem\u003eYAP1\u003c/em\u003e overactivation, this study not only enhances our understanding of fibrosis pathogenesis but also opens new avenues for precision therapies targeting the intersection of metabolism and mechanobiology.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExperimental reagents\u003c/h2\u003e \u003cp\u003eGSK621 (cat. # HY-100548), Dorsomorphin (cat. #HY-13418A) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, a photoinitiator, cat. #HY-44076) were purchased from MedChem Express (NJ, USA). Gelatin methacryloyl (GelMA, cat. #EFL-GM-90) was purchased from Engforlife Tech (Suzhou, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell lines and cell culture\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eHuman dermal fibroblasts (HDFs) were obtained from Pricella (CM-H103, Wuhan, China). The cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (11885084, ThermoScientific, US) supplemented with 10% fetal bovine serum (A5256701, ThermoScientific, US) and 1% penicillin-streptomycin (C100C5, NCM Biotech, China) at 37\u0026deg;C in a humidified incubator with 5% CO₂.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGelMA hydrogel preparation\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eGelMA was dissolved in sterile phosphate-buffered saline (PBS) containing 0.25% LAP, under dark conditions at 60\u0026deg;C until fully dissolved. The solutions were then adjusted to concentrations of 5%, 10%, or 15% (w/v) and sterilized by filtration. For hydrogel formation, 50\u0026ndash;100 \u0026micro;L of the GelMA precursor was added to each well of a 6-well plate or 10 cm culture dish, exposed to 405 nm light for 1 minute for crosslinking, and allowed to hydrate in complete medium for 2 hours at 37\u0026deg;C with 5% CO₂ before cell seeding.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell seeding and drug treatment\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eHDFs were seeded onto GelMA hydrogels at a density of 1\u0026times;10⁵ cells per well (6-well plate) or 1\u0026times;10⁶ cells per dish (10 cm plate). After 24 hours of adhesion, cells were treated with fresh medium containing vehicle (DMSO) or test compounds. Drug solutions were prepared as 10 mM stock solutions in DMSO and diluted to working concentrations in PBS or DMEM before use. The final concentrations were: 30 \u0026micro;M GSK621 (a PRKAA1 activator) and 10 \u0026micro;M Dorsomorphin (a PRKAA1 inhibitor).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmid/siRNA transfection\u003c/h3\u003e\n\u003cp\u003ePlasmids and siRNAs were designed and validated by GeneChem (Shanghai, China). For transfection, HDFs were seeded in 6-well plates and transfected with Lipofectamine Lipo6000 (C0526; Beyotime, China) according to the manufacturer\u0026rsquo;s protocol. GFP plasmids were used for transfection efficiency validation in pilot experiments.\u003c/p\u003e\n\u003ch3\u003eLentiviral infection and stable cell line construction\u003c/h3\u003e\n\u003cp\u003eLentiviruses encoding shRNAs or overexpression constructs were generated by GeneChem (Shanghai, China). For infection, HDFs were seeded at 50% confluence and treated with lentivirus (MOI\u0026thinsp;=\u0026thinsp;20) plus 8 \u0026micro;g/mL polybrene. Stable cells were selected with puromycin (2 \u0026micro;g/mL) for 7 days.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSite-directed mutagenesis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSer127\u003c/em\u003e, \u003cem\u003eSer128\u003c/em\u003e, and \u003cem\u003eThr156\u003c/em\u003e mutants of \u003cem\u003eYAP1\u003c/em\u003e were generated with primers designed by GeneChem (Shanghai, China). Mutant plasmids were validated by Sanger sequencing.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnimals\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eMale \u003cem\u003eC57BL/6\u003c/em\u003e mice (6 weeks old, ~\u0026thinsp;25 g) were purchased from Gempharmatech (Jiangsu, China). Heterozygous \u003cem\u003ePRKAA1\u003c/em\u003e knockout mice (\u003cem\u003ePRKAA1⁺/⁻\u003c/em\u003e) were generated and genotyped by Beijing Jieyalife Biotechnology Co., Ltd. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Yishang Biotechnology (approval NO. IACUC-2024-Mi-059) and conducted in accordance with the 3Rs principles. The mice were housed in specific pathogen-free (SPF) facilities with controlled temperature (22\u0026ndash;25\u0026deg;C), humidity (40\u0026ndash;60%), and a 12-hour light-dark cycle, with free access to standard chow and water.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFull-thickness wound model\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eThe mice were anesthetized via isoflurane inhalation. The back was removed using electric clippers followed by depilatory cream (Veet\u0026reg;). Two 8 mm diameter full-thickness skin wounds were created on the back using a biopsy punch. Wounds were secured with 12 mm polyvinyl chloride (PVC) rings and dressed with petroleum gauze, moist chlorhexidine gauze, and iodine film. Dressings were changed on days 3, 7, 10, 14, and 17, with hydrogel application (5% or 15% w/v GelMA) and wound photography. The contraction rings were removed on day 10. Six wound tissue samples were collected on days 10 and 20. For mechanotransduction studies, drugs (100 \u0026micro;L) were injected into the wound bed and surrounding area at day 0 and every 2 days (same concentrations as in vitro).