Phosphorylation-inducing molecules for regulating dynamic cellular processes

Phosphorylation-inducing molecules for regulating dynamic cellular processes | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Phosphorylation-inducing molecules for regulating dynamic cellular processes View ORCID Profile Rajaiah Pergu , View ORCID Profile Sreekanth Vedagopuram , View ORCID Profile Praveen Kokkonda , View ORCID Profile Sophia Lai , View ORCID Profile Praveen K. Tiwari , View ORCID Profile Sameek Singh , View ORCID Profile Junya Kawai , View ORCID Profile Santanu Singha , View ORCID Profile Wenzhi Tian , View ORCID Profile Vikram Thimaradka , View ORCID Profile Kien Tran , View ORCID Profile Amit Choudhary doi: https://doi.org/10.1101/2025.05.10.652895 Rajaiah Pergu 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rajaiah Pergu Sreekanth Vedagopuram 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA 2 Department of Medicine, Harvard Medical School , Boston, MA 02115, USA 3 Divisons of Renal Medicine and Engineering, Brigham and Women’s Hospital , Boston, MA 02115, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sreekanth Vedagopuram Praveen Kokkonda 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Praveen Kokkonda Sophia Lai 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA 4 Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, MA 02138, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sophia Lai Praveen K. Tiwari 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA 2 Department of Medicine, Harvard Medical School , Boston, MA 02115, USA 3 Divisons of Renal Medicine and Engineering, Brigham and Women’s Hospital , Boston, MA 02115, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Praveen K. Tiwari Sameek Singh 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sameek Singh Junya Kawai 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Junya Kawai Santanu Singha 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Santanu Singha Wenzhi Tian 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Wenzhi Tian Vikram Thimaradka 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA 2 Department of Medicine, Harvard Medical School , Boston, MA 02115, USA 3 Divisons of Renal Medicine and Engineering, Brigham and Women’s Hospital , Boston, MA 02115, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Vikram Thimaradka Kien Tran 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA 2 Department of Medicine, Harvard Medical School , Boston, MA 02115, USA 3 Divisons of Renal Medicine and Engineering, Brigham and Women’s Hospital , Boston, MA 02115, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kien Tran Amit Choudhary 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA 2 Department of Medicine, Harvard Medical School , Boston, MA 02115, USA 3 Divisons of Renal Medicine and Engineering, Brigham and Women’s Hospital , Boston, MA 02115, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Amit Choudhary For correspondence: achoudhary{at}bwh.harvard.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Dynamic cellular processes often employ protein phosphorylation for rapid information transfer within and between cells. Phosphorylation-inducing chimeric small molecules (PHICS) have been developed for targeted protein phosphorylation by on-demand inducing a kinase-protein pairing. However, widespread application of PHICS has been limited as previously reported PHICS that recruited AMP-activated protein kinase (AMPK) required serum starvation and target protein overexpression, recruited only a few of the potential AMPK complexes, and exhibited poor dose- and temporal control. Herein, we report an AMPK PHICS platform that operates under physiological conditions (i.e., no serum starvation or target overexpression), recruits multiple AMPK complexes, and can induce target protein phosphorylation with dose and temporal control. We demonstrated the utility of this platform for controlling phosphorylations that underlie two dynamic cellular processes, namely oncogenic signaling and phase separation. An AMPK PHICS directed against Bruton’s Tyrosine Kinase (BTK), which is a driver of several B cell malignancies, was effective at inducing the death of drug-resistant cancer cells. Here, PHICS induced inhibitory phosphorylations on BTK and attenuated its oncogenic pathway. Phosphorylation of Liprin-α3 induces phase separation in neurons and is critical for neurotransmitter release. Using AMPK PHICS that phosphorylated Liprin-α3, we induced Liprin-α3 phase separation with dose and temporal control. We envision this PHICS platform to find utility in inducing and controlling protein phosphorylations for basic research and biomedicine. Download figure Open in new tab Introduction Nature employs posttranslational modifications (PTMs) to regulate highly dynamic processes using specific enzymes that either “write” or “erase” PTMs on a target protein. 