TWH19 scaffolds AURKB to phosphorylate C-RAF and activate MAPK signaling

TWH19 scaffolds AURKB to phosphorylate C-RAF and activate MAPK signaling | 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 TWH19 scaffolds AURKB to phosphorylate C-RAF and activate MAPK signaling Qingxiang Sun, Yuling Li, Yujuan Li, Peng Chen, Chenhui Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9362063/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 The MAPK signaling cascade is a central driver of cell proliferation, yet the mechanisms governing its spatial organization and catalytic amplification remain incompletely understood. Here, we identify TWH19 as a scaffold that nucleates MAPK complex assembly at membranes by simultaneously binding NRAS-GTP and the kinase AURKB. This signaling module enables AURKB to directly phosphorylate C-RAF at T258, thereby weakening inhibitory 14-3-3 interactions, promoting C-RAF dimerization, and activating ERK signaling. Genetic ablation of TWH19 in mice causes embryonic lethality, whereas adult deletion leads to weight loss and profound ERK signaling deficits without detectable γH2AX accumulation in the tissues examined. Disruption of the TWH19-AURKB interface abolishes NRAS recruitment, ERK activation, and cell proliferation. Together, our findings establish TWH19 and AURKB as unanticipated yet critical regulators of RAS-to-ERK signaling and uncover a mechanism governing MAPK pathway activation that is critical for cell proliferation and embryonic development. Biological sciences/Cell biology/Cell signalling Biological sciences/Developmental biology/Cell proliferation Biological sciences/Structural biology/Molecular modelling Biological sciences/Biochemistry/Kinases Biological sciences/Cancer/Oncogenes NRAS phosphorylation AURKB MAPK structure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The small GTPase RAS is a central player in the regulation of cell proliferation, functioning primarily through the Mitogen-Activated Protein Kinase (MAPK) pathway. Mutations in RAS are frequently observed in a wide range of cancers, driving tumor progression and playing a significant role in the malignancy of many cancer types 1,2 . Structurally, RAS contains a guanine nucleotide-binding G-domain and a hypervariable C-terminal region (HVR). The HVR, upon farnesylation, enables RAS to localize to cellular membranes 2 , where it interacts with downstream effectors like RAF. This interaction sets off a cascade of phosphorylation events involving RAF, MEK, and ERK within the MAPK pathway. ERK phosphorylation, in particular, is a crucial step that drives cell proliferation 3,4 . While RAS-RAF-MEK-ERK represents the core MAPK cascade, how this pathway is spatially organized and catalytically amplified remains incompletely understood. Aurora B kinase (AURKB), a catalytic subunit of the chromosomal passenger complex (CPC), is essential for chromosome segregation and cell cycle progression, making it an attractive target for anti-cancer drugs 5-8 . Intriguingly, AURKB inhibition reduces ERK phosphorylation in certain contexts 9,10 , but the mechanism remains unknown, and whether AURKB participates in MAPK signaling independently of mitosis is unexplored. TWH19, previously known as STK19, was identified in the 1990s as a serine-threonine kinase 11,12 and implicated as a melanoma driver 13-15 . In 2023, our work revealed that it lacks a kinase domain and instead, contains three tandem winged helix domains, supporting its role in DNA binding 16 . Subsequent studies have elucidated how TWH19 functions in DNA damage repair 17-19 . We also observed that TWH19 promotes cell proliferation even in the absence of UV-induced DNA damage, suggesting additional functions beyond genome maintenance. Here, we uncover an unexpected convergence of these two proteins in regulating the core MAPK cascade. We show that TWH19 serves as a molecular scaffold that simultaneously engages NRAS-GTP and AURKB at cellular membranes, nucleating assembly of the C-RAF-MEK-ERK module. This spatial organization enables AURKB to directly phosphorylate C-RAF at threonine 258, a modification that weakens inhibitory 14-3-3 binding, promotes C-RAF dimerization, and activates ERK signaling. Genetic ablation of TWH19 in mice causes embryonic lethality and adult tissue homeostasis defects characterized by profound ERK signaling deficits, uncoupling this function from its established DNA repair role. These findings identify TWH19 and AURKB as unanticipated but critical regulators of RAS-to-ERK signaling, revealing a fundamental mechanism controlling cell proliferation. Results TWH19 is required for embryonic development, adult weight maintenance, and ERK signaling To investigate the physiological role of TWH19 in vivo, we crossed male and female mice (CAG + , Flox +/- ) to generate global TWH19 knockout mice. No viable homozygous TWH19 knockout pups were obtained, and the ratio of Flox +/- to Flox -/- mice (20:25) deviated significantly from the expected Mendelian ratio of 2:1 (Figure 1A), indicating that homozygous TWH19 deletion is embryonic lethal and suggesting reduced viability in heterozygous mice. Indeed, three of nine embryos from intercrossed Flox +/- mice examined at embryonic day 12.5 (E12.5) exhibited developmental arrest or resorption (Figure 1B), a phenotype not observed in control littermates. These results establish TWH19 as essential for murine embryonic development. To assess TWH19 function in adult tissues, we generated tamoxifen-inducible TWH19 knockout mice (Cre +/- ; Flox +/+ ) using a CreERT2 system (Figure S1A). Following tamoxifen administration, TWH19 deletion was efficiently induced but was not lethal in adult mice (Figure S1B). However, knockout mice exhibited progressive and sustained body weight loss compared to littermate controls (Cre -/- ; Flox +/+ ) (Figures 1C). Cellular TWH19 depletion leads to single-strand breaks that can convert to double-strand breaks upon replication, thereby increasing γH2AX levels 16,17,20 , a marker of double-strand DNA damage. We therefore tested whether DNA damage accumulation could explain the observed phenotypes. Unexpectedly, γH2AX levels remained unchanged in liver, spleen, and bone marrow tissue sections following TWH19 deletion (Figure 1D and 1E), which was confirmed by western blotting of tissue homogenates (Figure 1F and 1G). Therefore, the observed phenotypes seem independent of TWH19’s role in DNA damage driven but rather by alternative mechanisms. Given that MAPK signaling is a central regulator of cell survival and proliferation, and that C-RAF KO mouse also die around embryonic day 10.5-12.5 21,22 , we hypothesized that TWH19 loss may impair this pathway. Indeed, phosphorylated ERK (pERK) levels were markedly reduced in liver, spleen, and bone marrow of TWH19-deficient mice (Figure 1H and 1I). Furthermore, primary cells from these organs of Cre +/- Flox +/+ mice exhibited decreased pERK upon 4-hydroxytamoxifen (4-OHT)-induced deletion of TWH19 (Figure 1J). These findings highlight an indispensable role for TWH19 in maintaining tissue integrity and cellular proliferation, likely through the regulation of ERK signaling. The TWH19-associated kinase AURKB is critical for ERK phosphorylation Having established that TWH19 deletion impairs ERK signaling in vivo without causing DNA damage, we sought to understand the underlying mechanism. Both endogenous and overexpressed TWH19 localized to the cytoplasm and nucleus (Figures S2A and S2B), suggesting potential roles for TWH19 in the cytoplasm, beyond DNA damage repair. Consistent with its involvement in MAPK regulation, TWH19 silencing in 293T and HeLa cells significantly reduced phosphorylated ERK levels, while overexpression of TWH19 enhanced ERK phosphorylation (Figure 2A and S2C). In contrast, AKT phosphorylation remained unaffected, indicating that TWH19 specifically regulates ERK signaling (Figure 2A and S2C). Our previous immunoprecipitation-mass spectrometry (IP-MS) data showed that TWH19 associated with a high number of kinases 16 . Among them, PIP5K1A, SRPK2, and AURKB were selected based on prior links to MAPK signaling (Figure 2B) 23-25 . Co-immunoprecipitation confirmed that both the 29-kD and 41-kD TWH19 isoforms interacted with these three kinases (Figure 2C). Knockdown of each kinase with siRNA significantly reduced ERK phosphorylation (Figure 2D and 2E). Among the three kinases, only AURKB robustly associated with endogenous NRAS in co-immunoprecipitation assays (Figure 2F). AURKB also bound KRAS (Figure 2G), suggesting broader engagement with RAS family members. These findings position AURKB as a potential mediator of TWH19-dependent MAPK activation, acting through direct engagement with RAS. TWH19 scaffolds a ternary complex with AURKB and NRAS to drive MAPK signaling The selective association of AURKB with NRAS (Figure 2F) prompted us to investigate whether TWH19 facilitates this interaction. We first reconstituted the complex in vitro using purified proteins. MBP pull-down assays demonstrated that MBP-tagged AURKB directly bound His-TWH19 (residues 25-C) and His-NRAS (residues 1-169) simultaneously, indicating formation of a stable ternary complex (Figure 3A). Reciprocal pull-downs using GST-NRAS confirmed direct interactions with both MBP-TWH19 and MBP-AURKB (Figure 3B). These results establish that TWH19 and AURKB can directly engage NRAS without requiring additional cellular factors. In cellular contexts, TWH19 overexpression enhanced the association between endogenous NRAS and Flag-AURKB (Figure 3C and S3), while TWH19 knockdown reduced their cytoplasmic co-localization (Figure 3D). These results demonstrate TWH19's role as a molecular scaffold that recruits AURKB to NRAS in cells. Functional studies revealed that TWH19 was required for AURKB-mediated ERK activation. While AURKB overexpression increased pERK levels and cell viability in 293T and HeLa cells, these effects were abolished by TWH19 knockdown (Figures 3E and 3F). Conversely, TWH19 overexpression synergized with AURKB to further enhance ERK phosphorylation and proliferation (Figures 3G and 3H). These reciprocal gain- and loss-of-function experiments establish that TWH19 and AURKB cooperate to promote MAPK signaling and cell growth. The TWH19-AURKB interface is essential for NRAS recruitment and ERK activation To elucidate the molecular basis of TWH19-AURKB interaction, we employed AlphaFold 26 structural modeling combined with biochemical validation. The top-ranked AlphaFold model (RMSD 1.1-3.8 Å) revealed an extensive interface between the third winged-helix (WH3) domain of TWH19 and the inter-lobal cavity of AURKB (Figure 4A and S4). Key interacting residues included TWH19 L97, Q147, T162, N166, R173, and D174, which engaged with AURKB Q121, S222, M233, M246, E246, H250, and Y282 (Figure 4B). These interface residues are generally well-conserved across species, suggesting potential functional importance (Figure 4C). Biochemical validation demonstrated that all alanine mutations of predicted interface residues in AURKB significantly reduced binding to TWH19 in co-IP assays (Figure 4D). On the other hand, five of eight TWH19 interface mutants showed impaired AURKB binding in pull-down assays (Figure 4E). The interaction-disrupting AURKB mutants Q121A and S222A reduced NRAS recruitment (Figure 4F and 4G), severely abrogating the ERK activation and growth-promoting effects of the TWH19-AURKB complex (Figures 4H and 4I). Collectively, these structure-function studies validate the AlphaFold-predicted TWH19-AURKB