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMechanical property testing\u003c/h3\u003e\n\u003cp\u003eGelMA hydrogels (5%, 10%, and 15% w/w) were cast into cylindrical molds (diameter: 26.5 mm, height: 5 mm). The compressive modulus at 40% strain was measured using a computer-controlled universal testing machine (HD-B602, Haida, China) after 2 hours and 50 hours of immersion in culture medium. Young\u0026rsquo;s modulus was calculated from the linear elastic region of the stress-strain curve.\u003c/p\u003e\n\u003ch3\u003eCell viability assay (CCK-8)\u003c/h3\u003e\n\u003cp\u003eHDFs were seeded in 96-well plates at 3,000 cells per well and cultured for 12 hours. After being washed with PBS, the cells were treated with gradient concentrations of GelMA (0.1%, 0.5%, 1%, and 5%) dissolved in DMEM containing 2% FBS. A blank control (no cells) was included. At 0, 12 and 24 hour post-treatment, 10 \u0026micro;L of CCK-8 reagent (CK04, Dojindo, Japan) was added to each well, followed by incubation at 37\u0026deg;C with 5% CO₂ for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Relative cell viability was normalized to that of the blank control.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-time PCR (qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol reagent (RC112-01, Vazyme, China) and reverse-transcribed into cDNA with a PrimeScript\u0026trade; RT Reagent Kit (RR036A, Takara, Japan). qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using TB Green Premix Ex Taq II (RR820A, Takara, Japan). The cycling program was as follows: 95\u0026deg;C for 30 seconds; 40 cycles of 95\u0026deg;C for 5 seconds, 60\u0026deg;C for 30 seconds; and a melting curve analysis. Gene expression levels were normalized to \u003cem\u003eTUBULIN\u003c/em\u003e using the 2\u003csup\u003e⁻ΔΔCt\u003c/sup\u003e method. The primer sequences are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003e​\u003cb\u003eImmunophenotyping\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eFor surface marker analysis, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Antibodies against \u003cem\u003eVimentin\u003c/em\u003e (ab8069, Abcam, UK) and \u003cem\u003eα-SMA\u003c/em\u003e (#19245, CST, US) were added and the samples were incubated for 1 hour at room temperature. Fluorescent secondary antibodies (12-4739-81, 48-4015-82, ThermoScientific, US) were added and the samples were incubated for 1 hour at room temperature. Data were acquired on a Beckman CytoFlex and analyzed using Cytexpert2.5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting (WB)\u003c/h2\u003e \u003cp\u003eCells were lysed in RIPA buffer (WB3100, NCM Biotech, China) containing protlytic protease and phosphatase inhibitor (P002, NCM Biotech, China) on ice for 1 hour. The lysates were subsequently were centrifuged at 12,000 \u0026times; g for 20 minutes at 4\u0026deg;C, and protein concentrations were determined using a NanoDrop (Oxford, US). Equal amounts of protein (10\u0026ndash;20 \u0026micro;g) were separated by SDS-PAGE (8\u0026ndash;12% gels) and transferred to PVDF membranes (Millipore, US). Membranes were blocked with 5% BSA in TBST for 2 hours and probed with primary antibodies overnight at 4\u0026deg;C. After being washed, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. The protein bands were visualized using ECL Plus (P2100, NCM Biotech, China) and quantified with ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCo-iImmunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eHDFs (1\u0026times;10⁶) were seeded in 10 cm plates, treated with GSK621 (30 \u0026micro;M) or vehicle for 48 hours, and lysed in IP lysis buffer (P0013, Beyotime, China) containing protease/phosphatase inhibitors. Lysates were incubated with anti-PRKAA1 or IgG antibodies (5 \u0026micro;g/mL) overnight at 4\u0026deg;C, followed by incubation with Protein A/G magnetic beads (P2179S, Beyotime, China) for 2 hours. The beads were subsequently were washed, and bound proteins were eluted with elution buffer. Input and IP samples were analyzed by WB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGST pull-down assay\u003c/h2\u003e \u003cp\u003eGST, GST-\u003cem\u003ePRKAA1\u003c/em\u003e, and Flag-\u003cem\u003eYAP1\u003c/em\u003e plasmids were transformed into Rosetta competent cells (D1065S, Beyotime, China). Bacterial cultures were induced with 1 mM IPTG (ST098, Beyotime, China) at 37\u0026deg;C for 6 hours. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing lysozyme and protease inhibitors. Cleared lysates were incubated with Anti-Flag or anti-GST magnetic beads (P2115, P2138, Beyotime, China) overnight at 4\u0026deg;C. The beads were subsequently washed, after which the bound proteins were eluted. For kinase assays, GST-\u003cem\u003ePRKAA1\u003c/em\u003e and Flag-\u003cem\u003eYAP1\u003c/em\u003e were co-incubated in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) supplemented with 200 \u0026micro;M ATP at 37\u0026deg;C for 30 minutes. Interactions were validated by WB using anti-GST, anti-Flag, anti-\u003cem\u003ePRKAA1\u003c/em\u003e, and anti-\u003cem\u003eYAP1\u003c/em\u003e antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP)\u003c/h2\u003e \u003cp\u003eHDFs were crosslinked with 1% formaldehyde for 10 minutes at room temperature, quenched with 0.125 M glycine, and lysed in ChIP lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 10 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100). Nuclei were isolated and resuspended in sonication buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA). Chromatin was sheared using a Covaris S220 sonicator (175 W, 10% duty factor, 200 cycles, 10 minutes) to generate 200\u0026ndash;500 bp fragments. Immunoprecipitation was performed with anti-\u003cem\u003eYAP1\u003c/em\u003e or IgG antibodies, followed by protein A/G bead incubation. DNA was purified and quantified by qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePrediction of Protein-Protein interaction\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePRKAA1\u003c/em\u003e-\u003cem\u003eYAP1\u003c/em\u003e interactions were predicted using the RosettaDock and AlphaFold 3. Structural models of \u003cem\u003ePRKAA1\u003c/em\u003e (Q13131-1) and \u003cem\u003eYAP1\u003c/em\u003e (P46937-1) were downloaded from UniProt and visualized with PyMOL.