Athira Perunelly Gopalakrishnan

With-No-Lysine Kinase 1 (WNK1) is an atypical serine/threonine kinase involved in ion homeostasis, blood pressure regulation, migration and proliferation. Its intrinsically disordered regions (IDRs) support dynamic phosphorylation patterns. Here, we compiled 895 human phosphoproteomic profiling datasets and 352 differential phosphoproteomic datasets to systematically characterise the phosphorylation landscape associated with WNK1. Among reported WNK1 phosphosites, S2027, S2029, and S2032, located within an IDR, were the most frequently detected. Using a one-sided Fisher’s Exact Test followed by three sequential bias-correction filters, we identified 387 phosphosites in other proteins (PsOPs) showing positive co-differential association and 34 showing negative co-differential association with these three WNK1 sites. Computational approaches and database integration identified two candidate upstream kinases (EIF2AK1 and MAPK3), eight binary interactors, and 26 candidate substrate proteins. GO enrichment revealed site-specific functional categories: S2027-associated proteins were enriched in Ras/small GTPase signaling, chromatin organization, and cell-cycle-related processes; S2029-associated proteins in chromosome organization and transcriptional regulation; and S2032-associated proteins mainly in GTPase-related molecular functions. These findings provide a phosphosite-centric resource and computationally supported candidate associations that generate testable hypotheses for further experimental investigation of WNK1-associated signaling.

Unc-51-like autophagy-activating kinase 1 (ULK1), a serine/threonine kinase, is the central regulator of autophagy initiation and plays a crucial role in cellular homeostasis, metabolic adaptation, and stress responses. We performed a large-scale phosphoproteomic data analysis of ULK1 by systematically integrating and analyzing 891 profiling and 245 differential human phosphoproteomic datasets. S450, S623, and S638 emerged as the predominant phosphosites in ULK1, with the highest frequency of detection across multiple cellular phosphoproteomics datasets. Conservation analysis showed that these phosphosites are conserved across mammalian species and human ULK1 and ULK2 (Unc-51-like autophagy-activating kinase 2). The co-regulatory networks associated with these phosphosites, including interacting proteins, upstream kinases, and downstream substrates, were systematically analyzed. The phosphosite co-regulated ULK1 interactome revealed a network of associations linking ubiquitin-dependent cargo recognition to autophagosome biogenesis, trafficking, and maturation, indicating that ULK1 phosphoregulation may extend beyond its established role in autophagy initiation and be associated with multiple stages of selective autophagy. Notably, ULK1 phosphosites were linked to key metabolic enzymes in glycogen, sphingolipid, phospholipid, and glycolytic pathways, as well as classical lipolysis. Protein-normalized analysis across 12 cancer cohorts showed significant upregulation of the predominant ULK1 phosphosites, most frequently at S450 and S623. Comparative analysis with an independent ULK1/2 knockout phosphoproteomic dataset further supported the relevance of these identified co-regulated protein phosphosites. Collectively, this study provides a phosphosite-resolved view of ULK1 regulation and identifies candidate phosphosites and associated proteins for future functional and mechanistic studies.

Background: Mitogen-activated protein kinase kinase kinase 3 (MAP3K3) is a serine/threonine kinase that exhibits oncogenic activity in various cancers. Despite the established role of MAP3K3 in signal transduction, its phosphosignaling landscape, including the mechanisms underlying its activation and the identification of its upstream kinases and downstream substrates, remains poorly characterized. Methods: To explore this network, we analyzed the global phosphoproteomics datasets to identify predominant sites of MAP3K3. Co-concordance and co-occurrence analyses were conducted to characterize the functional significance of site-specific phosphorylation of MAP3K3. Results: From the 588 profiling datasets and 124 differential datasets, we identified S340, S337, and S166 as predominant phosphosites. We then categorized the phosphosites in other proteins (PsOPs), which were positively and negatively co-concordant with the predominant sites of MAP3K3. From the known and predicted upstream kinases, substrates, and interactors, we isolated the PsOPs that have a role in carcinogenesis. Conclusions: Finally, by integrating these co-concordance data with the currently known and predicted upstream kinases, substrates, and interactors, we constructed a phosphosignaling network of MAP3K3-driven carcinogenesis. This network provides insights into the potential upstream kinases and substrates of MAP3K3 in the context of carcinogenesis.