interface and establish that this interaction is required for NRAS recruitment, MAPK activation, and the proliferative output of the complex. TWH19 recruits MAPK cascade components at membranes in an NRAS-GTP-dependent manner We next examined whether TWH19 regulates the canonical NRAS-RAF interaction. Immunoprecipitation assays revealed that TWH19 depletion weakened the interaction between NRAS and C-RAF (Figures 5A and S5A), whereas TWH19 overexpression enhanced this interaction (Figures 5B and S5B). In addition to C-RAF, the interactions of NRAS with MEK and ERK were markedly enhanced by AURKB and further potentiated by TWH19 overexpression (Figure 5C and S5C). Similarly, AURKB interacted with NRAS and MAPK components (C-RAF, MEK, ERK), and TWH19 overexpression further strengthened these interactions (Figures 5D and S5D). This scaffolding effect was specific to the MAPK pathway, as no enhancement was observed for PI3K-AKT components (Figure S5E and S5F). Collectively, these findings suggest that TWH19 stabilizes a multiprotein complex encompassing the core RAS-MAPK cascade. Further analysis revealed that although TWH19 bound both GDP- and GTP-loaded NRAS in vitro (Figure S5G), its promotion of AURKB engagement with MAPK cascade components was restricted to GTP-bound active NRAS (Q61R) and not observed with WT or inactive NRAS (S17N) (Figure 5E), indicating signaling specificity for the active form of NRAS. Given that membrane localization of NRAS is critical for downstream pathway activation, we disrupted NRAS membrane anchoring with the farnesyltransferase inhibitor Salirasib. Salirasib abrogated the assembly of TWH19, AURKB, and MAPK components (Figures 5F and 5G), arguing that NRAS membrane localization is a prerequisite for complex formation. This result also explains why TWH19 preferentially assembled with active NRAS in cells, as only GTP-bound NRAS localizes to the membrane. Together, these findings establish that TWH19 scaffolds AURKB and active NRAS at the membrane to nucleate assembly of the C-RAF-MEK-ERK cascade, thereby creating a dedicated signaling module for MAPK activation. AURKB directly phosphorylates C-RAF at T258 to drive ERK phosphorylation The recruitment of AURKB suggested that it might phosphorylate one or more components of the MAPK cascade to activate the pathway. To investigate this possibility, we purified ectopically expressed NRAS and individual MAPK cascade proteins (C-RAF, MEK, and ERK) in the presence or absence of TWH19 and AURKB, then analyzed their phosphorylation status. In contrast to NRAS which did not show phosphorylation on serine/threonine (S/T) or tyrosine (Y) residues, the phosphorylation levels of C-RAF, MEK, and ERK were all increased upon TWH19/AURKB overexpression (Figure 6A). To identify the direct substrate of AURKB, we performed in vitro phosphorylation assays by incubating immunoprecipitated C-RAF, MEK, and ERK with E. coli -purified AURKB. The results revealed that C-RAF was directly phosphorylated by AURKB, specifically at S/T residues, while ERK and MEK were not phosphorylated at either Y or S/T residues (Figure 6B). In 293T cells, C-RAF S/T phosphorylation was inhibited by both Salirasib treatment and mutation of the AURKB active site (K106A) (Figure 6C), which indicates that both the recruitment and the kinase activity of AURKB are essential for C-RAF S/T phosphorylation in cells. To identify the specific phosphorylation sites, C-RAF was purified from 293T cells with or without co-expression of TWH19 and AURKB and analyzed by mass spectrometry. The results revealed enhanced phosphorylation at multiple sites upon co-expression of TWH19 and AURKB, with T258 exhibiting the greatest fold change (Table S1, Figure 6D). T258 phosphorylation was recorded in PhosphoSitePlus database, but the function is unclear. To assess the functional impact of these sites, each phosphorylated residue was mutated to aspartate to mimic phosphorylation, and the mutants were expressed in 293T cells. Strikingly, the T258D and T260D significantly increased ERK phosphorylation levels, whereas the other mutants did not (Figure 6E). These results indicate that phosphorylation at T258 or T260 in C-RAF is sufficient to activate MAPK. To assess the functional requirement of T258 and T260 phosphorylation in MAPK activation, we generated alanine mutants to block phosphorylation at these sites (Figure 6F). While wild-type C-RAF markedly enhanced ERK phosphorylation in the presence of TWH19 and AURKB, the T258A mutant and the T258A/T260A double mutant (2TA) showed only minimal effects (Figure 6G). Consistently, in TWH19-depleted cells, C-RAF T258D rescued ERK phosphorylation more potently than WT (Figure S6A). In line with these signaling changes, T258D promoted 293T cell proliferation relative to WT C-RAF, whereas the 2TA mutant impaired cell growth (Figure 6H). At the transcriptional level, T258D selectively increased expression of the canonical ERK target genes, including EGR1 and FOS, while CCND1 and MYC remained unchanged (Figure 6I). To directly assess endogenous T258 phosphorylation, we generated a phospho-specific antibody against C-RAF pT258. The antibody recognized C-RAF T258D but showed minimal reactivity toward wild-type C-RAF, confirming its selectivity for the phosphorylated epitope (Figure 6J, S6B and S6C). Using this tool, we surveyed endogenous C-RAF pT258 levels across multiple cancer cell lines and observed considerable variations, which somewhat correlated with pERK intensity (Figure 6K). Importantly, overexpression of the phosphomimetic C-RAF T258D mutant enhanced proliferation relative to wild-type C-RAF in these cell lines, albeit to different degrees (Figure 6L). Taken together, these findings establish that AURKB directly phosphorylates C-RAF at T258, a modification that drives ERK activation and cell proliferation. C-RAF T258 phosphorylation inhibits 14-3-3 binding and promotes C-RAF dimerization We next investigated how T258 phosphorylation drives ERK activation. It is well-established that S259 phosphorylation recruits 14-3-3 and suppress C-RAF dimerization and subsequent cascade phosphorylation 27,28 . T258 (and T260) is adjacent to S259, therefore, its phosphorylation may inhibit C-RAF’s binding to 14-3-3 and promote C-RAF dimerization. Indeed, we showed that T258D binding to 14-3-3 was significantly reduced, whereas 2TA slightly enhanced it (Figure 7A). Using IP, we further showed that T258D not only enhanced C-RAF dimerization with itself, but also its dimerization with A-RAF and B-RAF (Figure 7B, 7C and 7D), indicating that T258 phosphorylation promotes formation of signaling-competent C-RAF dimers. To determine whether C-RAF dimerization is required for TWH19-driven MAPK activation, we pharmacologically inhibited RAF dimerization using Tovorafenib. Tovorafenib suppressed TWH19-induced ERK phosphorylation (Figure 7E), reduced expression of ERK target genes (EGR1, FOS, CCND1) (Figure 7F), and abolished the growth-promoting effect of TWH19 overexpression (Figure 7G). These results establish that C-RAF dimerization is a critical functional output downstream of C-RAF T258 phosphorylation. We next examined whether this regulatory mechanism extends to other RAF isoforms. Sequence alignment shows that T258 in C-RAF is conserved in A-RAF but corresponds to a S in B-RAF (Figure S7A). We therefore investigated whether a phosphorylation mimicking D mutation also activate A-RAF and B-RAF. Unexpectedly, T213D in A-RAF and S364D in B-RAF enhanced 14-3-3 interaction (Figure S7B and S7C). Their dimerization with different RAF isoforms also did not increase upon mutation (Figure S7D-S7I). In contrast C-RAF, phosphorylation mimetic mutants in A-RAF and B-RAF also failed to enhance ERK phosphorylation (Figure S7J). Thus, the T258-dependent release from 14-3-3 and promotion of dimerization appears to be specific to C-RAF. Together, these results support a working model in which TWH19 scaffolds NRAS and AURKB on membranes to assemble the MAPK module; AURKB phosphorylates C-RAF at T258, which weakens inhibitory 14-3-3 binding, promotes C-RAF dimerization, and thereby enables ERK activation and cell proliferation (Figure 7H). Discussion Through an integrated approach combining mouse genetics, biochemical reconstitution, structural modeling, and phospho-proteomics, we have uncovered a previously unrecognized mechanism by which TWH19 and AURKB coordinate to regulate the core MAPK cascade. Our findings establish TWH19 as a molecular scaffold that simultaneously engages NRAS-GTP and AURKB at cellular membranes, nucleating assembly of the C-RAF-MEK-ERK module. This spatial organization enables AURKB to directly phosphorylate C-RAF at T258, a modification that weakens inhibitory 14-3-3 binding, promotes C-RAF dimerization, and thereby drives ERK activation. The physiological importance of this axis is underscored by the embryonic lethality and adult tissue homeostasis defects observed upon TWH19 ablation. The absence of detectable γH2AX accumulation in the examined tissues may be explained by the low proliferation rate of cells in adult tissues, where single-strand breaks are less likely converted into double-strand breaks. Nonetheless, the clear reduction in ERK phosphorylation in the same tissues indicates that this signaling function operates in cells where DNA damage is not readily detectable, arguing for a direct role for TWH19 in MAPK regulation independent of its DNA repair function. Our findings resolve several outstanding questions in the field. First, they explain the long-standing observation that AURKB inhibition reduces ERK phosphorylation in certain cancer contexts 9,10,29,30 , providing a mechanistic basis for a phenomenon that had remained mysterious. Second, our work also necessitates revision of a prior model proposing that TWH19 (STK19) directly phosphorylates NRAS at S89 to promote its oncogenic activity 31,32 . We demonstrate that TWH19 lacks kinase activity 16 and instead functions as a scaffold that recruits the authentic kinase AURKB, which does not phosphorylate NRAS itself but rather the associated C-RAF to regulate ERK signaling. This revised model aligns with the concept that RAS signaling fidelity is achieved through dynamic nanocluster formation 33,34 , and identifies TWH19 as a critical organizer of such assemblies. The clinical implications of our findings are manifold. The detection of T258 phosphorylation in human cancers, together with the observation that T258D overexpression enhances cell proliferation, suggests that this axis may contribute to cancer progression. Tumors with high TWH19 and AURKB expression might be particularly vulnerable to strategies that disrupt their interaction or inhibit AURKB catalytic activity. Notably, there are several reports linking AURKB to MAPK-targeted therapy resistance in cancers 9,10,29,30 . Our work provides a mechanistic rationale for combining AURKB inhibitors with MAPK pathway-targeted therapies, or for directly disrupting the TWH19-AURKB interaction as a therapeutic strategy in RAS-driven cancers. Several questions remain unanswered. Why the RAS-AURKB-TWH19 complex selectively assembles the C-RAF-MEK-ERK cascade but not the PI3K-AKT cascade? Additionally, the contribution of PIP5K1A and SRPK2, two other TWH19-associated kinases (Figure 2D), to MAPK activation warrants further exploration, given their reported role in KRAS signaling 24,25 . Our observation that T258 phosphorylation promotes C-RAF dimerization but the analogous mutations in A-RAF and B-RAF produce distinct effects