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrediction of in silico phosphorylation site\u003c/b\u003e​\u003c/p\u003e \u003cp\u003ePotential phosphorylation sites on \u003cem\u003eYAP1\u003c/em\u003e targeted by \u003cem\u003ePRKAA1\u003c/em\u003e were predicted using PhosphoSitePlus (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.phosphosite.org/\u003c/span\u003e\u003cspan address=\"https://www.phosphosite.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and GPS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gps.biocuckoo.cn/\u003c/span\u003e\u003cspan address=\"https://gps.biocuckoo.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Public phosphoproteomics data were mined to validate candidate sites.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence Staining\u003c/h2\u003e \u003cp\u003e​\u003cb\u003eIn vitro\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eCells grown on coverslips were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.3% Triton X-100 for 10 minutes, and blocked with 5% BSA. Primary antibodies (e.g., anti-YAP1, anti-PRKAA1) were added and the samples were incubated overnight at 4\u0026deg;C. Fluorescent secondary antibodies were added and incubated for 1 hour at room temperature. The nuclei were counterstained with DAPI. Images were captured using a Leica confocal microscope.\u003c/p\u003e \u003cp\u003e​\u003cb\u003eIn vivo\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eParaffin-embedded tissues were sectioned (4\u0026ndash;6 \u0026micro;m), deparaffinized, and rehydrated. Antigen retrieval was performed using citrate buffer (pH 6.0) via microwave heating. Subsequent steps followed the in vitro protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHistological staining\u003c/h2\u003e \u003cp\u003e​\u003cb\u003eMasson\u0026rsquo;s trichrome staining\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eSections were stained with Weigert\u0026rsquo;s iron hematoxylin (nuclei), acid fuchsin (cytoplasm/muscle), and aniline blue (collagen). Images were analyzed for collagen content.\u003c/p\u003e \u003cp\u003e​\u003cb\u003eSirius Red Staining\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eThe sections were incubated with 0.1% Sirius Red (in saturated picric acid) for 1 hour, rinsed with acidic ethanol, and dehydrated. Collagen fibers were visualized under polarized light.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and were analyzed using GraphPad Prism 9.0. Normality was assessed using the Shapiro-Wilk test. Differences between groups were evaluated by one-way ANOVA with Tukey\u0026rsquo;s post hoc test for multiple comparisons. P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ​P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ​\u003cb\u003e​\u003c/b\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; ns, not significant).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the data from UniProt, PhosphoSitePlus (https://www.phosphosite.org/) and GPS (https://gps.biocuckoo.cn/). We express our gratitude to Cosmos Wisdom (Hangzhou, China) for its support in bioinformatics sequencing and analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYX: Conceptualization, Formal analysis, Investigation, Validation, Visualization, Writing-original draft. YyC: Data curation, Formal analysis, Methodology, Validation, Writing-original draft. JZ: Conceptualization, Methodology, Resources, Validation, Writing-original draft. BhP: Data curation, Investigation, Methodology, Writing- review \u0026amp; editing. JyL: Investigation, Methodology, Validation, Writing-review \u0026amp; editing. CrW Investigation, Writing- review \u0026amp; editing. JqC: Investigation, Writing- review \u0026amp; editing. CL: Investigation, Writing-review \u0026amp; editing.\u0026nbsp;YyY: Funding acquisition, Investigation, Supervision, review \u0026amp; editing. SzJ: Funding acquisition,\u0026nbsp;Supervision, Writing-review \u0026amp; editing.\u0026nbsp;ShZ: Funding acquisition, Project administration, Supervision, Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare\u0026nbsp;that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Key Research Project (2019YSL010), National Key R\u0026amp;D Program of China (2024YFA1108405), National Natural Science Foundation of China (No.82473580 and No. 82574032), and the Academic General project (No.2023MS006).