Background: Myocardin-related transcription factor B (MRTFB) acts as a transcriptional coactivator for serum response factor (SRF) and regulates actin cytoskeletal dynamics. Despite its biological significance, the phosphoregulatory network, upstream kinases, and interactors remain poorly understood. Methods: To explore this, we analyzed global phosphoproteomic datasets to identify predominant phosphosites. We categorized phosphosites in other proteins as positively or negatively co-regulated with the predominant phosphosites of MRTFB and also integrated predicted upstream kinases and interactors of MRTFB. Further, phosphosite conservation, co-occurrence patterns, and functional and pathway enrichment analyses of co-regulated proteins were also performed. Results: We identified S66 and S921 as predominant phosphosites in MRTFB, with the highest detection frequency across multiple experimental conditions. These phosphosites were found to be conserved within the MRTF family and across multiple species. From the predicted upstream kinases, MAPK1, MAPK3, MAST3, and CSNK1A1 were identified as predicted upstream kinases. Co-occurrence analysis revealed high positive co-occurrence between the predominant phosphosites, S66 and S921, suggesting similar functional roles. Functional enrichment analysis highlighted the involvement of co-regulated proteins in the actin cytoskeleton pathway. Conclusions: This study provides the detailed phosphoproteomic landscape of MRTFB, mapping its upstream kinases and co-regulated proteins involved in actin cytoskeleton regulation.

KIF21A, a member of the Kinesin-4 family of motor proteins, is involved in the regulation of microtubule dynamics and intracellular transport, with emerging evidence suggesting its potential role in cancer progression. In this study, we performed an integrative analysis of over 3825 human phosphoproteomics studies to characterize site-specific phosphorylation of KIF21A. Three predominant phosphosites were identified in KIF21A (S853, S1212, and S1239) with the highest detection frequency across phosphoproteomics studies, and were analyzed for co-regulation patterns to identify potential kinase associations and functional networks. Phosphosite S853 showed a strong association with cytoskeletal organization and cortical microtubule stabilization complexes (CMSCs) components, including KANK1 (S186), PHLDB2 (S513, S42) and CLASP1 (S600, S572, S646), indicating its role in cytoskeletal organization. Upstream kinase analysis identified potential regulators, such as PAK2, RPS6KA1/A3, RPS6KB1, CHEK1/2 and CDK18/16 with site-specific variability in their associations with KIF21A predominant sites. Interestingly, phosphosite-specific correlation analysis between KIF21A and candidate kinases revealed that the KIF21A S1239 phosphosite exhibited tumor-specific correlations with CDK18 across multiple cancer types. Functional enrichment revealed that co-regulated phosphoproteins were involved in cytoskeleton regulation, cell cycle regulation, and carcinogenesis. Pan-cancer analysis demonstrated dysregulated expression of KIF21A in multiple tumor types, with stage-associated upregulation in selected cancers. Gene-level validation further supported these findings, showing consistent positive correlations between KIF21A and key regulators such as CTNND1 and PTK2, as well as other cytoskeleton and cancer-associated genes. Overall, this study highlights site-specific phosphorylation as a key regulatory mechanism of KIF21A and suggests its involvement in cytoskeleton-associated signaling networks in cancer.

Tousled-like kinases 1 and 2 (TLK1 and TLK2) are paralogous serine/threonine kinases that share high sequence similarity yet exhibit functional divergence in cellular processes such as DNA replication, damage response, and chromatin organization. This study elucidates the paralog-specific co-phosphoregulatory networks underlying this divergence through a comprehensive analysis of 3825 human phosphoproteomic articles. Predominant phosphosites were identified as S134 and T38 for TLK1 and S73, S99, and S111 for TLK2, revealing context-dependent regulation across cancers and perturbations. Co-phosphoregulation analyses uncovered distinct networks: TLK1 associates with DNA damage signaling via proteins like ABRAXAS1, PML, and RAD9A, while TLK2 integrates with chromatin remodeling and replication through CHD4, DOT1L, NASP, and RNF20. Upstream kinases for TLK2, predominantly CDKs, link it to cell-cycle progression, whereas downstream substrates and binary interactors converge on genome stability pathways with paralog-specific nuances. These findings highlight the potential role of TLK1 on checkpoint activation and TLK2 on replication-coupled chromatin maintenance, providing insights into their roles in cancer amplification and therapeutic resistance, as well as neurodevelopmental disorders, where emerging evidence also support the involvement of TLK1 alongside TLK2.