on 14-3-3 binding adds to the growing understanding that RAF paralogs have distinct functions despite their synergy in ERK signaling 35,36 . How the same phosphorylation event elicits nearly opposite outcomes in different isoforms remains unclear and will require structural studies to resolve. In summary, our study establishes TWH19 and AURKB as unanticipated but critical regulators of RAS-to-ERK signaling, revealing a fundamental mechanism that controls cell proliferation and organismal survival. By expanding the functional repertoire of both proteins beyond their canonical roles in DNA repair and mitosis, respectively, these findings open new avenues for understanding normal physiology and for therapeutic intervention in diseases where MAPK signaling is dysregulated. Materials and Methods Animal studies C57BL/6J wild-type, CAG-iCre transgenic, and TWH19-floxed mice (TWH19-Flox +/- ) were obtained from GemPharmatech (Nanjing, China). To generate constitutive whole-body TWH19 knockout mice, TWH19-Flox +/- mice were crossed with CAG-Cre mice. Offspring genotypes were determined by tail-tip PCR using vendor-provided primers. For embryonic analysis, pregnant females from heterozygous intercrosses were sacrificed at embryonic day 12.5 (E12.5), and embryos were examined for developmental abnormalities or resorption. For inducible deletion, Cre-ERT2 mice were crossed with TWH19-floxed mice to generate inducible knockout mice (Cre +/- ; Flox +/+ ) and littermate controls (Cre -/- ; Flox +/+ ). Mice were administered tamoxifen (100 mg/kg) once daily for 7 consecutive days, followed by a 1-week washout and a second 7-day tamoxifen course to activate Cre recombinase and induce systemic TWH19 deletion. Body weight was recorded daily, and tissues (liver, spleen, bone marrow) were collected after completion of the regimen to assess deletion efficiency and downstream signaling. Tamoxifen-treated Cre +/- ; Flox +/+ mice and controls were monitored for survival, body weight, and tissue phenotypes. All animal experiments were approved by the Animal Ethics Committee of Sichuan Provincial People's Hospital (approval no. 2025-496). Protein expression and purification Human NRAS (residues 1-169) was cloned into the pET-15b expression vector with an N-terminal His-tag fusion. Human TWH19, TWH19-41 kD, AURKB and their mutants were cloned into a psyno-1 vector expressing an N-terminal cleavable 6xHis-MBP fusion to facilitate protein expression and purification. His-TWH19 25-C (the 29-kD isoform with residues 1-24 deleted to enhance stability) and its mutants were cloned into a psyno-1 vector expressing an N-terminal 6xHis tag. All constructs were transformed into E. coli BL21 (DE3). Bacterial cultures were grown in LB medium and induced with 0.5 mM IPTG at 22 °C for 12 h. Cells were harvested and sonicated in a lysis buffer containing 50 mM Tris pH 8.0, 350 mM NaCl, 10% glycerol, 10 mM imidazole, 2 mM MgCl 2 , and 1 mM PMSF. The proteins were first purified on a Ni-NTA column and eluted in a buffer containing 50 mM Tris pH 8.0, 350 mM NaCl, 300 mM imidazole, 10% glycerol, 2 mM MgCl 2 . Eluted proteins were concentrated and further purified by a Superdex 200 Increase gel filtration column on ÄktaPure (GE Healthcare) using a gel filtration buffer containing 20 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM MgCl 2 , and 2 mM DTT. Eluted proteins were frozen at -80 °C at 5-10 mg/mL. For GST-fusion proteins, C-RAF-RBD (C-RAF residues 50-131) and NRAS were cloned into pGEX-4T-1 expression vector incorporating an N-terminal TEV cleavable GST tag. After transformation into E. coli BL21 (DE3), protein expression was induced with 0.5 mM IPTG at 22 °C overnight. Cells were harvested and sonicated in lysis buffer containing 50 mM Tris pH 8.0, 200 mM NaCl, 10% glycerol, 2 mM MgCl 2 , 1 mM PMSF, and 1 mM DTT. After centrifugation, the supernatant was passed through glutathione sepharose beads and eluted in a buffer containing 20 mM Tris pH 8.0, 200 mM NaCl, 10% glycerol, 2 mM MgCl 2 , 10 mM GSH and 1 mM DTT. The proteins were further purified by size exclusion chromatography using a Superdex 200 column (GE Healthcare) in a buffer containing 20 mM Tris pH 8.0, 200 mM NaCl, 10% glycerol, 2 mM MgCl 2 , and 2 mM DTT. Pull-down assays For MBP pull-down assays, MBP-TWH19, MBP-AURKB, or MBP was immobilized on anti-MBP magnetic beads. For GST pull-down assays, GST-C-RAF-RBD, GST-NRAS or GST was immobilized on glutathione Sepharose beads. Cell lysates or purified proteins (3.0 μM) were incubated with the protein-immobilized beads in a total volume of 500 μL with gentle rotation for 2 h at 4 °C. After incubation, beads were washed three times with pull-down buffer containing 20 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM MgCl 2 , 2 mM DTT, and 0.005% Triton X-100. Bound proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining or immunoblotting. Cell culture, siRNA transfection and plasmid transfection 293T and HeLa cells were cultured in DMEM (Gibco) with 10% fetal bovine serum (Newzerum) at 37 °C in a humidified incubator with 5% CO 2 . For gene knockdown experiments, cells were transfected with the indicated siRNAs (sequences listed in Table S2) using jetPRIME® transfection reagent (Polyplus), and knockdown efficiency was evaluated 72 h after transfection. For plasmid transfection, cells were seeded at approximately 50% confluence and transfected with expression constructs or the corresponding empty vector controls using jetPRIME® (Polyplus) or liposome transfection reagent (Yeasen). After 24 h, the transfected cells were used for subsequent studies. Immunofluorescence HeLa cells grown on coverslips were fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100. After blocking with 5% bovine serum albumin, cells were incubated with primary antibodies overnight at 4 °C, followed by incubation with fluorophore-conjugated secondary antibodies for 2 h at room temperature. Images were acquired using a Zeiss LSM 900 confocal microscope and analyzed with ImageJ software. Immunoprecipitation and co-immunoprecipitation 293T or HeLa cells were harvested and lysed in lysis buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EGTA, 2 mM MgCl 2 , 0.5% NP-40, 1% (v/v) protease inhibitors cocktail (Selleck, B14001) and 1% phosphatase inhibitor cocktail (B15001, Selleck) at 4 °C for 30 minutes. Lysates were centrifuged at 14,000 rpm for 10 minutes. Supernatants were incubated with glutathione Sepharose beads, anti-Flag affinity gel (Selleck, B23101) or anti-HA magnetic beads (MCE, HY-K0201) for 3 h at 4 °C. After incubation, the beads were washed 3 times with lysis buffer, and the bound proteins were analyzed by western blotting. Western blotting Protein samples were separated on 10% SDS-PAGE gels (Vazyme) and transferred onto Immobilon®-P PVDF membranes (Millipore, IPVH00010). Membranes were blocked with 5% (w/v) skimmed milk in TBS-T (TBS, 0.1% (v/v) Tween 20) for 1 h at room temperature and incubated with primary antibody overnight at 4 °C. After washing with TBS-T, the membranes were incubated with HRP-conjugated secondary antibodies in 5% (w/v) skimmed milk in TBS-T and visualized using Super ECL Detection Reagent (Yeasen). Antibodies used in this study are listed in Table S3. Histology and immunohistochemistry For histological analysis, liver, spleen, and bone marrow tissues were collected from tamoxifen-treated mice and fixed according to standard procedures. Tissue processing, paraffin embedding, sectioning, and immunohistochemical (IHC) staining for γ·H2AX were performed by a commercial service provider (FBersw Biotechnology Co., Ltd., China). Stained sections were scanned using a digital slide scanner, and high-resolution images were provided by the vendor. Quantification of γ·H2AX staining intensity was performed using ImageJ software under identical analysis parameters, and values were normalized to the corresponding control group. Cell proliferation assay Sulforhodamine B (SRB) assay was used to assess cell proliferation. After seeding in 96-well plates (5000 cells/well) overnight, cells were transfected with indicated plasmid or si-RNA and cultured for 3 days. After fixing the cells with 100 µL of 10% TCA for 2 hours, the cells were stained with 0.4% SRB solution (in 1% acetic acid) at room temperature for 20 minutes. After removing the unbound SRB and drying the plate completely, 100 µL of 10 mM Tris Base (pH 10.5) was added to each well to dissolve the bound SRB dye. Finally, the absorbance at 570 nm was measured using a microplate reader. In vitro kinase assays and phosphoproteomic analysis For in vitro kinase assays, Flag-C-RAF, Flag-MEK1 and Flag-ERK2 proteins were transiently expressed in 293T cells and purified using anti-Flag beads. Bead-bound substrates were incubated with 5.0 μM purified MBP or MBP-AURKB for 2 h at 4 °C with gentle rotation. After incubation, beads were washed and the bound proteins were separated by SDS-PAGE. The kinase reaction buffer contained 20 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 2 mM MgCl 2 , 2 mM DTT, 300 μM ATP and 0.005% Triton X-100. Phosphorylation signals were initially assessed by immunoblotting using phospho-specific antibodies. For phosphoproteomic analysis, C-RAF samples purified from 293T cells under the indicated conditions were subjected to mass spectrometry analysis performed by a commercial service provider (SpecAlly Life Technology Co., Ltd., China). Phosphorylation sites were identified and quantified based on peptide signal intensity, and fold changes were calculated relative to control conditions. A summary of identified phosphosites is provided in Table S1. Generation of a phospho-specific antibody against C-RAF pT258 A phosphopeptide corresponding to the region surrounding C-RAF pT258 (RQRSpTSTPN) was synthesized by GL Biochem (Shanghai, China). The peptide was emulsified in complete Freund's adjuvant (0.5 mg/mL) and used to immunize mice by subcutaneous injection at three sites (100 μL per site). Two weeks later, mice received a second immunization with the peptide emulsified in incomplete Freund's adjuvant (0.75 mg/mL; three sites, 100 μL per site), followed by a third boost three weeks later (incomplete Freund's adjuvant, 1.0 mg/mL; three sites, 100 μL per site). Serum was collected two weeks after the final boost. Antibodies were purified by anion-exchange chromatography, and fractions (300 μL per tube) were screened using C-RAF proteins to identify fractions that selectively recognized the phosphorylated T258 epitope. Specificity was further evaluated using lysates from 293T cells expressing wild-type C-RAF or the phosphomimetic T258D mutant. Statistical analysis All quantitative data were analyzed and plotted using GraphPad Prism. Unless otherwise stated, data are presented as mean ± SD from independent biological replicates. Statistical significance between two groups was evaluated using a two-tailed unpaired Student’s t test. For experiments involving more than two groups, statistical tests are described in the corresponding figure legends (e.g., one-way or two-way ANOVA). P value < 0.05 was considered statistically significant. Significance is denoted as: ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Declarations Funding: National Natural Science Foundation of China (NSFC #82273850, #82403683); China Postdoctoral Science Foundation (CPSF #2024M760361, #GZC20240208). Author contributions: Conceptualization: YL1, QS Methodology: YL1, YL2, PC, QS Investigation: YL1, YL2, PC Visualization: YL1, QS Supervision: CW, LG, QS Writing-original draft: YL1, QS Writing-review & editing: YL1, LG, QS Competing interests: The authors declare no conflicts of interest. Ethical Statement Not applicable. Data Availability Statement All data are available in the main text or the supplementary materials. References Moore, A. R., Rosenberg, S. C., McCormick, F. & Malek, S. RAS-targeted therapies: is the undruggable drugged? Nat Rev Drug Discov 19 , 533-552, doi:10.1038/s41573-020-0068-6 (2020). Simanshu, D. K., Nissley, D. V. & McCormick, F. RAS Proteins and Their Regulators in Human Disease. Cell 170 , 17-33, doi:10.1016/j.cell.2017.06.009 (2017). Imperial, R., Toor, O. 