1 , 2 Leveraging the ability of small molecules that afford dose and temporal control of enzymic activity, 3 several groups have reported synthetic chimeric molecules for writing or erasing PTMs. These chimeric molecules, 4 , 5 formed by connecting binders of a target and an endogenous PTM-writing or -erasing enzyme, induce proximity between the enzyme and target, resulting in PTM induction or removal even when the target protein is not the enzyme’s natural substrate. For example, proteolysis-targeting chimeras (PROTACs) 6 have been developed to induce proximity between the target protein and E3 ubiquitin ligase, causing ubiquitination and subsequent protein degradation. Chimeras have also been reported for writing or erasing PTMs, such as deubiquitinase-targeting chimeras (DUBTACs) that remove ubiquitin, 7 phosphorylation-targeting chimeras (PhosTACs) and phosphatase-targeting chimeras (PHORCs) that remove phosphoryl group, 8 , 9 heterobifunctional molecules that induce acetylation (AceTAGs), 10 and bifunctional nanobody conjugates that add or remove glycosylation. 11 , 12 We recently described phosphorylation-inducing chimeric small molecules (PHICS) that allow kinases to phosphorylate target proteins, including non-natural substrates. 13 - 15 These chimeras operate on the principle of chemically-induced proximity: linking binders of the kinase and target protein together can artificially induce an enzyme-substrate pairing that results in targeted protein phosphorylation. While the reported kinase binder in PHICS to recruit AMP-activated protein kinase (AMPK) performed well in proof-of-concept systems, the binder suffers from serious flaws preventing the widespread application of PHICS. For instance, previous AMPK ligands required overexpression of the target protein and serum starvation of cells, and they exhibited poor dose- and temporal control, all of which prevent PHICS applications in realistic biological scenarios. Additionally, the reported AMPK ligands recruited only β1 isoforms of AMPK, which is a complex comprising three heterotrimeric subunits, each with various isoforms—the α subunit has two isoforms, β has 2, and γ has 3, for 12 possible AMPK complexes 16 in mammalian cells. As these complexes vary in expression across cell types, an AMPK binder that can recruit all complexes can be useful in myriad scenarios. 17 Herein, we describe a new AMPK PHICS platform that uses a pan-AMPK binder capable of recruiting multiple AMPK complexes ( Figure 1A ) and that functions on endogenous proteins without cell starvation, allowing precise dose and temporal control of biological pathways. With this platform, we showed the first examples of AMPK-induced phosphorylation in non-native targets, including endogenous BCR-ABL, an aberrant, constitutively active fusion protein of BCR and ABL tyrosine kinase commonly expressed in CML patients. 18 We also applied this platform to two relevant biological scenarios. Bruton Tyrosine Kinase (BTK) is a driver of the proliferation of malignant B cells in chronic lymphocytic leukemia and mantle cell lymphoma and resistance has emerged for drugs targeting BTK. 19 We developed PHICS that induce inhibitory phosphorylations on BTK and these PHICS also impeded growth of drug-resistant cancer cell lines. The active zone in neurons, which plays a critical role in neurotransmitter release, is a highly dynamic and specialized structure controlled by phosphorylation-induced phase separation of Liprin-α3. 20 Using AMPK PHICS targeting Liprin-α3, we induced its phase separation with dose- and temporal control. Using global phosphoproteomics and genetic methods, we identified and validated the phosphorylation site. These studies led to discovery of a Liprin-α3 variant with a two-fold higher degree of phosphorylation-mediated phase separation. Overall, these studies describe a new PHICS platform to induce serine/threonine phosphorylation with dose and temporal control that enabled induction of phase-separation triggering phosphorylation and inhibitory phosphorylation to block an oncogenic signaling pathway. Download figure Open in new tab Figure 1. Design of AMPK PHICS. ( A ) Binders for β1-specific AMPK and pan-AMPK recruitment. ( B ) Molecular docking showing the solvent-accessible end of the modified MK8722 analog. ( C ) Treatment of BRD4-GST with 1.1 or 1.2 and five AMPK complexes revealed differences in BRD4 phosphorylation. Asterisk refers to GST-tagged AMPK subunit, and the open circle refers to the phosphorylated target protein (p-BRD4-GST). BRD4 and two of the AMPK subunits (α1/β2/γ3 and α2/β1/γ2) have GST-tag. ( D ) Chemical structures of the BCR-ABL PHICS and its analogs. ( E ) Induction of BCR-ABL phosphorylation by PHICS. ( F ) Comparison of phosphorylation-inducing activities of active and inactive PHICS analogs n K562 cells. ( G ) Competition experiment using MK8722 competition (1:1 at 5 µM) against 1.6 in K562 cells. Results and Discussion Development of AMPK PHICS platform The efficacy of AMPK PHICS would be increased via the capacity to recruit various AMPK complexes with differing intrinsic activities. To develop a pan-AMPK PHICS, we used molecular docking of the reported pan-AMPK binder MK8722 to identify the linker attachment site as the para position of the terminal phenyl ring of MK8722 that is located on the opposite end of the isomannide ring ( Figure 1B and Figure S1A ). 