Alimath Sambreena

RNA-binding proteins (RBPs) are crucial regulators of post-transcriptional gene expression, orchestrating essential processes including splicing, mRNA transport, translation modulation, and mRNA decay. Phosphorylation of RBPs is crucial for regulatory mechanisms, mediating how extracellular stimuli control intracellular signaling and cellular decisions. In this study, mass spectrometry-based phosphoproteomic data were systematically integrated and harmonized, and Fisher’s exact test (p-Value < 0.05) was applied to quantify conserved co-differential phosphorylation across pairs of phosphorylation sites in comparison to a chosen set of RNA-binding proteins (HNRNPM, SLU7, and SRSF5). For each such pair, data were benchmarked across >800 phosphorylation profiling and 200 differential phosphorylation datasets, identifying predominant co-differential phosphorylation patterns that accounted for >75% of all observed phosphorylation events. Major phosphorylation hotspots were identified in HNRNPM, SLU7, and SRSF5 RNA-binding proteins. High-confidence pairs were further refined using secondary filters, yielding a focused set of functionally relevant relationships in mRNA and alternative splicing. As the RNA-binding activity is mediated by RNA-binding proteins (RBDs), co-differentially regulated phosphorylation sites were mapped to RBDs, and upstream kinases modulating such phosphorylation were derived from experimental, semi-experimental and predictive sources. Overall analysis confirmed phosphorylation of RBPs on their RBDs through the upstream kinases, modulating mRNA and alternative splicing through a conserved network of co-differential phosphorylation events. With further validation, the data imply a platform for identifying relevant targets and markers for studying mRNA splicing and RNA-binding activity, central to RNA processing.

Nucleolin (NCL) is a highly phosphorylated, multilocalized, pleiotropic nucleolar protein involved in chromatin organization, DNA damage response, the cell cycle, RNA metabolism, and ribosome biogenesis. A large-scale integration of publicly available human cellular phosphoproteome datasets led to the identification of 18 frequently detected/reported phosphosites across 1266 profiling and 507 differential datasets. Amidst various strategies currently being considered to target NCL in cancers, multi-domain distribution of these phosphosites differentially regulated across distinct cancers highlighted its plausible involvement in tumor-associated regulatory mechanisms. This demands the exploration of the potential kinases and interactors associated with these phosphorylation events. We performed a co-occurrence analysis to explore potential coordinated phosphorylation events among them and across other phosphoproteins. An integrative analysis of phosphosite-specific co-regulation, upstream kinases, and interaction networks was employed to explore potential phosphosite-specific signaling associations. Intriguingly, our analysis inferred the association of the predominant phosphosites within the acidic tract with chromatin organization and histone-related functions, whereas TPxKK motif phosphosites were linked to NCL localization, chromosome segregation, and nucleolar reorganization. Phosphosites in the RRM domain showed a potential association with RNA-binding proteins and metabolic pathways. Collectively, these findings suggest a domain-specific phosphosite-centric regulatory network of NCL, providing a valuable reference framework for future experimental studies and phosphosite-directed therapeutic strategies in cancers.

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.

Many kinases are known to homo- or hetero-merize and phosphorylate themselves or each other. Phosphorylation by itself (autophosphorylation) is mechanistically perceived as either cis- or trans-interactions. Identification of autophosphorylation sites in kinases provides an opportunity to assess the activity of the kinome in phosphoproteome datasets as an efficient indicator of kinase activity. In order to predict kinase activity-associated autophosphorylation sites, we first encompassed the currently reported homodimeric interactions between kinases from various sources. Subsequently, we accounted for the autophosphorylation sites in kinases that are predicted by phosphomotif screening approaches, in vitro kinase assays, and those perceived based on phosphomotif mutant analysis. Together, we identified 4184 autophosphorylation sites in 386 kinases, and among them, 315 kinases were reported with homodimeric protein–protein interactions. Furthermore, we analysed 361 kinases that harbor multiple autophosphorylation sites and 25 kinases with a single autophosphosite based on contemporary kinase–substrate interactions. Among the 386 kinases, autophosphosites for 17 kinases were derived through high-throughput or low-throughput approaches, and for 80 kinases, they were predicted based on their phosphomotif analysis. The remaining 289 were based on predictions by multiple tools or were reported in more than one dataset. There were many instances in which, although one auto-phosphorylation site was reported, multiple other sites were also predicted. In this regard, we analysed the instances of phosphosites in kinases that are already associated with the induction or inhibition of kinase activity and identified 223 kinases with one or more sites with functional association. To evaluate their potential as auto-phosphorylation sites, we systematically assembled 1572 profiles and 978 differential cellular phosphoproteome datasets. Subsequently, we accounted for the frequency of co-detection or co-differential regulation of these autophosphosites with multiple kinase activity-associated sites within each kinase and further accounted for their known/predicted substrate phosphorylation sites. Together, we provide a reference compendium of autophosphorylation sites secluded in the human kinome and their co-regulation with substrates based on global phosphoproteome datasets, AutoPhosDb. As indicators of kinase activity, these phosphosites in kinases would serve as a surrogate to enrich the active kinases and their potential substrate-specificity in phosphoproteome datasets.

Background: Fibroblast growth factor receptors (FGFRs) play a crucial role in tissue homeostasis and organ development by regulating cellular processes, including proliferation, differentiation, and survival. Dysregulation of FGFRs contributes to developmental disorders and carcinogenesis. As membrane-bound receptors, they represent promising targets for therapeutic intervention and drug development. Methods: This study employed a systematic in silico analysis of publicly available phosphoproteomics datasets to provide a comprehensive overview of the phosphorylation regulatory network of the FGFR family. Results: We identified predominant phosphosites in FGFR1-4 that exhibited differential abundance across diverse experimental conditions, specifically, Y653 in FGFR1; S453, Y586, Y656, and Y657 in FGFR2; S444 and S445 in FGFR3; and S573 in FGFR4. Our analysis identified 32 and 89 significantly co-modulated phosphosites on other proteins with FGFR3 and FGFR4, respectively. Beyond the upstream kinases from the FGFR family, we also identified MAPK1 as a potential upstream kinase of FGFR4. Furthermore, disease enrichment analysis revealed that proteins co-modulated with FGFR3 were primarily involved in skeletal developmental disorders, such as brachydactyly, short toe, and syndactyly of fingers, whereas those associated with FGFR4 were linked to various cancers. Conclusions: Our findings highlight key disease-associated phosphosites within the FGFRs and offer a foundation for advancing phosphosite-focused therapeutic research.