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Sekiya, S. et al. Drosophila Screening Identifies Dual Inhibition of MEK and AURKB as an Effective Therapy for Pancreatic Ductal Adenocarcinoma. Cancer Res 83 , 2704-2715, doi:10.1158/0008-5472.CAN-22-3762 (2023). Yin, C. et al. Pharmacological Targeting of STK19 Inhibits Oncogenic NRAS-Driven Melanomagenesis. Cell 176 , 1113-1127 e1116, doi:10.1016/j.cell.2019.01.002 (2019). Qian, L. et al. Targeting NRAS-Mutant Cancers with the Selective STK19 Kinase Inhibitor Chelidonine. Clin Cancer Res 26 , 3408-3419, doi:10.1158/1078-0432.CCR-19-2604 (2020). Tian, T. et al. Plasma membrane nanoswitches generate high-fidelity Ras signal transduction. Nat Cell Biol 9 , 905-914, doi:10.1038/ncb1615 (2007). Lee, S. Y. & Lee, K. Y. Conditional Cooperativity in RAS Assembly Pathways on Nanodiscs and Altered GTPase Cycling. Angew Chem Int Ed Engl 63 , e202316942, doi:10.1002/anie.202316942 (2024). Rezaei Adariani, S. et al. Structural snapshots of RAF kinase interactions. Biochem Soc Trans 46 , 1393-1406, doi:10.1042/BST20170528 (2018). Desideri, E., Cavallo, A. L. & Baccarini, M. Alike but Different: RAF Paralogs and Their Signaling Outputs. Cell 161 , 967-970, doi:10.1016/j.cell.2015.04.045 (2015). Additional Declarations There is NO Competing Interest. Supplementary Files suptablesandfigures260408.docx supplemental figures and tables 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-9362063","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":631428413,"identity":"a0e5de4b-f0a4-498f-9424-73b4eae88de8","order_by":0,"name":"Qingxiang Sun","email":"data:image/png;base64,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","orcid":"","institution":"University of Electronic Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Qingxiang","middleName":"","lastName":"Sun","suffix":""},{"id":631428414,"identity":"8c2316e3-6d57-4b2c-89f4-ee119a3ae530","order_by":1,"name":"Yuling Li","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yuling","middleName":"","lastName":"Li","suffix":""},{"id":631428415,"identity":"8b92e68a-da82-4f6d-822d-c377b4a0ae45","order_by":2,"name":"Yujuan Li","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yujuan","middleName":"","lastName":"Li","suffix":""},{"id":631428416,"identity":"df2719c7-6cb0-442e-a356-753990d4ce70","order_by":3,"name":"Peng Chen","email":"","orcid":"","institution":"Sichuan Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Chen","suffix":""},{"id":631428417,"identity":"29b25e64-f46a-41db-b47e-3745110055c7","order_by":4,"name":"Chenhui Wang","email":"","orcid":"","institution":"University of Electronic Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Chenhui","middleName":"","lastName":"Wang","suffix":""},{"id":631428418,"identity":"931ac339-6159-4851-a4f8-dbffcbbe6686","order_by":5,"name":"Lu Guo","email":"","orcid":"","institution":"University of Electronic Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2026-04-09 02:15:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9362063/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9362063/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108491340,"identity":"cd20c3cd-c62e-4f0e-b517-22462db844b6","added_by":"auto","created_at":"2026-05-05 09:53:22","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":220571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTWH19 is required for embryonic development, adult weight maintenance, and ERK signaling.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eCrossing TWH19-Flox\u003csup\u003e+/+\u003c/sup\u003e mice with CAG-Cre mice yielded no viable homozygous knockout pups at birth, and the proportion of heterozygous offspring was also reduced, indicating embryonic lethality. \u003cstrong\u003e(B)\u003c/strong\u003e Representative images of E12.5 embryos from the indicated crosses in panel (A) reveal developmental arrest in a subset of embryos. \u003cstrong\u003e(C)\u003c/strong\u003eTamoxifen (Tamo.)-induced deletion of TWH19 in adult mice caused progressive body weight loss (two-way repeated-measures ANOVA, interaction P \u0026lt; 0.0001). \u003cstrong\u003e(D)\u003c/strong\u003e Representative immunohistochemical staining for γ·H2AX in liver, spleen, and bone marrow from tamoxifen-treated TWH19 wild-type (WT) and knockout (KO) mice. Scale bars, 200 μm. \u003cstrong\u003e(E)\u003c/strong\u003e Quantification of γ·H2AX levels shown in (D) revealed no significant difference between groups. \u003cstrong\u003e(F)\u003c/strong\u003e Representative immunoblot images of γ·H2AX in tissue homogenates from liver, spleen, and bone marrow of WT and KO mice. \u003cstrong\u003e(G)\u003c/strong\u003e Quantification of γ·H2AX levels shown in (F) showed no significant difference between groups.\u003cstrong\u003e (H) \u003c/strong\u003eImmunoblot analysis shows reduced phosphorylated ERK (pERK) levels in liver, spleen, and bone marrow of TWH19-deficient mice. \u003cstrong\u003e(I)\u003c/strong\u003e Quantification of pERK levels shown in (H), normalized to total ERK. \u003cstrong\u003e(J)\u003c/strong\u003e Levels of pERK are reduced in primary cells isolated from the indicated tissues following 4-hydroxytamoxifen (4-OHT)-induced TWH19 deletion ex vivo. In panel (E), (G), and (I), data are shown as mean ± SD (n ≥ 4) with unpaired two-tailed Student's \u003cem\u003et\u003c/em\u003e-test. *, P \u0026lt; 0.05; ***, P \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9362063/v1/b4ea011d80b51b6d34a4a0fb.jpeg"},{"id":108185385,"identity":"e997e9be-47c7-4d4c-94bf-7f49ea4308da","added_by":"auto","created_at":"2026-04-30 09:05:41","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":225103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTWH19-associated kinases, particularly AURKB, promote ERK phosphorylation. (A)\u003c/strong\u003e In 293T cells, siRNA-mediated TWH19 knockdown reduced ERK phosphorylation (left), whereas HA-TWH19 overexpression enhanced it (right). NC, nontargeting control; EV, empty vector. \u003cstrong\u003e(B)\u003c/strong\u003e Volcano plot of kinases identified by TWH19 IP-MS analysis (≥ 5-fold enrichment, p \u0026lt; 0.05). PIP5K1A, SRPK2, and AURKB (highlighted) were selected for further validation. \u003cstrong\u003e(C)\u003c/strong\u003e HA-tagged TWH19 isoforms (29 kDa and 41 kDa) were co-immunoprecipitated (co-IP) with Flag-tagged PIP5K1A, AURKB, or SRPK2 using an anti-Flag antibody in 293T cells. \u003cstrong\u003e(D)\u003c/strong\u003e ERK phosphorylation in 293T cells was reduced by siRNAs targeting PIP5K1A, SRPK2, or AURKB, respectively. \u003cstrong\u003e(E)\u003c/strong\u003e Statistical analysis of pERK levels from (D), normalized to total ERK (mean ± SD, n = 3; ***p \u0026lt; 0.001, *p \u0026lt; 0.05, unpaired Student’s \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003e(F)\u003c/strong\u003eAURKB, but not PIP5K1A or SRPK2, exhibited a robust association with endogenous NRAS in co-IP assays in 293T cells. \u003cstrong\u003e(G)\u003c/strong\u003e Flag-AURKB also bound to HA-KRAS in 293T cells.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9362063/v1/74c2c1af68084912ca7b089b.jpeg"},{"id":108185383,"identity":"1eb9b878-5052-43e4-bcaf-10925010f052","added_by":"auto","created_at":"2026-04-30 09:05:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":152037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTWH19 scaffolds a\u003c/strong\u003e \u003cstrong\u003eternary complex with AURKB and NRAS to promote MAPK signaling.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eImmobilized MBP-AURKB simultaneously bound to His-TWH19 (25-C fragment) and His-NRAS (1-169) in MBP pull-down assays using purified recombinant proteins. \u003cstrong\u003e(B)\u003c/strong\u003e Ternary complex formation was verified with GST pull-down assays using GST-NRAS (1-169) and MBP-TWH19 and MBP-AURKB. \u003cstrong\u003e(C)\u003c/strong\u003eTWH19 overexpression enhanced the association between AURKB and endogenous NRAS, as demonstrated by Flag-AURKB co-IP in 293T cells. \u003cstrong\u003e(D)\u003c/strong\u003e TWH19 depletion reduced cytoplasmic co-localization of AURKB and RAS (stained with pan-RAS antibody) in HeLa cells. Left: representative confocal immunofluorescence images; right: quantification of Pearson’s correlation coefficient (PCC) (≥30 cells per group). ***P \u0026lt; 0.001, unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. \u003cstrong\u003e(E)\u003c/strong\u003e TWH19 siRNA (#1) abrogated the increase in pERK induced by Flag-AURKB overexpression in both 293T and HeLa cells. \u003cstrong\u003e(F) \u003c/strong\u003eTWH19 siRNA (#1) abrogated the enhancement of cell proliferation (SRB assay) induced by Flag-AURKB overexpression in 293T and HeLa cells.\u003cstrong\u003e (G) \u003c/strong\u003eCo-expression of TWH19 and AURKB further increased ERK phosphorylation in 293T cells. Numbers indicate pERK intensity normalized to total ERK. \u003cstrong\u003e(H)\u003c/strong\u003e SRB proliferation assay shows that co-expression of TWH19 and AURKB further enhanced cell growth in 293T cells. Data in panels (F) and (H) are representative of three independent experiments. Mean ± SD. Unpaired Student’s \u003cem\u003et\u003c/em\u003e-test, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9362063/v1/2455eead4934f7a2b5276c4a.jpeg"},{"id":108185388,"identity":"fe870d59-ad68-427a-99ce-55a28fb1ee95","added_by":"auto","created_at":"2026-04-30 09:05:41","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":226879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe TWH19-AURKB interface is required for NRAS recruitment and ERK activation.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e AlphaFold model of the TWH19-AURKB complex. TWH19 is positioned within the inter-lobal cavity of AURKB, and the predicted binding interface is boxed. \u003cstrong\u003e(B)\u003c/strong\u003e Enlarged view of the predicted TWH19-AURKB interface showing putative interacting residues (sticks) and polar contacts (dashed lines). \u003cstrong\u003e(C)\u003c/strong\u003e Interface residues (circled) are conserved across species. Conserved residues are shown in blue, divergent residues in red. \u003cstrong\u003e(D)\u003c/strong\u003e Alanine mutations in predicted AURKB interface residues reduced binding to TWH19 in co-IP assays in 293T cells. \u003cstrong\u003e(E)\u003c/strong\u003e Alanine substitution of TWH19 interface residues impaired its association with AURKB to varying degrees in binding assays with recombinant proteins. \u003cstrong\u003e(F, G)\u003c/strong\u003e Interface-disrupting AURKB mutations (Q121A and S222A) reduced recruitment of NRAS in co-IP assays in 293T cells. \u003cstrong\u003e(H)\u003c/strong\u003e Alanine mutations in AURKB (Q121A, S222A) reduced ERK phosphorylation in 293T and HeLa cells expressing HA-TWH19 and Flag-AURKB. \u003cstrong\u003e(I)\u003c/strong\u003e Alanine mutations in AURKB (Q121A, S222A) reduced cell proliferation in 293T and HeLa cells expressing HA-TWH19 and Flag-AURKB, as measured by SRB assays. Data are presented as mean ± SD, n=3. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 vs WT; unpaired two-tailed Student's \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9362063/v1/5369b6e0b5913df5551eabcb.jpeg"},{"id":108185384,"identity":"b96aed0d-cc1e-4506-97f4-c05c286ff421","added_by":"auto","created_at":"2026-04-30 09:05:41","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":187627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTWH19 recruits MAPK cascade components in an NRAS-GTP-dependent manner at membranes.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e GST-C-RAF RBD pull-down assays in 293T cells, showing that TWH19 knockdown reduced NRAS-C-RAF interaction. \u003cstrong\u003e(B)\u003c/strong\u003e GST-C-RAF RBD pull-down assays in 293T cells, showing that TWH19 overexpression enhanced NRAS-C-RAF association. \u003cstrong\u003e(C)\u003c/strong\u003e Co-immunoprecipitation of Flag-NRAS with C-RAF, MEK, and ERK in 293T cells under the indicated expression conditions, showing that TWH19 promoted assembly of the NRAS-associated MAPK signaling complex. \u003cstrong\u003e(D)\u003c/strong\u003e Co-immunoprecipitation of Flag-AURKB with NRAS, C-RAF, MEK, and ERK in 293T cells, showing that TWH19 further strengthened AURKB-associated MAPK complex formation. \u003cstrong\u003e(E)\u003c/strong\u003e Co-immunoprecipitation analysis comparing wild-type NRAS with constitutively active NRAS (Q61R) and inactive NRAS (S17N) in 293T cells, showing preferential assembly of the TWH19-AURKB-MAPK complex with active NRAS. \u003cstrong\u003e(F)\u003c/strong\u003e Co-immunoprecipitation analysis after treatment with the RAS membrane-localization inhibitor Salirasib (50 μM, 12 h) in 293T cells, showing that membrane disruption weakened AURKB-associated MAPK complex formation. \u003cstrong\u003e(G)\u003c/strong\u003e Co-immunoprecipitation analysis under Salirasib treatment, confirming reduced association of TWH19, AURKB, and MAPK components with NRAS. Data are representative of at least three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9362063/v1/92e7a037b93d963625f40b92.jpeg"},{"id":108185393,"identity":"a4469076-56a7-451b-b2e1-2300f7df956f","added_by":"auto","created_at":"2026-04-30 09:05:42","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":439450,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAURKB directly phosphorylates C-RAF at T258 to drive ERK phosphorylation.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Immunoblot analysis of purified Flag-tagged NRAS, C-RAF, MEK1, and ERK2 from 293T cells expressing TWH19 and/or AURKB, showing increased phosphorylation of C-RAF, MEK1, and ERK2 but not NRAS upon TWH19 and AURKB co-expression. \u003cstrong\u003e(B)\u003c/strong\u003e In vitro kinase assay using purified MBP-AURKB and immunopurified Flag-C-RAF, MEK1, or ERK2, showing direct phosphorylation of C-RAF by AURKB on S/T residues. \u003cstrong\u003e(C)\u003c/strong\u003e Immunoblot analysis of Flag-C-RAF phosphorylation in 293T cells, showing that Salirasib treatment and kinase-dead AURKB (K106A) abolished C-RAF S/T phosphorylation. \u003cstrong\u003e(D)\u003c/strong\u003e Phosphoproteomic analysis of C-RAF from 293T cells, highlighting T258 as the site with the greatest phosphorylation fold increase upon TWH19 and AURKB co-expression. \u003cstrong\u003e(E)\u003c/strong\u003e Immunoblot analysis of pERK in 293T cells expressing phosphomimetic C-RAF mutants, showing that T258D and T260D increased ERK phosphorylation. \u003cstrong\u003e(F)\u003c/strong\u003e Immunoblot analysis showing reduced S/T phosphorylation of the C-RAF T258A/T260A double mutant (2TA) compared to wild-type. \u003cstrong\u003e(G)\u003c/strong\u003e Immunoblot analysis showing that the T258A and 2TA mutants impaired ERK activation in cells expressing TWH19 and AURKB. \u003cstrong\u003e(H)\u003c/strong\u003e SRB proliferation assay showing that T258D promoted cell proliferation whereas 2TA did not (mean ± SD, n = 4). \u003cstrong\u003e(I)\u003c/strong\u003e RT-qPCR analysis showing increased EGR1 and FOS expression in T258D-expressing cells. \u003cstrong\u003e(J)\u003c/strong\u003e Validation of the phospho-specific anti-C-RAF pT258 antibody using wild-type and T258D lysates. \u003cstrong\u003e(K)\u003c/strong\u003e Immunoblot analysis of endogenous C-RAF pT258 across multiple cancer cell lines, showing variable abundance. \u003cstrong\u003e(L)\u003c/strong\u003e SRB proliferation assays showing that T258D enhanced proliferation relative to wild-type C-RAF across multiple cell lines (mean ± SD, n = 3). Unpaired two-tailed Student's \u003cem\u003et\u003c/em\u003e-test. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 vs wild-type.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9362063/v1/140ec2473ed6363e98e4342b.jpeg"},{"id":108492142,"identity":"1075aec0-de46-45f7-a88b-28bacfe87ed9","added_by":"auto","created_at":"2026-05-05 09:56:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1798077,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9362063/v1/56e1830a-00b0-46b8-b041-78f3f4bb0a47.pdf"},{"id":108185386,"identity":"c95b1c3a-da93-463c-820b-884634fbe462","added_by":"auto","created_at":"2026-04-30 09:05:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1099311,"visible":true,"origin":"","legend":"supplemental figures and tables","description":"","filename":"suptablesandfigures260408.docx","url":"https://assets-eu.researchsquare.com/files/rs-9362063/v1/9e34fa39332cbf6b557557ba.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"TWH19 scaffolds AURKB to phosphorylate C-RAF and activate MAPK signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe small GTPase RAS is a central player in the regulation of cell proliferation, functioning primarily through the Mitogen-Activated Protein Kinase (MAPK) pathway. Mutations in RAS are frequently observed in a wide range of cancers, driving tumor progression and playing a significant role in the malignancy of many cancer types \u003csup\u003e1,2\u003c/sup\u003e. Structurally, RAS contains a guanine nucleotide-binding G-domain and a hypervariable C-terminal region (HVR). The HVR, upon farnesylation, enables RAS to localize to cellular membranes \u003csup\u003e2\u003c/sup\u003e, where it interacts with downstream effectors like RAF. This interaction sets off a cascade of phosphorylation events involving RAF, MEK, and ERK within the MAPK pathway. ERK phosphorylation, in particular, is a crucial step that drives cell proliferation \u003csup\u003e3,4\u003c/sup\u003e. While RAS-RAF-MEK-ERK represents the core MAPK cascade, how this pathway is spatially organized and catalytically amplified remains incompletely understood.\u003c/p\u003e\n\u003cp\u003eAurora B kinase (AURKB), a catalytic subunit of the chromosomal passenger complex (CPC), is essential for chromosome segregation and cell cycle progression, making it an attractive target for anti-cancer drugs \u003csup\u003e5-8\u003c/sup\u003e. Intriguingly, AURKB inhibition reduces ERK phosphorylation in certain contexts \u003csup\u003e9,10\u003c/sup\u003e, but the mechanism remains unknown, and whether AURKB participates in MAPK signaling independently of mitosis is unexplored.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTWH19, previously known as STK19, was identified in the 1990s as a serine-threonine kinase \u003csup\u003e11,12\u003c/sup\u003e and implicated as a melanoma driver \u003csup\u003e13-15\u003c/sup\u003e. In 2023, our work revealed that it lacks a kinase domain and instead, contains three tandem winged helix domains, supporting its role in DNA binding \u003csup\u003e16\u003c/sup\u003e. Subsequent studies have elucidated how TWH19 functions in DNA damage repair \u003csup\u003e17-19\u003c/sup\u003e. We also observed that TWH19 promotes cell proliferation even in the absence of UV-induced DNA damage, suggesting additional functions beyond genome maintenance.\u003c/p\u003e\n\u003cp\u003eHere, we uncover an unexpected convergence of these two proteins in regulating the core MAPK cascade. We show that TWH19 serves as a molecular scaffold that simultaneously engages NRAS-GTP and AURKB at cellular membranes, nucleating assembly of the C-RAF-MEK-ERK module. This spatial organization enables AURKB to directly phosphorylate C-RAF at threonine 258, a modification that weakens inhibitory 14-3-3 binding, promotes C-RAF dimerization, and activates ERK signaling. Genetic ablation of TWH19 in mice causes embryonic lethality and adult tissue homeostasis defects characterized by profound ERK signaling deficits, uncoupling this function from its established DNA repair role. These findings identify TWH19 and AURKB as unanticipated but critical regulators of RAS-to-ERK signaling, revealing a fundamental mechanism controlling cell proliferation.\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003eTWH19 is required for embryonic development, adult weight maintenance, and ERK signaling\u003c/h3\u003e\n\u003cp\u003eTo investigate the physiological role of TWH19 in vivo, we crossed male and female mice (CAG\u003csup\u003e+\u003c/sup\u003e, Flox\u003csup\u003e+/-\u003c/sup\u003e) to generate global TWH19 knockout mice. No viable homozygous TWH19 knockout pups were obtained, and the ratio of Flox\u003csup\u003e+/-\u003c/sup\u003e to Flox\u003csup\u003e-/-\u003c/sup\u003e mice (20:25) deviated significantly from the expected Mendelian ratio of 2:1 (Figure 1A), indicating that homozygous TWH19 deletion is embryonic lethal and suggesting reduced viability in heterozygous mice. Indeed, three of nine embryos from intercrossed Flox\u003csup\u003e+/-\u003c/sup\u003e mice examined at embryonic day 12.5 (E12.5) exhibited developmental arrest or resorption (Figure 1B), a phenotype not observed in control littermates. These results establish TWH19 as essential for murine embryonic development.\u003c/p\u003e\n\u003cp\u003eTo assess TWH19 function in adult tissues, we generated tamoxifen-inducible TWH19 knockout mice (Cre\u003csup\u003e+/-\u003c/sup\u003e; Flox\u003csup\u003e+/+\u003c/sup\u003e) using a CreERT2\u0026nbsp;system (Figure S1A).\u0026nbsp;Following tamoxifen administration, TWH19 deletion was efficiently induced but was not lethal in adult mice (Figure S1B). However, knockout mice exhibited progressive and sustained body weight loss compared to littermate controls (Cre\u003csup\u003e-/-\u003c/sup\u003e; Flox\u003csup\u003e+/+\u003c/sup\u003e) (Figures 1C).\u0026nbsp;Cellular TWH19 depletion leads to single-strand breaks that can convert to double-strand breaks upon replication, thereby increasing \u0026gamma;H2AX levels \u003csup\u003e16,17,20\u003c/sup\u003e, a marker of double-strand DNA damage. We therefore tested whether DNA damage accumulation could explain the observed phenotypes. Unexpectedly, \u0026gamma;H2AX levels remained unchanged in liver, spleen, and bone marrow tissue sections following TWH19 deletion (Figure 1D and 1E), which was confirmed by western blotting of tissue homogenates (Figure 1F and 1G). Therefore, the observed phenotypes seem independent of TWH19\u0026rsquo;s role in DNA damage driven but rather by alternative mechanisms.