21 , 22 In addition to being solvent inaccessible, the free hydroxyl on the isomannide forms hydrogen bonds to Asn48 and Ile46, which should not be perturbed to maximize binding affinity ( Figure S1B ). With the chosen attachment site, the para-appended 2-phenoxylethylamino linker emerges between the α (catalytic domain) and β subunits ( Figure S2 ). To test the ability of MK8722-based PHICS to recruit multiple isoforms of AMPK, we measured the phosphorylation induced in the target bromodomain-containing protein 4 (BRD4) 13 in the presence of various PHICS and isoforms of AMPK. Since the AMPK complexes differ in their intrinsic kinase activities, it is not meaningful to directly compare the phosphorylation levels between the various complexes but rather to observe the relative phosphorylation induced via various PHICS. To thus generate a direct comparison, we synthesized two PHICS with different AMPK ligands, with 1.1 containing the β1-specific AMPK binder from our previous work and 1.2 containing our modified MK8722 ( Figure S3 ). Using immunoblotting, we measured the ability of these compounds to phosphorylate recombinant truncated BRD4-GST via five different AMPK heterotrimers— α1/β1/γ1, α1/β2/γ1, α1/β1/γ3, α1/β2/γ3, and α2/β1/γ2 ( Figure 1C ). Comparing the relative activities of 1.1 versus 1.2 , isoforms α1/β1/γ1, α1/β2/γ1, and α1/β1/γ3 saw similar BRD4 phosphorylation levels between the two PHICS, while isoforms α1/β2/γ3, and α2/β1/γ2 only induced phosphorylation in the presence of 1.2 . Thus, we have demonstrated that this pan-specific AMPK binder can recruit more complexes to its target protein of interest, expanding the potential generalizability of the AMPK PHICS platform. Next, to test our pan-AMPK PHICS against an endogenous protein, we targeted BCR-ABL, which is a constitutively active fusion of BCR and ABL, and driver of CML pathogenesis. 23 To design PHICS against BCR-ABL, we used the allosteric and non-ATP competitive inhibitor GNF-2 because of its selectivity against BCR-ABL compared to other tyrosine kinases, including wild-type ABL. 24 We generated PHICS with various linker lengths ( Figure 1D ) to identify the analog that induced the highest phosphorylation level. Among the PHICS tested, 1.6 generated the highest level of endogenous BCR-ABL phosphorylation in K562 cells ( Figure 1E , S4A ). We generated a control compound 1.7 that has reduced affinity 25 for BCR-ABL and this control compound induced less phosphorylation ( Figure 1F , S4B ). To demonstrate whether MK8722 could compete with 1.6 and reduce phosphorylation levels, we co-treated 1.6 with MK8722 and observed reduced BCR-ABL phosphorylation ( Figure 1G , S4C ). We also observed dose-dependent phosphorylation induction of endogenous BCR-ABL phosphorylation in K562 cells by 1.6 ( Figure S4D ). Importantly, phosphorylation of BCR-ABL was observed in as little as 0.5–1 µM 1.6 without needing to overexpress either BCR-ABL or AMPK. Overall, these results suggest that PHICS can induce phosphorylation on the endogenous target protein. To determine if the new PHICS platform can induce phosphorylation without serum starvation of cells, we used BTK as the target protein whose phosphorylation (pS180) by previous PHICS required serum starvation ( Figure S5A ). 13 We synthesized several BTK PHICS containing various linker lengths and chemotypes ( Figure 2E ) and tested them in HEK293T cells transiently expressing BTK. From the compounds tested, the newly generated PHICS 2.3, 2.4 , and 2.5 yielded a higher phosphorylation (pS180) level as compared to previously generated 2.1 and 2.2 , though interestingly, PHICS did not require the HEK293T cells to be serum starved to induce phosphorylation ( Figure 2B , S5B, C ). In addition, the BTK PHICS generated herein induced phosphorylation on BTK (pS180) at lower concentrations than had been required in the previous work ( Figure S5B ). This might be due to the ability of a pan-AMPK binder to recruit many different AMPK complexes, making it statistically more likely to induce a ternary complex between AMPK and the target protein BTK. We note that individual components of BTK PHICS (AMPK or BTK binder) did not induce S180 phosphorylation ( Figure S5C ). Overall, these studies suggest that the AMPK PHICS platform can induce phosphorylation of target proteins without serum starvation. Download figure Open in new tab Figure 2. Development and functional characterization of BTK PHICS. ( A ) Chemical structures of BTK PHICS with varying linkers and chemotypes. ( B ) Induction of BTK S180 phosphorylation expressed in HEK293T cells treated with 5 µM of PHICS in serum-containing media. Results represent mean ± sd, relative pBTK S180 levels (fold-change with DMSO control). ( C ) Schematic representation of BTK domains (PH: Pleckstrin Homology; TH: Tec Homology; BH: BTK Homology region; PPR: PolyProline Region; SH3: Src-Homology 3; SH2: Src-Homology 2; Sh1: Src-Homology 1(Kinase domain). ( D ) Quantification of the effect of phospho-resistant and phospho-mimetic variants of BTK on its kinase activity measured by the autophosphorylation (pY223) levels. ( E ) CellTiter-Glo viability assay demonstrating the dose-dependent effects of PHICS 2.5 on Raji (EC 50 = 3.2µM), and HEK293T cells viability. BTK PHICS induces death of ibrutinib-resistant cells S180 phosphorylation is known to inhibit BTK activity 26 and we, indeed, observed a decrease in BTK autophosphorylation at pY223 (a metric for kinase activity) in PHICS-treated cells ( Figure S5D ). The aforementioned studies focused on S180, and we were interested in determining if other sites were being phosphorylated. We treated HEK293T cells transiently expressing BTK with PHICS and performed mass spectrometry on immunoprecipitated BTK using DMSO-treated cells as a control. We used a previously reported method 27 to determine the relative phosphorylation levels in PHICS and DMSO-treated cells and observed increased phosphorylation at S366, S378, T410, and S604 but not at T191 or T316 ( Figure S5E-J, S6 ). T410 is located near the ATP binding pocket, while S366 and S378 are on the loops of the SH2 domain, which is critical for BTK activity. To determine the impact of these phosphorylations on BTK activity, we generated phospho-mimetic (Ser/Thr→Asp) and phospho-resistant (Ser/Thr→Ala) BTK variants at these sites. We assessed the activity of these variants by measuring the Y223 autophosphorylation and observed that the phospho-mimetic variants at S366, S378, T410, or S604 had reduced Y223 autophosphorylation ( Figure 2D , S5L ), but the control mutations (Ser→Ala) at these sites did not reduce Y223 phosphorylation. These studies suggest that inhibition of BTK activity by PHICS could arise via multiple mechanisms in addition to induction of S180 phosphorylation. Motivated by the new inhibitory mechanisms of PHICS, we explored PHICS activity in the context of drug-resistant cancer cells. The FDA-approved BTK inhibitor, ibrutinib, prevents B-cell proliferation by blocking the aberrant BTK activity in B-cell cancers, such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) 28 but the emergence of ibrutinib resistance is a challenge. 29 We hypothesize that despite utilizing the same BTK binder (ibrutinib), chimeric molecules can circumvent resistance arising from binding pocket mutations, owing to their event-driven mechanism of action rather than the conventional occupancy-driven approach. 30 To test this hypothesis, we tested PHICS in reported 31 BTK cell lines (Raji and Mino) against which Ibrutinib exhibits weak antiproliferative activity (EC 50 = 14.5 µM for Raji and 8.4 μM for Mino cells). Strikingly, 2.5 inhibited the proliferation of Raji and Mino cells but not HEK293T cells (BTK negative cells), with an EC 50 of 3.2 µM and 5.0 µM, respectively ( Figure 2E , S5M ). Since the dose-response curve’s baselines differ between cell lines, we computed area under the curve (AUC) and observed similar selectivity of 2.5 for BTK cell lines over HEK293T cells ( Figure S5N ). 32 We note that the tested PHICS still exhibit moderate potency (low micromolar), and further structure-activity optimization will be needed for deep mechanistic investigations, an avenue we will pursue in the future. PHICS induces Liprin-α3 phase separation Charged PTMs, like phosphorylation, play an essential role in regulating the formation and function of protein condensates that have a crucial role in processes such as metabolism, 33 signaling complexes, 34 gene transcription and translation, 35 and synaptic transmission. 36 Phosphorylation-induced phase separation of Liprin-α3, a component of specialized presynaptic structures, is responsible for regulating synaptic vesicle exocytosis ( Figure 3A ) . 20 Since small-molecule binders of Liprin-α3 are unavailable, we used a chemogenetic tag (FKBP12 F36V ) 37 fused to Liprin-α3 for recruiting AMPK to Liprin-α3 using PHICS. We synthesized several bifunctional molecules using pan-AMPK binder and FKBP12 F36V binder (AP1867) connected by linkers of various length and chemotype ( Figure 3B ) . In parallel, we generated HEK293T cell lines stably expressing mVenus–Liprin-α3 fused to FKBP12 F36V at either N- or C-termini ( Figure 3C ) . A two-dimensional screening of Liprin fusions and bifunctional molecules using automated imaging showed that only the C-terminal FKBP fusion (hereafter referred to as mVenus–Liprin-α3–FKBP12 F36V ) exhibited phase separation ( Figure 3D ) . Notably, neither the individual binders (MK8722 or AP1867) nor their combination exhibited phase separation. PHICS exhibited linker length dependence and PHICS treatment did not induce phase separation in cells expressing mVenus–Liprin-α3 that lacks FKBP12 F36V chemogenetic tag ( Figure 3D ) . Download figure Open in new tab Figure 3. Phase separation of Liprin-α3. ( A ) Schematic representation of Liprin-α3–PHICS–AMPK ternary complex. ( B ) Chemical