PRDM2 is a histone methyltransferase that regulates gene expression through histone H3 lysine 9 methylation. It is involved in the DNA damage response by controlling chromatin remodeling and maintaining genomic integrity. However, the functional relevance of its phosphorylation remains poorly understood. To address this gap, we systematically characterized PRDM2 phosphosites and their associated phospho-signaling networks using large-scale cellular phosphoproteomics data curated from PubMed-indexed articles. Frequency-based ranking revealed Ser643 and Ser421 as predominant phosphosites, detected across 334 and 141 qualitative datasets and 70 and 47 differential cellular phosphoproteomics datasets, respectively. To explore PRDM2-associated phospho-signaling, expression co-regulation analysis was performed to identify phosphosites in other proteins exhibiting consistently similar or opposing expression patterns relative to the predominant PRDM2 phosphosites. This analysis identified 1,251 phosphosites in other proteins showing high-confidence expression co-regulation with PRDM2 Ser421 and 715 phosphosites with PRDM2 Ser643. Functional enrichment revealed significant associations with cell cycle regulation, chromatin organization, RNA processing, and DNA damage response (DDR) pathways. Notably, phosphosites in 30 DDR-related proteins positively co-regulated with Ser643 and 28 with Ser421. Additionally, ATR was identified as a potential kinase predicted to phosphorylate the predominant PRDM2 sites, and its phosphosites exhibited consistent expression co-regulation with PRDM2 sites. Collectively, this study establishes a comprehensive phospho-signaling framework for PRDM2, uncovering its strong association with DNA damage response pathways and providing mechanistic insights into its regulatory network. Clinical trial registration: This study is not part of any clinical trial. The online version contains supplementary material available at 10.1007/s13205-026-04782-3.

Sucrose non-fermenting 1-related kinase (SNRK) is an understudied serine/threonine kinase of the CAMKL family, known for its role in metabolic regulation and cell signaling. Despite its emerging relevance in various biological processes and diseases, the phosphoregulatory landscape of human SNRK (valid substrates or role of its phosphosites) remains unexplored and demands robust, large-scale, data-oriented approaches to predict the potential substrates. A comprehensive analysis of global human phosphoproteomics datasets was performed to systematically identify class I phosphosites on SNRK, along with their predicted upstream kinases, potential downstream substrates, and coregulated phosphoproteins. Our analysis resulted in the identification of 33 dark SNRK phosphosites, of which 19 were differentially regulated across an array of experimental conditions. Among them, S518 and S569, outside their kinase domain, were the most frequently regulated and co-occurred phosphosites under diverse conditions. Notably, S569 is predicted as a candidate autophosphorylation site of SNRK. In these contexts, coregulation analysis of proteins and their phosphorylation sites suggested associations of phospho-SNRK in cell cycle progression, chromatin organization, and DNA replication. Uncovering candidate upstream kinases and potential substrates for prioritized validation, this study provides the first comprehensive phosphoproteomic map of SNRK, serving as a foundation for future investigations into its signaling network associations and therapeutic approaches.

RAVER1 is a conserved PTBP1-associated regulator of alternative splicing with emerging roles in cytoskeletal organisation and proliferative signaling. Despite the central role of phosphorylation in intracellular signaling, phospho-dependent regulation of RAVER1 remains poorly defined. To address this gap, we performed a comprehensive meta-analysis of global human cellular phosphoproteome datasets to characterise RAVER1 phosphorylation and its associated signaling networks. Class I phosphosites were systematically compiled, and phosphosite-centric analyses were conducted to evaluate site predominance, co-occurrence patterns, phosphoproteome-wide co-regulation, and predicted upstream kinase associations. Thirteen Class I phosphosites were identified, of which S14, S17, and T463 emerged as predominant based on consistent detection across profiling and differential datasets. Significant phosphosite co-occurrence among S6, T8, and S14 within the N-terminal region suggests coordinated, context-dependent regulation and functional association of these sites. Phosphoproteome-wide co-regulation analysis linked RAVER1 phosphorylation states to proteins involved in RNA processing, cell-cycle regulation, and DNA repair. Kinase-oriented analyses identified CDK family members (CDK9, CDK12, and CDK13) as high-confidence predicted upstream kinases for RAVER1 S14. Notably, phosphosites in several epithelial–mesenchymal transition (EMT)-associated regulators, including KDM1A, VIM, and PTK2, showed positive co-regulation with RAVER1 phosphosites, consistent with multi-omics perturbation datasets reporting EMT-associated changes upon RAVER1 depletion. To our knowledge, this study provides the first phospho-signaling map of RAVER1 and establishes a phosphosite-centric analytical framework for functional investigation of RAVER1 phosphorylation, with potential implications for targeted therapeutic strategies.