\u003c/p\u003e\n\u003cp\u003eGiven that MAPK signaling is a central regulator of cell survival and proliferation, and that C-RAF KO mouse also die around embryonic day 10.5-12.5 \u003csup\u003e21,22\u003c/sup\u003e, we hypothesized that TWH19 loss may impair this pathway. Indeed, phosphorylated ERK (pERK) levels were markedly reduced in liver, spleen, and bone marrow of TWH19-deficient mice (Figure 1H and 1I). Furthermore, primary cells from these organs of Cre\u003csup\u003e+/-\u003c/sup\u003e Flox\u003csup\u003e+/+\u003c/sup\u003e mice exhibited decreased pERK upon 4-hydroxytamoxifen (4-OHT)-induced deletion of TWH19 (Figure 1J). These findings highlight an indispensable role for TWH19 in maintaining tissue integrity and cellular proliferation, likely through the regulation of ERK signaling.\u003c/p\u003e\n\u003ch3\u003eThe TWH19-associated kinase AURKB is critical for ERK phosphorylation\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eHaving established that TWH19 deletion impairs ERK signaling in vivo without causing DNA damage, we sought to understand the underlying mechanism. Both endogenous and overexpressed TWH19 localized to the cytoplasm and nucleus (Figures S2A and S2B), suggesting potential roles for TWH19 in the cytoplasm, beyond DNA damage repair. Consistent with its involvement in MAPK regulation, TWH19 silencing in 293T and HeLa cells significantly reduced phosphorylated ERK levels, while overexpression of TWH19 enhanced ERK phosphorylation (Figure 2A and S2C). In contrast, AKT phosphorylation remained unaffected, indicating that TWH19 specifically regulates ERK signaling (Figure 2A and S2C).\u003c/p\u003e\n\u003cp\u003eOur previous immunoprecipitation-mass spectrometry (IP-MS) data showed that TWH19 associated with a high number of kinases \u003csup\u003e16\u003c/sup\u003e. Among them, PIP5K1A, SRPK2, and AURKB were selected based on prior links to MAPK signaling (Figure 2B) \u003csup\u003e23-25\u003c/sup\u003e. Co-immunoprecipitation confirmed that both the 29-kD and 41-kD TWH19 isoforms interacted with these three kinases (Figure 2C). Knockdown of each kinase with siRNA significantly reduced ERK phosphorylation (Figure 2D and 2E). Among the three kinases, only AURKB robustly associated with endogenous NRAS in co-immunoprecipitation assays (Figure 2F). AURKB also bound KRAS (Figure 2G), suggesting broader engagement with RAS family members. These findings position AURKB as a potential mediator of TWH19-dependent MAPK activation, acting through direct engagement with RAS.\u003c/p\u003e\n\u003ch3\u003eTWH19 scaffolds a ternary complex with AURKB and NRAS to drive MAPK signaling\u003c/h3\u003e\n\u003cp\u003eThe selective association of AURKB with NRAS (Figure 2F) prompted us to investigate whether TWH19 facilitates this interaction. We first reconstituted the complex in vitro using purified proteins. MBP pull-down assays demonstrated that MBP-tagged AURKB directly bound His-TWH19 (residues 25-C) and His-NRAS (residues 1-169) simultaneously, indicating formation of a stable ternary complex (Figure 3A). Reciprocal pull-downs using GST-NRAS confirmed direct interactions with both MBP-TWH19 and MBP-AURKB (Figure 3B). These results establish that TWH19 and AURKB can directly engage NRAS without requiring additional cellular factors. In cellular contexts, TWH19 overexpression enhanced the association between endogenous NRAS and Flag-AURKB (Figure 3C and S3), while TWH19 knockdown reduced their cytoplasmic co-localization (Figure 3D). These results demonstrate TWH19\u0026apos;s role as a molecular scaffold that recruits AURKB to NRAS in cells.\u003c/p\u003e\n\u003cp\u003eFunctional studies revealed that TWH19 was required for AURKB-mediated ERK activation. While AURKB overexpression increased pERK levels and cell viability in 293T and HeLa cells, these effects were abolished by TWH19 knockdown (Figures 3E and 3F). Conversely, TWH19 overexpression synergized with AURKB to further enhance ERK phosphorylation and proliferation (Figures 3G and 3H). These reciprocal gain- and loss-of-function experiments establish that TWH19 and AURKB cooperate to promote MAPK signaling and cell growth.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eThe TWH19-AURKB interface is essential for NRAS recruitment and ERK activation\u003c/h3\u003e\n\u003cp\u003eTo elucidate the molecular basis of TWH19-AURKB interaction, we employed AlphaFold \u003csup\u003e26\u003c/sup\u003e structural modeling combined with biochemical validation. The top-ranked AlphaFold model (RMSD 1.1-3.8 \u0026Aring;) revealed an extensive interface between the third winged-helix (WH3) domain of TWH19 and the inter-lobal cavity of AURKB (Figure 4A and S4). Key interacting residues included TWH19 L97, Q147, T162, N166, R173, and D174, which engaged with AURKB Q121, S222, M233, M246, E246, H250, and Y282 (Figure 4B). These interface residues are generally well-conserved across species, suggesting potential functional importance (Figure 4C).\u003c/p\u003e\n\u003cp\u003eBiochemical validation demonstrated that all alanine mutations of predicted interface residues in AURKB significantly reduced binding to TWH19 in co-IP assays (Figure 4D). On the other hand, five of eight TWH19 interface mutants showed impaired AURKB binding in pull-down assays (Figure 4E). The interaction-disrupting AURKB mutants Q121A and S222A reduced NRAS recruitment (Figure 4F and 4G), severely abrogating the ERK activation and growth-promoting effects of the TWH19-AURKB complex (Figures 4H and 4I). Collectively, these structure-function studies validate the AlphaFold-predicted TWH19-AURKB interface and establish that this interaction is required for NRAS recruitment, MAPK activation, and the proliferative output of the complex.\u003c/p\u003e\n\u003ch3\u003eTWH19 recruits MAPK cascade components at membranes in an NRAS-GTP-dependent manner\u003c/h3\u003e\n\u003cp\u003eWe next examined whether TWH19 regulates the canonical NRAS-RAF interaction. Immunoprecipitation assays revealed that TWH19 depletion weakened the interaction between NRAS and C-RAF (Figures 5A and S5A), whereas TWH19 overexpression enhanced this interaction (Figures 5B and S5B). In addition to C-RAF, the interactions of NRAS with MEK and ERK were markedly enhanced by AURKB and further potentiated by TWH19 overexpression (Figure 5C and S5C). Similarly, AURKB interacted with NRAS and MAPK components (C-RAF, MEK, ERK), and TWH19 overexpression further strengthened these interactions (Figures 5D and S5D). This scaffolding effect was specific to the MAPK pathway, as no enhancement was observed for PI3K-AKT components (Figure S5E and S5F). Collectively, these findings suggest that TWH19 stabilizes a multiprotein complex encompassing the core RAS-MAPK cascade.\u003c/p\u003e\n\u003cp\u003eFurther analysis revealed that although TWH19 bound both GDP- and GTP-loaded NRAS in vitro (Figure S5G), its promotion of AURKB engagement with MAPK cascade components was restricted to GTP-bound active NRAS (Q61R) and not observed with WT or inactive NRAS (S17N) (Figure 5E), indicating signaling specificity for the active form of NRAS. Given that membrane localization of NRAS is critical for downstream pathway activation, we disrupted NRAS membrane anchoring with the farnesyltransferase inhibitor Salirasib. Salirasib abrogated the assembly of TWH19, AURKB, and MAPK components (Figures 5F and 5G), arguing that NRAS membrane localization is a prerequisite for complex formation. This result also explains why TWH19 preferentially assembled with active NRAS in cells, as only GTP-bound NRAS localizes to the membrane. Together, these findings establish that TWH19 scaffolds AURKB and active NRAS at the membrane to nucleate assembly of the C-RAF-MEK-ERK cascade, thereby creating a dedicated signaling module for MAPK activation.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eAURKB directly phosphorylates C-RAF at T258 to drive ERK phosphorylation\u003c/h3\u003e\n\u003cp\u003eThe recruitment of AURKB suggested that it might phosphorylate one or more components of the MAPK cascade to activate the pathway. To investigate this possibility, we purified ectopically expressed NRAS and individual MAPK cascade proteins (C-RAF, MEK, and ERK) in the presence or absence of TWH19 and AURKB, then analyzed their phosphorylation status. In contrast to NRAS which did not show phosphorylation on serine/threonine (S/T) or tyrosine (Y) residues, the phosphorylation levels of C-RAF, MEK, and ERK were all increased upon TWH19/AURKB overexpression (Figure 6A). To identify the direct substrate of AURKB, we performed in vitro phosphorylation assays by incubating immunoprecipitated C-RAF, MEK, and ERK with \u003cem\u003eE. coli\u003c/em\u003e-purified AURKB. The results revealed that C-RAF was directly phosphorylated by AURKB, specifically at S/T residues, while ERK and MEK were not phosphorylated at either Y or S/T residues (Figure 6B). In 293T cells, C-RAF S/T phosphorylation was inhibited by both Salirasib treatment and mutation of the AURKB active site (K106A) (Figure 6C), which indicates that both the recruitment and the kinase activity of AURKB are essential for C-RAF S/T phosphorylation in cells.\u003c/p\u003e\n\u003cp\u003eTo identify the specific phosphorylation sites, C-RAF was purified from 293T cells with or without co-expression of TWH19 and AURKB and analyzed by mass spectrometry. The results revealed enhanced phosphorylation at multiple sites upon co-expression of TWH19 and AURKB, with T258 exhibiting the greatest fold change (Table S1, Figure 6D). T258 phosphorylation was recorded in PhosphoSitePlus database, but the function is unclear. To assess the functional impact of these sites, each phosphorylated residue was mutated to aspartate to mimic phosphorylation, and the mutants were expressed in 293T cells. Strikingly, the T258D and T260D significantly increased ERK phosphorylation levels, whereas the other mutants did not (Figure 6E). These results indicate that phosphorylation at T258 or T260 in C-RAF is sufficient to activate MAPK.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the functional requirement of T258 and T260 phosphorylation in MAPK activation, we generated alanine mutants to block phosphorylation at these sites (Figure 6F). While wild-type C-RAF markedly enhanced ERK phosphorylation in the presence of TWH19 and AURKB, the T258A mutant and the T258A/T260A double mutant (2TA) showed only minimal effects (Figure 6G). Consistently, in TWH19-depleted cells, C-RAF T258D rescued ERK phosphorylation more potently than WT (Figure S6A). In line with these signaling changes, T258D promoted 293T cell proliferation relative to WT C-RAF, whereas the 2TA mutant impaired cell growth (Figure 6H). At the transcriptional level, T258D selectively increased expression of the canonical ERK target genes, including EGR1 and FOS, while CCND1 and MYC remained unchanged (Figure 6I).\u003c/p\u003e\n\u003cp\u003eTo directly assess endogenous T258 phosphorylation, we generated a phospho-specific antibody against C-RAF pT258. The antibody recognized C-RAF T258D but showed minimal reactivity toward wild-type C-RAF, confirming its selectivity for the phosphorylated epitope (Figure 6J, S6B and S6C). Using this tool, we surveyed endogenous C-RAF pT258 levels across multiple cancer cell lines and observed considerable variations, which somewhat correlated with pERK intensity (Figure 6K). Importantly, overexpression of the phosphomimetic C-RAF T258D mutant enhanced proliferation relative to wild-type C-RAF in these cell lines, albeit to different degrees (Figure 6L). Taken together, these findings establish that AURKB directly phosphorylates C-RAF at T258, a modification that drives ERK activation and cell proliferation.