structures of Liprin-α3 PHICS with varying linker lengths and chemotypes. ( C ) Schematic representation of Liprin-α3 constructs showing N- or C-terminal fusion of FKBP12 F36V . ( D ) Screening of Liprin-α3 constructs for the formation of Liprin-α3 droplets in the presence of PHICS (5 µM) or controls. ( E ) Confocal microscopic images (63x) of Liprin-α3 droplets in HEK293T cells stably expressing mVenus–Liprin-α3–FKBP12 F36V . ( F, G ) Quantification of dose and time-dependent formation of Liprin-α3 droplets induced by 3.3 . ( H ) Competition experiment with FKBP12 F36V binder AP1867 (5 µM) with 3.3 (5 µM). Among the PHICS tested compound 3.3 exhibited the most pronounced induction of Liprin-α3 phase separation, with robust droplet formation observed across a 1–10 µM concentration range ( Figures 3E, F , S7A) . Importantly, neither individual binders (MK8722 or AP1867) alone nor their equimolar combination (Figure S7A) induced phase separation, reinforcing the necessity of optimizing linker length and chemotype to achieve the appropriate kinase–substrate proximity and orientation for phase separation. Additionally, the phase separation process induced by 3.3 was rapid, with detectable droplet formation within 15 minutes of treatment and saturation observed at approximately 45 minutes ( Figure 3G ) . Competition experiment with co-treatment with AP1867 abrogated PHICS activity, confirming that 3.3 triggered droplet formation depends on its bifunctional nature ( Figure 3H ) . Identification of phosphorylation sites responsible for Liprin-α3 droplet formation Following confirmation of the bifunctional mechanism, we performed global phosphoproteomics to evaluate the selectivity of 3.3 and to identify the phosphosite responsible for PHICS-induced phase separation. Cells treated with 3.3 had similar global protein expression profile as cells treated with either DMSO ( Figure S7B ) or equimolar combination of MK87222 and AP1867 ( Figure S7C ). Furthermore, out of ≈42,000 identified phosphopeptides, only 17 phosphopeptides showed significant increase (fold-change >2.0, p < 0.05) in 3.3 -treated samples ( Figure 4A ). Of these 17 phosphopeptides, 14 are on mVenus-Liprin-α3 fusion, 2 on CASK (a Liprin-α3 interactor), and 1 on ELF4G, pointing to the exquisite specificity of PHICS. Finally, MK8722 treated cells showed an increase in phosphopeptides from known AMPK substrates 38 (fold-change >2.0, p <0.05), but not on Liprin-α3 ( Figure S7D ), further suggesting that 3.3 is operating via bifunctional mechanism. Download figure Open in new tab Figure 4. Global phosphoproteomics and validation of phosphosites responsible for Liprin-α3 droplet formation ( A ) Volcano plots displaying the −log 10 ( P -value) versus log 2 fold-change of phosphopeptides for 3.3 vs . combination of MK8722 and AP1867. ( B ) Schematic representation and alphafold2 structure of Liprin-α3 marked with potential phosphosites (red). Coiled-coil regions and sterile alpha motifs (SAM) are also highlighted in the schematic. ( C ) Confocal microscopic images (40x) of HEK293T stable cells expressing single alanine variant (i.e., S/T→A) of mVenus–Liprin-α3– FKBP12 F36V . ( D ) Screening and validation of 3.3 -induced Liprin-α3 droplets in HEK293T stable cell lines expressing single alanine variants of mVenus–Liprin-α3–FKBP12 F36V . ( E-H ) Confocal microscopic images (E, G) and quantification (F, H) of 3.3 -induced Liprin-α3 droplets in HEK293T stable cell lines expressing double and multiple alanine variants of mVenus–Liprin-α3–FKBP12 F36V . While global phosphoproteomics identified several 3.3 induced phosphosites that mostly reside in the disordered regions of the Liprin-α3, we did not observe phosphorylation at S760 that has been shown to trigger phase separation. 20 The S760 is a basophilic site (Ser/Thr are surrounded by Arg/Lys), which complicates global phosphoproteomics studies as the digestion enzymes used (Trypsin/Lys-C) also cleave at Arg/Lys. Furthermore, AMPK has a higher propensity to phosphorylate basophilic site. Hence, we created an exhaustive list of potential phosphorylation sites considering not just the aforementioned phosphoproteomics data but also potential AMPK phosphosites 38 , 40 , 41 and literature reports ( Figure 4B ). 39 , 42 , 43 To validate these sites, we created 14 cell lines stably expressing the alanine variant of the phosphosite (i.e., S/T→A) ( Figure 4B , S7D) . Of all the variants tested, S764A variant showed the most resistance to 3.3 -mediated droplet formation ( Figure 4C, D ). Interestingly, S760A or S763A variants alone were not resistant ( Figure 4C, D ), but composite mutations with S764A (i.e., double mutants S760A/S764A or S763A/S764A, or triple mutant S764A/S763A/S760A) were resistant ( Figure 4E-H ), further validating that S764 is the target phosphosite. Finally, the S760A variant showed two-fold higher propensity to form droplets than wild-type in presence of PHICS, opening avenues to examine the role of enhanced droplet formation on neuronal function. Overall, these results suggest that