\u0026nbsp;We thank Biorender (https://biorender.com) for the production of\u0026nbsp;the figures in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptome sequencing and metabolomics data used in the study have been uploaded to the National Genomics Data Center (https://www.cncb.ac.cn/; BioProject Number: PRJCA051113). The data are expected to be released in November 2027. Further inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Yishang Biotechnology (approval NO. IACUC-2024-Mi-059) and conducted in accordance with the 3Rs principles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePe\u0026ntilde;a, O. A. \u0026amp; Martin, P. Cellular and molecular mechanisms of skin wound healing. \u003cem\u003eNat Rev Mol Cell Biol\u003c/em\u003e 25, 599\u0026ndash;616 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalbott, H. E., Mascharak, S., Griffin, M., Wan, D. C. \u0026amp; Longaker, M. T. Wound healing, fibroblast heterogeneity, and fibrosis. \u003cem\u003eCell Stem Cell\u003c/em\u003e 29, 1161\u0026ndash;1180 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y. Y., Ji, S. F., Fu, X. B., Jiang, Y. F. \u0026amp; Sun, X. Y. Biomaterial-based mechanical regulation facilitates scarless wound healing with functional skin appendage regeneration. \u003cem\u003eMil Med Res\u003c/em\u003e 11, 13 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMascharak, S., desJardins-Park, H. E., Davitt, M. F., Griffin, M., Borrelli, M. R., Moore, A. 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M., Zhan, M., Yang, M. \u003cem\u003eet al.\u003c/em\u003e AMPK agonist alleviate renal tubulointerstitial fibrosis via activating mitophagy in high fat and streptozotocin induced diabetic mice. \u003cem\u003eCell Death Dis\u003c/em\u003e 12, 925 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable style=\"border: none;border-collapse: collapse;width: 100px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width:100.0%;border:none;border-bottom: solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eTable 1 Mass spectrometry results of protein phosphorylation modification\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:10.96%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003ePRKAA1 function\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.82%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003ePhosphorylatable sites within the sequence\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:9.62%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eConfidence\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:32.34%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eSequence\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:7.26%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eXcorr score\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:10.96%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp 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style='font-family:\"Arial\",sans-serif;color:black;'\u003euntreated\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.82%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eY188-P\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:9.62%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eHigh\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:32.34%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eYFLNHIDQTTTWQDPR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:7.26%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e1.95\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:10.96%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eInhibited\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.82%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eY188-P\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:9.62%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eHigh\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:32.34%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eYFLNHIDQTTTWQDPR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:7.26%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e1.79\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:10.96%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eActivated\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.82%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eY188-P\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:9.62%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eHigh\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:32.34%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eYFLNHIDQTTTWQDPR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:7.26%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e1.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"margin:0cm;text-align:justify;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:16px;font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border: none;width:100.0%;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width:100.0%;border:none;border-bottom: solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eTable 2 Predictions from the PhosphoSitePlus Platform\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003esite\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.46%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003esite sequence\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:14.88%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003elog2(score)*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.98%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003esite