\u003c/p\u003e\n\u003ch3\u003eC-RAF T258 phosphorylation inhibits 14-3-3 binding and promotes C-RAF dimerization\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eWe next investigated how T258 phosphorylation drives ERK activation. It is well-established that S259 phosphorylation recruits 14-3-3 and suppress C-RAF dimerization and subsequent cascade phosphorylation \u003csup\u003e27,28\u003c/sup\u003e. T258 (and T260) is adjacent to S259, therefore, its phosphorylation may inhibit C-RAF\u0026rsquo;s binding to 14-3-3 and promote C-RAF dimerization. Indeed, we showed that T258D binding to 14-3-3 was significantly reduced, whereas 2TA slightly enhanced it (Figure 7A). Using IP, we further showed that T258D not only enhanced C-RAF dimerization with itself, but also its dimerization with A-RAF and B-RAF (Figure 7B, 7C and 7D), indicating that T258 phosphorylation promotes formation of signaling-competent C-RAF dimers.\u003c/p\u003e\n\u003cp\u003eTo determine whether C-RAF dimerization is required for TWH19-driven MAPK activation, we pharmacologically inhibited RAF dimerization using Tovorafenib. Tovorafenib suppressed TWH19-induced ERK phosphorylation (Figure 7E), reduced expression of ERK target genes (EGR1, FOS, CCND1) (Figure 7F), and abolished the growth-promoting effect of TWH19 overexpression (Figure 7G). These results establish that C-RAF dimerization is a critical functional output downstream of C-RAF T258 phosphorylation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next examined whether this regulatory mechanism extends to other RAF isoforms.\u0026nbsp;Sequence alignment shows that T258 in C-RAF is conserved in A-RAF but corresponds to a S in B-RAF (Figure S7A). We therefore investigated whether a phosphorylation mimicking D mutation also activate A-RAF and B-RAF. Unexpectedly, T213D in A-RAF and S364D in B-RAF enhanced 14-3-3 interaction (Figure S7B and S7C). Their dimerization with different RAF isoforms also did not increase upon mutation (Figure S7D-S7I). In contrast C-RAF, phosphorylation mimetic mutants in A-RAF and B-RAF also failed to enhance ERK phosphorylation (Figure S7J). Thus, the T258-dependent release from 14-3-3 and promotion of dimerization appears to be specific to C-RAF.\u003c/p\u003e\n\u003cp\u003eTogether, these results support a working model in which TWH19 scaffolds NRAS and AURKB on membranes to assemble the MAPK module; AURKB phosphorylates C-RAF at T258, which weakens inhibitory 14-3-3 binding, promotes C-RAF dimerization, and thereby enables ERK activation and cell proliferation (Figure 7H).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThrough an integrated approach combining mouse genetics, biochemical reconstitution, structural modeling, and phospho-proteomics, we have uncovered a previously unrecognized mechanism by which TWH19 and AURKB coordinate to regulate the core MAPK cascade. Our findings establish TWH19 as a molecular scaffold that simultaneously engages NRAS-GTP and AURKB at cellular membranes, nucleating assembly of the C-RAF-MEK-ERK module. This spatial organization enables AURKB to directly phosphorylate C-RAF at T258, a modification that weakens inhibitory 14-3-3 binding, promotes C-RAF dimerization, and thereby drives ERK activation. The physiological importance of this axis is underscored by the embryonic lethality and adult tissue homeostasis defects observed upon TWH19 ablation. The absence of detectable \u0026gamma;H2AX accumulation in the examined tissues may be explained by the low proliferation rate of cells in adult tissues, where single-strand breaks are less likely converted into double-strand breaks. Nonetheless, the clear reduction in ERK phosphorylation in the same tissues indicates that this signaling function operates in cells where DNA damage is not readily detectable, arguing for a direct role for TWH19 in MAPK regulation independent of its DNA repair function.\u003c/p\u003e\n\u003cp\u003eOur findings resolve several outstanding questions in the field. First, they explain the long-standing observation that AURKB inhibition reduces ERK phosphorylation in certain cancer contexts \u003csup\u003e9,10,29,30\u003c/sup\u003e, providing a mechanistic basis for a phenomenon that had remained mysterious. Second, our work also necessitates revision of a prior model proposing that TWH19 (STK19) directly phosphorylates NRAS at S89 to promote its oncogenic activity \u003csup\u003e31,32\u003c/sup\u003e. We demonstrate that TWH19 lacks kinase activity \u003csup\u003e16\u003c/sup\u003e and instead functions as a scaffold that recruits the authentic kinase AURKB, which does not phosphorylate NRAS itself but rather the associated C-RAF to regulate ERK signaling. This revised model aligns with the concept that RAS signaling fidelity is achieved through dynamic nanocluster formation \u003csup\u003e33,34\u003c/sup\u003e, and identifies TWH19 as a critical organizer of such assemblies.\u003c/p\u003e\n\u003cp\u003eThe clinical implications of our findings are manifold. The detection of T258 phosphorylation in human cancers, together with the observation that T258D overexpression enhances cell proliferation, suggests that this axis may contribute to cancer progression. Tumors with high TWH19 and AURKB expression might be particularly vulnerable to strategies that disrupt their interaction or inhibit AURKB catalytic activity. Notably, there are several reports linking AURKB to MAPK-targeted therapy resistance in cancers \u003csup\u003e9,10,29,30\u003c/sup\u003e. Our work provides a mechanistic rationale for combining AURKB inhibitors with MAPK pathway-targeted therapies, or for directly disrupting the TWH19-AURKB interaction as a therapeutic strategy in RAS-driven cancers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral questions remain unanswered. Why the RAS-AURKB-TWH19 complex selectively assembles the C-RAF-MEK-ERK cascade but not the PI3K-AKT cascade? Additionally, the contribution of PIP5K1A and SRPK2, two other TWH19-associated kinases (Figure 2D), to MAPK activation warrants further exploration, given their reported role in KRAS signaling \u003csup\u003e24,25\u003c/sup\u003e. Our observation that T258 phosphorylation promotes C-RAF dimerization but the analogous mutations in A-RAF and B-RAF produce distinct effects on 14-3-3 binding adds to the growing understanding that RAF paralogs have distinct functions despite their synergy in ERK signaling \u003csup\u003e35,36\u003c/sup\u003e. How the same phosphorylation event elicits nearly opposite outcomes in different isoforms remains unclear and will require structural studies to resolve.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, our study establishes TWH19 and AURKB as unanticipated but critical regulators of RAS-to-ERK signaling, revealing a fundamental mechanism that controls cell proliferation and organismal survival. By expanding the functional repertoire of both proteins beyond their canonical roles in DNA repair and mitosis, respectively, these findings open new avenues for understanding normal physiology and for therapeutic intervention in diseases where MAPK signaling is dysregulated.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6J wild-type, CAG-iCre transgenic, and TWH19-floxed mice (TWH19-Flox\u003csup\u003e+/-\u003c/sup\u003e) were obtained from GemPharmatech (Nanjing, China). To generate constitutive whole-body TWH19 knockout mice, TWH19-Flox\u003csup\u003e+/-\u003c/sup\u003e mice were crossed with CAG-Cre mice. Offspring genotypes were determined by tail-tip PCR using vendor-provided primers. For embryonic analysis, pregnant females from heterozygous intercrosses were sacrificed at embryonic day 12.5 (E12.5), and embryos were examined for developmental abnormalities or resorption.\u003c/p\u003e\n\u003cp\u003eFor inducible deletion, Cre-ERT2 mice were crossed with TWH19-floxed mice to generate inducible knockout mice (Cre\u003csup\u003e+/-\u003c/sup\u003e ; Flox\u003csup\u003e+/+\u003c/sup\u003e) and littermate controls (Cre\u003csup\u003e-/-\u003c/sup\u003e; Flox\u003csup\u003e+/+\u003c/sup\u003e). Mice were administered tamoxifen (100 mg/kg) once daily for 7 consecutive days, followed by a 1-week washout and a second 7-day tamoxifen course to activate Cre recombinase and induce systemic TWH19 deletion. Body weight was recorded daily, and tissues (liver, spleen, bone marrow) were collected after completion of the regimen to assess deletion efficiency and downstream signaling. Tamoxifen-treated Cre\u003csup\u003e+/-\u003c/sup\u003e; Flox\u003csup\u003e+/+\u003c/sup\u003e mice and controls were monitored for survival, body weight, and tissue phenotypes. All animal experiments were approved by the Animal Ethics Committee of Sichuan Provincial People\u0026apos;s Hospital (approval no. 2025-496).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman NRAS (residues 1-169) was cloned into the pET-15b expression vector with an N-terminal His-tag fusion. Human TWH19, TWH19-41 kD, AURKB and their mutants were cloned into a psyno-1 vector expressing an N-terminal cleavable 6xHis-MBP fusion to facilitate protein expression and purification. His-TWH19 25-C (the 29-kD isoform with residues 1-24 deleted to enhance stability) and its mutants were cloned into a psyno-1 vector expressing an N-terminal 6xHis tag.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll constructs were transformed into \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eBL21 (DE3). Bacterial cultures were grown in LB medium and induced with 0.5 mM IPTG at 22 \u0026deg;C for 12 h. Cells were harvested and sonicated in a lysis buffer containing 50\u0026thinsp;mM Tris pH 8.0, 350\u0026thinsp;mM NaCl, 10% glycerol, 10 mM imidazole, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 1\u0026thinsp;mM PMSF. The proteins were first purified on a Ni-NTA column and eluted in a buffer containing 50\u0026thinsp;mM Tris pH 8.0, 350\u0026thinsp;mM NaCl, 300\u0026thinsp;mM imidazole, 10% glycerol, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e. Eluted proteins were concentrated and further purified by a Superdex 200 Increase gel filtration column on \u0026Auml;ktaPure (GE Healthcare) using a gel filtration buffer containing 20\u0026thinsp;mM Tris pH 8.0, 150\u0026thinsp;mM NaCl, 10% glycerol, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 2\u0026thinsp;mM DTT. Eluted proteins were frozen at -80 \u0026deg;C at 5-10 mg/mL.