PHICS can redirect AMPK to phosphorylate S764 and induce phase-separation of Liprin-α3. Conclusions We addressed issues with the previous PHICS platform that limited its applicability by designing, synthesizing, and validating the PHICS platform that recruits AMPK to target proteins. To do this, we used a pan-AMPK binder to generate AMPK PHICS for different target proteins and demonstrated their recruitment of multiple types of AMPK complexes. The AMPK PHICS can induce phosphorylation without serum starvation of cells and has demonstrated phosphorylation of an endogenous protein without the need for target overexpression. Application of AMPK PHICS to biologically relevant models revealed its potent inhibitory effects on cell proliferation in ibrutinib-resistant cells. Finally, further investigations using AMPK PHICS demonstrated its ability to initiate Liprin-α3 phosphorylation-mediated phase separation. Overall, these findings suggest that PHICS can be designed to induce proximity and the correct orientation of the kinase and the target protein for effective phosphorylation, emphasizing the potential utility of this bifunctional system in inducing biologically relevant phosphorylations on target proteins. The ability of these bifunctional compounds to modify the phosphorylation of native and disease-relevant proteins has therapeutic implications in health and disease. Author Contributions R.P., S.V., P.K., S.L., P.K.T., and S.S. contributed equally to this paper. COMPETING FINANCIAL INTERESTS Broad Institute has filed patent applications for the work described herein, some of which were licensed to Photys Therapeutics. A.C. is the scientific founder and is on the scientific advisory board of Photys Therapeutics. J.K. is an employee of Daiichi Sankyo Co., Ltd. Acknowledgments This work was supported by the Merkin Institute of Transformative Technologies in Healthcare, The Mark Foundation for Cancer Research, DARPA (HR00112120010), and NIH (1U01DK137242, R01GM137606, R01DK129464, R01GM132825). S.L. was supported by the NSF Graduate Research Fellowship (DGE-1745303). We thank Prof. Pascal Kaeser for providing Liprin plasmids. Funder Information Declared Merkin Institute of Transformative Technologies in Healthcare The Mark Foundation for Cancer Research DARPA , HR00112120010 NIH , 1U01DK137242 , R01GM137606 , R01DK129464 , R01GM132825 References 1. ↵ Cohen , P. The origins of protein phosphorylation . Nat Cell Biol 2002 , 4 ( 5 ), E127 – 130 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Walsh , C. Posttranslational modification of proteins: expanding nature’s inventory ; Roberts and Company Publishers , 2006 . 3. ↵ Weiss , W. A. ; Taylor , S. S. ; Shokat , K. M. Recognizing and exploiting differences between RNAi and small-molecule inhibitors . Nature chemical biology 2007 , 3 ( 12 ), 739 – 744 . OpenUrl CrossRef PubMed 4. ↵ Gerry , C. J. ; Schreiber , S. L. Unifying principles of bifunctional, proximity-inducing small molecules . Nat Chem Biol 2020 , 16 ( 4 ), 369 – 378 . OpenUrl CrossRef PubMed 5. ↵ Singh , S. ; Tian , W. ; Severance , Z. C. ; Chaudhary , S. K. ; Anokhina , V. ; Mondal , B. ; Pergu , R. ; Singh , P. ; Dhawa , U. ; Singha , S. ; et al. Proximity-inducing modalities: the past, present, and future . Chemical Society Reviews 2023 , 52 ( 16 ), 5485 - 5515 , 10.1039/D2CS00943A. OpenUrl CrossRef PubMed 6. ↵ Chirnomas , D. ; Hornberger , K. R. ; Crews , C. M. Protein degraders enter the clinic - a new approach to cancer therapy . Nat Rev Clin Oncol 2023 , 20 ( 4 ), 265 – 278 . OpenUrl CrossRef PubMed 7. ↵ Henning , N. J. ; Boike , L. ; Spradlin , J. N. ; Ward , C. C. ; Liu , G. ; Zhang , E. ; Belcher , B. P. ; Brittain , S. M. ; Hesse , M. J. ; Dovala , D. ; et al. Deubiquitinase-targeting chimeras for targeted protein stabilization . Nature Chemical Biology 2022 , 18 ( 4 ), 412 – 421 . OpenUrl CrossRef PubMed 8. ↵ Chen , P.-H. ; Hu , Z. ; An , E. ; Okeke , I. ; Zheng , S. ; Luo , X. ; Gong , A. ; Jaime-Figueroa , S. ; Crews , C. M. Modulation of Phosphoprotein Activity by Phosphorylation Targeting Chimeras (PhosTACs) . ACS Chemical Biology 2021 , 16 ( 12 ), 2808 – 2815 . OpenUrl CrossRef PubMed 9. ↵ Yamazoe , S. ; Tom , J. ; Fu , Y. ; Wu , W. ; Zeng , L. ; Sun , C. ; Liu , Q. ; Lin , J. ; Lin , K. ; Fairbrother , W. J. ; et al. Heterobifunctional Molecules Induce Dephosphorylation of Kinases-A Proof of Concept Study . J Med Chem 2020 , 63 ( 6 ), 2807 – 2813 . OpenUrl CrossRef PubMed 10. ↵ Wang , W. W. ; Chen , L.-Y. ; Wozniak , J. M. ; Jadhav , A. M. ; Anderson , H. ; Malone , T. E. ; Parker , C. G. Targeted Protein Acetylation in Cells Using Heterobifunctional Molecules . Journal of the American Chemical Society 2021 , 143 ( 40 ), 16700 – 16708 . OpenUrl CrossRef PubMed 11. ↵ Ramirez , D. H. ; Aonbangkhen , C. ; Wu , H.-Y. ; Naftaly , J. A. ; Tang , S. ; O’Meara , T. R. ; Woo , C. M. Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells . ACS Chemical Biology 2020 , 15 ( 4 ), 1059 – 1066 . OpenUrl CrossRef PubMed 12. ↵ Ge , Y. ; Ramirez , D. H. ; Yang , B. ; D’Souza , A. K. ; Aonbangkhen , C. ; Wong , S. ; Woo , C. M. Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase . Nat Chem Biol 2021 . 13. ↵ Siriwardena , S. U. ; Munkanatta Godage , D. N. P. ; Shoba , V. M. ; Lai , S. ; Shi , M. ; Wu , P. ; Chaudhary , S. K. ; Schreiber , S. L. ; Choudhary , A. Phosphorylation-Inducing Chimeric Small Molecules . J Am Chem Soc 2020 , 142 ( 33 ), 14052 – 14057 . OpenUrl CrossRef PubMed 14. Shoba , V. M. ; Munkanatta Godage , D. N. P. ; Chaudhary , S. K. ; Deb , A. ; Siriwardena , S. U. ; Choudhary , A. Synthetic Reprogramming of Kinases Expands Cellular Activities of Proteins . Angewandte Chemie International Edition 2022 , 61 ( 29 ), e202202770 . OpenUrl CrossRef 15. ↵ Pergu , R. ; Shoba , V. M. ; Chaudhary , S. K. ; Munkanatta Godage , D. N. P. ; Deb , A. ; Singha , S. ; Dhawa , U. ; Singh , P. ; Anokhina , V. ; Singh , S. ; et al. Development and Applications of Chimera Platforms for Tyrosine Phosphorylation . ACS Cent Sci 2023 , 9 ( 8 ), 1558 – 1566 . OpenUrl CrossRef PubMed 16. ↵ Herzig , S. ; Shaw , R. J. AMPK: guardian of metabolism and mitochondrial homeostasis . Nat Rev Mol Cell Biol 2018 , 19 ( 2 ), 121 – 135 . OpenUrl CrossRef PubMed 17. ↵ Aledavood , E. ; Forte , A. ; Estarellas , C. ; Javier Luque , F. Structural basis of the selective activation of enzyme isoforms: Allosteric response to activators of β1- and β2-containing AMPK complexes . Computational and Structural Biotechnology Journal 2021 , 19 , 3394 – 3406 . OpenUrl CrossRef 18. ↵ Nowell , P. C. Discovery of the Philadelphia chromosome: a personal perspective . J Clin Invest 2007 , 117 ( 8 ), 2033 – 2035 . OpenUrl CrossRef PubMed Web of Science 19. ↵ Hendriks , R. W. ; Yuvaraj , S. ; Kil , L. P. Targeting Bruton’s tyrosine kinase in B cell malignancies . Nat Rev Cancer 2014 , 14 ( 4 ), 219 – 232 . OpenUrl CrossRef PubMed 20. ↵ Emperador-Melero , J. ; Wong , M. Y. ; Wang , S. S. H. ; de Nola , G. ; Nyitrai , H. ; Kirchhausen , T. ; Kaeser , P. S. PKC-phosphorylation of Liprin-alpha3 triggers phase separation and controls presynaptic active zone structure . Nat Commun 2021 , 12 ( 1 ), 3057 . OpenUrl CrossRef PubMed 21. ↵ Feng , D. ; Biftu , T. ; Romero , F. A. ; Kekec , A. ; Dropinski , J. ; Kassick , A. ; Xu , S. ; Kurtz , M. M. ; Gollapudi , A. ; Shao , Q. ; et al. Discovery of MK-8722: A Systemic, Direct Pan-Activator of AMP-Activated Protein Kinase . ACS Medicinal Chemistry Letters 2018 , 9 ( 1 ), 39 – 44 . OpenUrl CrossRef PubMed 22. ↵ Myers , R. W. ; Guan , H.-P. ; Ehrhart , J. ; Petrov , A. ; Prahalada , S. ; Tozzo , E. ; Yang , X. ; Kurtz , M. M. ; Trujillo , M. ; Gonzalez Trotter , D. ; et al. Systemic pan-AMPK activator MK-8722 improves glucose homeostasis but induces cardiac hypertrophy . Science 2017 , 357 ( 6350 ), 507 – 511 . OpenUrl Abstract / FREE Full Text 23. ↵ Ren , R. Mechanisms of BCR–ABL in the pathogenesis of chronic myelogenous leukaemia . Nature Reviews Cancer 2005 , 5 ( 3 ), 172 – 183 . OpenUrl CrossRef PubMed Web of Science 24. ↵ Adrián , F. J. ; Ding , Q. ; Sim , T. ; Velentza , A. ; Sloan , C. ; Liu , Y. ; Zhang , G. ; Hur , W. ; Ding , S. ; Manley , P. ; et al. Allosteric inhibitors of Bcr-abl-dependent cell proliferation . Nature chemical biology 2006 , 2 ( 2 ), 95 – 102 . OpenUrl CrossRef PubMed 25. ↵ Jahnke , W. ; Grotzfeld , R. M. ; Pellé , X. ; Strauss , A. ; Fendrich , G. ; Cowan-Jacob , S. W. ; Cotesta , S. ; Fabbro , D. ; Furet , P. ; Mestan , J. ; et al. Binding or Bending: Distinction of Allosteric Abl Kinase Agonists from Antagonists by an NMR-Based Conformational Assay . Journal of the American Chemical Society 2010 , 132 ( 20 ), 7043 – 7048 . OpenUrl CrossRef PubMed Web of Science 26. ↵ Kang , S. W. ; Wahl , M. I. ; Chu , J. ; Kitaura , J. ; Kawakami , Y. ; Kato , R. M. ; Tabuchi , R. ; Tarakhovsky , A. ; Kawakami , T. ; Turck , C. W. ; et al. PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization . Embo j 2001 , 20 ( 20 ), 5692 – 5702 . OpenUrl Abstract / FREE Full Text 27. ↵ Bonenfant , D. ; Schmelzle , T. ; Jacinto , E. ; Crespo , J. L. ; Mini , T. ; Hall , M. N. ; Jenoe , P. Quantitation of changes in protein phosphorylation: A simple method based on stable isotope labeling and mass spectrometry . Proceedings of the National Academy of Sciences 2003 , 100 ( 3 ), 880 – 885 . OpenUrl Abstract / FREE Full Text 28. ↵ Wilson , W. H. ; Young , R. M. ; Schmitz , R. ; Yang , Y. ; Pittaluga , S. ; Wright , G. ; Lih , C. J. ; Williams , P. M. ; Shaffer , A. L. ; Gerecitano , J. ; et al. Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma . Nat Med 2015 , 21 ( 8 ), 922 – 926 . OpenUrl CrossRef PubMed 29. ↵ George , B. ; Mullick Chowdhury , S. ; Hart , A. ; Sircar , A. ; Singh , S. K. ; Nath , U. K. ; Mamgain , M. ; Singhal , N. K. ; Sehgal , L. ; Jain , N. Ibrutinib Resistance Mechanisms and Treatment Strategies for B-Cell Lymphomas . Cancers 2020 , 12 ( 5 ), 1328 – 1328 . OpenUrl CrossRef PubMed 30. ↵ Modell , A. E. ; Lai , S. ; Nguyen , T. M. ; Choudhary , A. Bifunctional modalities for repurposing protein function . Cell Chemical Biology 2021 , 28 ( 7 ), 1081 – 1089 . OpenUrl CrossRef PubMed 31. ↵ Yu , H. ; Wang , X. ; Li , J. ; Ye , Y. ; Wang , D. ; Fang , W. ; Mi , L. ; Ding , N. ; Wang , X. ; Song , Y. ; et al. Addition of BTK inhibitor orelabrutinib to rituximab improved anti-tumor effects in B cell lymphoma . Molecular Therapy - Oncolytics 2021 , 21 , 158 – 170 . OpenUrl CrossRef PubMed 32. ↵ Basu , A. ; Bodycombe , N. E. ; Cheah , J. H. ; Price , E. V. ; Liu , K. ; Schaefer , G. I. ; Ebright , R. Y. ; Stewart , M. L. ; Ito , D. ; Wang , S. ; et al. An interactive resource to identify cancer genetic and lineage dependencies targeted by small molecules . Cell 2013 , 154 ( 5 ), 1151 – 1161 . OpenUrl CrossRef PubMed Web of Science 33. ↵ Qian , Z. G. ; Huang , S. C. ; Xia , X. X. Synthetic protein condensates for cellular and metabolic engineering . Nat Chem Biol 2022 , 18 ( 12 ), 1330 – 1340 . OpenUrl CrossRef PubMed 34. ↵ Xiao , Q. ; McAtee , C. K. ; Su , X. Phase separation in immune signalling . Nat Rev Immunol 2022 , 22 ( 3 ), 188 – 199 . OpenUrl CrossRef PubMed 35. ↵ Hirose , T. ; Ninomiya , K. ; Nakagawa , S. ; Yamazaki , T. A guide to membraneless organelles and their various roles in gene regulation . Nat Rev Mol Cell Biol 2023 , 24 ( 4 ), 288 – 304 . OpenUrl CrossRef PubMed 36. ↵ Chen , X. ; Wu , X. ; Wu , H. ; Zhang , M. Phase separation at the synapse . Nat Neurosci 2020 , 23 ( 3 ), 301 – 310 . OpenUrl CrossRef PubMed 37. ↵ Clackson , T. ; Yang , W. ; Rozamus , L. W. ; Hatada , M. ; Amara , J. F. ; Rollins , C. T. ; Stevenson , L. F. ; Magari , S. R. ; Wood , S. A. ; Courage , N. L. ; et al. Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity . Proc Natl Acad Sci U S A 1998 , 95 ( 18 ), 10437 – 10442 . OpenUrl Abstract / FREE Full Text 38. ↵ Schaffer , B. E. ; Levin , R. S. ; Hertz , N. T. ; Maures , T. J. ; Schoof , M. L. ; Hollstein , P. E. ; Benayoun , B. A. ; Banko , M. R. ; Shaw , R. J. ; Shokat , K. M. ; et al. Identification of AMPK Phosphorylation Sites Reveals a Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction . Cell Metab 2015 , 22 ( 5 ), 907 – 921 . OpenUrl CrossRef PubMed 39. ↵ Tibbe , D. ; Ferle , P. ; Krisp , C. ; Nampoothiri , S. ; Mirzaa , G. ; Assaf , M. ; Parikh , S. ; Kutsche , K. ; Kreienkamp , H. J. Regulation of Liprin-α phase separation by CASK is disrupted by a mutation in its CaM kinase domain . Life Sci Alliance 2022 , 5 ( 10 ). 40. ↵ Chen , Z. ; Lei , C. ; Wang , C. ; Li , N. ; Srivastava , M. ; Tang , M. ; Zhang , H. ; Choi , J. M. ; Jung , S. Y. ; Qin , J. ; et al. Global phosphoproteomic analysis reveals ARMC10 as an AMPK substrate that regulates mitochondrial dynamics . Nat Commun 2019 , 10 ( 1 ), 104 . OpenUrl CrossRef PubMed 41. ↵ Johnson , J. L. ; Yaron , T. M. ; Huntsman , E. M. ; Kerelsky , A. ; Song , J. ; Regev , A. ; Lin , T. Y. ; Liberatore , K. ; Cizin , D. M. ; Cohen , B. M. ; et al. An atlas of substrate specificities for the human serine/threonine kinome . Nature 2023 , 613 ( 7945 ), 759 – 766 . OpenUrl CrossRef PubMed 42. ↵ Emperador-Melero , J. ; Wong , M. Y. ; Wang , S. S. H. ; de Nola , G. ; Nyitrai , H. ; Kirchhausen , T. ; Kaeser , P. S. PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure . Nat Commun 2021 , 12 ( 1 ), 3057 . OpenUrl CrossRef PubMed 43. ↵ Mayer , A. ; Derua , R. ; Spahn , E. ; Verbinnen , I. ; Zhang , Y. ; Wadzinski , B. ; Swingle , M. R. ; Honkanen , R. ; Janssens , V. ; Xia , H. The role of liprin-α1 phosphorylation in its liquid-liquid phase separation: regulation by PPP2R5D/PP2A holoenzyme . bioRxiv 2024 . View the discussion thread. Back to top Previous Next Posted May 15, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Phosphorylation-inducing molecules for regulating dynamic cellular processes Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Phosphorylation-inducing molecules for regulating dynamic cellular processes Rajaiah Pergu , Sreekanth Vedagopuram , Praveen Kokkonda , Sophia Lai , Praveen K. Tiwari , Sameek Singh , Junya Kawai , Santanu Singha , Wenzhi Tian , Vikram Thimaradka , Kien Tran , Amit Choudhary bioRxiv 2025.05.10.652895; doi: https://doi.org/10.1101/2025.05.10.652895 Share This Article: Copy Citation Tools Phosphorylation-inducing molecules for regulating dynamic cellular processes Rajaiah Pergu , Sreekanth Vedagopuram , Praveen Kokkonda , Sophia Lai , Praveen K. Tiwari , Sameek Singh , Junya Kawai , Santanu Singha , Wenzhi Tian , Vikram Thimaradka , Kien Tran , Amit Choudhary bioRxiv 2025.05.10.652895; doi: https://doi.org/10.1101/2025.05.10.652895 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biochemistry Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13894) Bioinformatics (41951) Biophysics (21455) Cancer Biology (18592) Cell Biology (25507) Clinical Trials (138) Developmental Biology (13380) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24321) Genetics (15610) Genomics (22509) Immunology (17737) Microbiology (40398) Molecular Biology (17182) Neuroscience (88618) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7641) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)