percentile*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eS127\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.46%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAHs*sPAsLQLGAVsPGtLtPtGVVsGPAAtPtAQHLR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:14.88%;background:#FFEFEF;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.07\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.98%;background:#FF2C2C;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e81.03%\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eS128\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.46%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAHss*PAsLQLGAVsPGtLtPtGVVsGPAAtPtAQHLR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:14.88%;background:#F4F7FB;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e-0.223\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.98%;background:#FF3C3C;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e77.88%\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n 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style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e38.61%\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eS138\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.46%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAHssPAsLQLGAVs*PGtLtPtGVVsGPAAtPtAQHLR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:14.88%;background:#7CA8D0;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e-2.654\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.98%;background:#FFFDFD;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e39.43%\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eT141\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.46%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAHssPAsLQLGAVsPGt*LtPtGVVsGPAAtPtAQHLR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:14.88%;background:#3679B7;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e-4.099\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.98%;background:#3E7FBA;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e17.13%\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eT143\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.46%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAHssPAsLQLGAVsPGtLt*PtGVVsGPAAtPtAQHLR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:14.88%;background:#2F75B5;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e-4.245\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.98%;background:#2F75B5;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e15.38%\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eT145\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.46%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAHssPAsLQLGAVsPGtLtPt*GVVsGPAAtPtAQHLR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:14.88%;background:#6E9ECB;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e-2.954\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.98%;background:#D5E3F0;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e34.26%\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eS149\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.46%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAHssPAsLQLGAVsPGtLtPtGVVs*GPAAtPtAQHLR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:14.88%;background:#A2C1DE;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e-1.891\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.98%;background:#FFBABA;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e52.89%\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eT154\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.46%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAHssPAsLQLGAVsPGtLtPtGVVsGPAAt*PtAQHLR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:14.88%;background:#5890C3;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e-3.407\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.98%;background:#93B7D8;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e26.79%\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:7.68%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eT156\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:60.46%;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAHssPAsLQLGAVsPGtLtPtGVVsGPAAtPt*AQHLR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:14.88%;border:none;border-bottom:solid windowtext 1.0pt;background:red;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e1.103\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:16.98%;border:none;border-bottom:solid windowtext 1.0pt;background:red;padding:0cm 5.4pt 0cm 5.4pt;height:14.5pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e89.64%\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width:100.0%;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:60.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e*in site sequence, phosphorylation site.\u003cbr\u003e\u0026nbsp;*log2(score)=0, neural; \u0026gt;0, favorable; \u0026lt;0, unfavorable.\u003cbr\u003e\u0026nbsp;*site percentile: percentage of sites in the phosphoproteome with lower scores, kinase specific.