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor GST-fusion proteins, C-RAF-RBD\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(C-RAF residues 50-131) and NRAS were cloned into pGEX-4T-1 expression vector incorporating an N-terminal TEV cleavable GST tag. After transformation into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3), protein expression was induced with 0.5 mM IPTG at 22 \u0026deg;C overnight. Cells were harvested and sonicated in lysis buffer containing 50\u0026thinsp;mM Tris pH 8.0, 200 mM NaCl, 10% glycerol, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1\u0026thinsp;mM PMSF, and 1 mM DTT. After centrifugation, the supernatant was passed through glutathione sepharose beads and eluted in a buffer containing 20 mM Tris pH 8.0, 200 mM NaCl, 10% glycerol, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM GSH and 1 mM DTT. The proteins were further purified by size exclusion chromatography using a Superdex 200 column (GE Healthcare) in a buffer containing 20 mM Tris pH 8.0, 200 mM NaCl, 10% glycerol, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 2 mM DTT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePull-down assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor MBP pull-down assays, MBP-TWH19, MBP-AURKB, or MBP was immobilized on anti-MBP magnetic beads. For GST pull-down assays, GST-C-RAF-RBD, GST-NRAS or GST was immobilized on glutathione Sepharose beads. Cell lysates or purified proteins (3.0 \u0026mu;M) were incubated with the protein-immobilized beads in a total volume of 500\u0026thinsp;\u0026mu;L with gentle rotation for 2\u0026thinsp;h at 4 \u0026deg;C. After incubation, beads were washed three times with pull-down buffer containing 20\u0026thinsp;mM Tris pH 8.0, 150\u0026thinsp;mM NaCl, 10% glycerol, 2\u0026thinsp;mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM DTT, and 0.005% Triton X-100. Bound proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining or immunoblotting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture, siRNA transfection and plasmid transfection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e293T and HeLa cells were cultured in DMEM (Gibco) with 10% fetal bovine serum (Newzerum) at 37 \u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. For gene knockdown experiments, cells were transfected with the indicated siRNAs (sequences listed in Table S2) using jetPRIME\u0026reg; transfection reagent (Polyplus), and knockdown efficiency was evaluated 72 h after transfection. For plasmid transfection, cells were seeded at approximately 50% confluence and transfected with expression constructs or the corresponding empty vector controls using jetPRIME\u0026reg; (Polyplus) or liposome transfection reagent (Yeasen). After 24 h, the transfected cells were used for subsequent studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells grown on coverslips were fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100. After blocking with 5% bovine serum albumin, cells were incubated with primary antibodies overnight at 4 \u0026deg;C, followed by incubation with fluorophore-conjugated secondary antibodies for 2 h at room temperature. Images were acquired using a Zeiss LSM 900 confocal microscope and analyzed with ImageJ software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoprecipitation and co-immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e293T or HeLa cells were harvested and lysed in lysis buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EGTA, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5% NP-40, 1% (v/v) protease inhibitors cocktail (Selleck, B14001) and 1% phosphatase inhibitor cocktail (B15001, Selleck) at 4 \u0026deg;C for 30 minutes. Lysates were centrifuged at 14,000 rpm for 10 minutes. Supernatants were incubated with glutathione Sepharose beads, anti-Flag affinity gel (Selleck, B23101) or anti-HA magnetic beads (MCE, HY-K0201) for 3 h at 4 \u0026deg;C. After incubation, the beads were washed 3 times with lysis buffer, and the bound proteins were analyzed by western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein samples were separated on 10% SDS-PAGE gels (Vazyme) and transferred onto Immobilon\u0026reg;-P PVDF membranes (Millipore, IPVH00010). Membranes were blocked with 5% (w/v) skimmed milk in TBS-T (TBS, 0.1% (v/v) Tween 20) for 1 h at room temperature and incubated with primary antibody overnight at 4 \u0026deg;C. After washing with TBS-T, the membranes were incubated with HRP-conjugated secondary antibodies in 5% (w/v) skimmed milk in TBS-T and visualized using Super ECL Detection Reagent (Yeasen). Antibodies used in this study are listed in Table S3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology and immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor histological analysis, liver, spleen, and bone marrow tissues were collected from tamoxifen-treated mice and fixed according to standard procedures. Tissue processing, paraffin embedding, sectioning, and immunohistochemical (IHC) staining for \u0026gamma;\u0026middot;H2AX were performed by a commercial service provider (FBersw Biotechnology Co., Ltd., China).\u003c/p\u003e\n\u003cp\u003eStained sections were scanned using a digital slide scanner, and high-resolution images were provided by the vendor. Quantification of \u0026gamma;\u0026middot;H2AX staining intensity was performed using ImageJ software under identical analysis parameters, and values were normalized to the corresponding control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSulforhodamine B (SRB) assay was used to assess cell proliferation. After seeding in 96-well plates (5000 cells/well) overnight, cells were transfected with indicated plasmid or si-RNA and cultured for 3 days. After fixing the cells with 100 \u0026micro;L of 10% TCA for 2 hours, the cells were stained with 0.4% SRB solution (in 1% acetic acid) at room temperature for 20 minutes. After removing the unbound SRB and drying the plate completely, 100 \u0026micro;L of 10 mM Tris Base (pH 10.5) was added to each well to dissolve the bound SRB dye. Finally, the absorbance at 570 nm was measured using a microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro kinase assays\u003c/strong\u003e \u003cstrong\u003eand phosphoproteomic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor in vitro kinase assays, Flag-C-RAF, Flag-MEK1 and Flag-ERK2 proteins were transiently expressed in 293T cells and purified using anti-Flag beads. Bead-bound substrates were incubated with 5.0 \u0026mu;M purified MBP or MBP-AURKB for 2\u0026thinsp;h at 4 \u0026deg;C with gentle rotation. After incubation, beads were washed and the bound proteins were separated by SDS-PAGE. The kinase reaction buffer contained 20\u0026thinsp;mM Tris pH 8.0, 100\u0026thinsp;mM NaCl, 10% glycerol, 2\u0026thinsp;mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM DTT, 300\u0026nbsp;\u0026mu;M ATP and 0.005% Triton X-100. Phosphorylation signals were initially assessed by immunoblotting using phospho-specific antibodies.\u003c/p\u003e\n\u003cp\u003eFor phosphoproteomic analysis, C-RAF samples purified from 293T cells under the indicated conditions were subjected to mass spectrometry analysis performed by a commercial service provider (SpecAlly Life Technology Co., Ltd., China). Phosphorylation sites were identified and quantified based on peptide signal intensity, and fold changes were calculated relative to control conditions. A summary of identified phosphosites is provided in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of a phospho-specific antibody against C-RAF pT258\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA phosphopeptide corresponding to the region surrounding C-RAF pT258 (RQRSpTSTPN) was synthesized by GL Biochem (Shanghai, China). The peptide was emulsified in complete Freund\u0026apos;s adjuvant (0.5 mg/mL) and used to immunize mice by subcutaneous injection at three sites (100 \u0026mu;L per site). Two weeks later, mice received a second immunization with the peptide emulsified in incomplete Freund\u0026apos;s adjuvant (0.75 mg/mL; three sites, 100 \u0026mu;L per site), followed by a third boost three weeks later (incomplete Freund\u0026apos;s adjuvant, 1.0 mg/mL; three sites, 100 \u0026mu;L per site). Serum was collected two weeks after the final boost. Antibodies were purified by anion-exchange chromatography, and fractions (300 \u0026mu;L per tube) were screened using C-RAF proteins to identify fractions that selectively recognized the phosphorylated T258 epitope. Specificity was further evaluated using lysates from 293T cells expressing wild-type C-RAF or the phosphomimetic T258D mutant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll quantitative data were analyzed and plotted using GraphPad Prism. Unless otherwise stated, data are presented as mean \u0026plusmn; SD from independent biological replicates. Statistical significance between two groups was evaluated using a two-tailed unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test. For experiments involving more than two groups, statistical tests are described in the corresponding figure legends (e.g., one-way or two-way ANOVA). P value \u0026lt; 0.05 was considered statistically significant. Significance is denoted as: ns, not significant; *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eNational Natural Science Foundation of China (NSFC #82273850, #82403683);\u003c/p\u003e\n\u003cp\u003eChina Postdoctoral Science Foundation (CPSF #2024M760361, #GZC20240208).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Conceptualization: YL1, QS\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Methodology: YL1, YL2, PC, QS\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Investigation: YL1, YL2, PC\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Visualization: YL1, QS\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Supervision: CW, LG, QS\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Writing-original draft: YL1, QS\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Writing-review \u0026amp; editing: YL1, LG, QS\u003c/p\u003e\n\u003ch2\u003eCompeting interests:\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMoore, A. 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Alike but Different: RAF Paralogs and Their Signaling Outputs. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e161\u003c/strong\u003e, 967-970, doi:10.1016/j.cell.2015.04.045 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"NRAS, phosphorylation, AURKB, MAPK, structure","lastPublishedDoi":"10.21203/rs.3.rs-9362063/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9362063/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The MAPK signaling cascade is a central driver of cell proliferation, yet the mechanisms governing its spatial organization and catalytic amplification remain incompletely understood. Here, we identify TWH19 as a scaffold that nucleates MAPK complex assembly at membranes by simultaneously binding NRAS-GTP and the kinase AURKB. This signaling module enables AURKB to directly phosphorylate C-RAF at T258, thereby weakening inhibitory 14-3-3 interactions, promoting C-RAF dimerization, and activating ERK signaling. Genetic ablation of TWH19 in mice causes embryonic lethality, whereas adult deletion leads to weight loss and profound ERK signaling deficits without detectable γH2AX accumulation in the tissues examined. Disruption of the TWH19-AURKB interface abolishes NRAS recruitment, ERK activation, and cell proliferation. Together, our findings establish TWH19 and AURKB as unanticipated yet critical regulators of RAS-to-ERK signaling and uncover a mechanism governing MAPK pathway activation that is critical for cell proliferation and embryonic development.","manuscriptTitle":"TWH19 scaffolds AURKB to phosphorylate C-RAF and activate MAPK signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 09:05:34","doi":"10.21203/rs.3.rs-9362063/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"66f9e7b4-752f-4ce3-9a79-fe32d81db172","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"3","date":"2026-04-29T03:26:13+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67205191,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":67205192,"name":"Biological sciences/Developmental biology/Cell proliferation"},{"id":67205193,"name":"Biological sciences/Structural biology/Molecular modelling"},{"id":67205194,"name":"Biological sciences/Biochemistry/Kinases"},{"id":67205195,"name":"Biological sciences/Cancer/Oncogenes"}],"tags":[],"updatedAt":"2026-04-30T09:05:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 09:05:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9362063","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9362063","identity":"rs-9362063","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}