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ctable style=\"border: none;width:102.68%;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width:99.98%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eTable 3 Predictions from the GPS 6.0 Platform\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:10.82%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003ePosition\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:8.1%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eCode\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.6%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eKinase\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:33.82%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003ePeptide sequence\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:9.66%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eScore\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:10.82%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e127\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:8.1%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eSer\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.6%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eCAMK/CAMKL/AMPK/AMPK\u0026alpha;1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:33.82%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003ePQHVRAHSSPASLQL\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:9.66%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.0075\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:10.82%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e128\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:8.1%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eSer\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.6%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eCAMK/CAMKL/AMPK/AMPK\u0026alpha;1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:33.82%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eQHVRAHSSPASLQLG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:9.66%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.0025\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:10.82%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e131\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:8.1%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eSer\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.6%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eCAMK/CAMKL/AMPK/AMPK\u0026alpha;1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:33.82%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:left;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eRAHSSPASLQLGAVS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:9.66%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.0014\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:10.82%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e138\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:8.1%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eSer\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.6%;padding:0cm 5.4pt 0cm 5.4pt;height:14.0pt;\"\u003e\n \u003cp style=\"margin:0cm;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan 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AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"skin, fibrosis, fibroblast, mechanical response, metabolism, PRKAA1, YAP1","lastPublishedDoi":"10.21203/rs.3.rs-8422435/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8422435/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eModulating mechanically mediated fibrosis to alleviate scar formation represents a critical frontier in current research. In previous fibrosis studies, metabolic reprogramming was largely viewed as a passive, secondary process. By combining biological experiments with multiomics analyses, our study reveals that pharmacological activation of AMP-activated catalytic subunit alpha1 (\u003cem\u003ePRKAA1/AMPKα1\u003c/em\u003e) affects mechanical signal transduction pathways and initiates regenerative metabolic rewiring. In dermal fibroblasts, elevated mechanical stress triggers a vicious cycle: insufficient \u003cem\u003ePRKAA1\u003c/em\u003e activation fails to supply adequate energy for \u003cem\u003eYAP1\u003c/em\u003e-driven fibrotic progression and increases the production of lactate and proinflammatory molecules. In this context, pharmacologically enhancing \u003cem\u003ePRKAA1\u003c/em\u003e activity results in a ​synergistic antifibrotic effect​ through two key mechanisms: First, \u003cem\u003ePRKAA1\u003c/em\u003e directly phosphorylates \u003cem\u003eYAP1\u003c/em\u003e to suppress hyperactive mechanical signaling, reducing the expression of fibrotic effector factors and thereby limiting their nuclear localization and transcriptional activity. Second, it reprograms cellular metabolism to fulfil the energy requirements for adapting to high stress, increasing the production of anti-inflammatory molecules and accelerating the synthesis of the extracellular matrix (ECM). This dual antifibrotic action rooted in \u003cem\u003ePRKAA1\u003c/em\u003e activity not only deepens our understanding of the regulatory role of metabolism in fibrosis but also offers a valuable translational framework for clinical scar management.\u003c/p\u003e","manuscriptTitle":"The synergistic inhibitory effect of PRKAA1 activation on ECM mechanical stress-induced skin fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 10:03:53","doi":"10.21203/rs.3.rs-8422435/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e122f85c-b6be-42e3-bd74-69744d9f80aa","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60591540,"name":"Biological sciences/Cell biology/Cell adhesion/Extracellular matrix"},{"id":60591541,"name":"Biological sciences/Cell biology/Proteolysis/Ubiquitylation"}],"tags":[],"updatedAt":"2026-01-29T10:03:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 10:03:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8422435","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8422435","identity":"rs-8422435","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}