Introduction: Histone-lysine N-methyltransferase 2D (KMT2D) is an H3K4 methyltransferase and a potential tumor suppressor with a crucial role in regulating gene expression. Its dysregulation has been implicated in developmental disorders and several types of cancers. Despite this, the molecular mechanisms that govern its activity remain largely elusive. Among these, post-translational modifications, especially phosphorylation, serve as an essential regulator, fine-tuning KMT2D stability, localization and functional interactions for maintaining cellular homeostasis. With over 173 phosphorylation sites reported, KMT2D is significantly regulated by kinases and exploring its phospho-regulatory network based on targeted in vitro approaches is challenging. Methods: We systematically curated and integrated the global phosphoproteomic datasets, along with their corresponding experimental conditions, to comprehensively identify the phosphorylation events reported for KMT2D. The site exhibiting the highest frequency of detection across these datasets is considered the predominant phosphorylation site. To investigate its functional significance, we analyzed the proteins and their phosphorylation sites that are differentially co-regulated with the predominant site, as well as its associated upstream kinases and interacting proteins. Results: Among the 173 reported phosphorylation sites representing KMT2D, Serine 2274 (S2274) emerged as the predominant site being detected in over 42% of diverse mass spectrometry-based phosphoproteomics datasets. This site lies within one of KMT2D’s unique “ LSPPP ” motifs, suggesting a potential regulatory role. Detailed investigation on the differentially co-regulated protein phosphosites revealed the phosphorylation of KMT2D at S2274 is consistently and positively co-regulated with MAPK1/ERK2 activation, as well as with the proteins involved in the MAPK cascade, epigenetic regulation and cell differentiation. Notably, ERK2 was predicted as an upstream kinase targeting S2274, suggesting that KMT2D S2274 functions as a potential downstream effector of MEK-ERK signaling pathway, potentially linking to epigenetic regulation and cell differentiation. Further, our results highlighted a potential mechanistic link between disrupted phosphorylation at S2274 and the pathogenesis of Kabuki syndrome. Discussion: This study delineates the phosphoregulatory network of KMT2D, positioning it as a dynamic epigenetic effector modulated by MEK-ERK signaling, with broader implications for cancer and developmental disorders.

MAST2 (Microtubule-Associated Serine/Threonine Kinase 2) is an enigmatic serine/threonine kinase that is considered to bridge microtubule-associated cytoskeletal architecture through its phospho-regulatory networks. Yet, MAST2 remains a dark horse in the human kinome, as the molecular details on its structure, upstream regulators, and downstream phosphorylation targets remain unknown. To interpret MAST2-linked phospho-signaling dynamics, PubMed-indexed articles were curated based on predefined MeSH terms to obtain global cellular phosphoproteomics datasets. Among 105 class I phosphosites in MAST2 identified across cellular phosphoproteomics datasets, 2 phosphosites, S74 and S148, were represented more abundantly compared to other phosphosites, making them predominant and functionally significant. Expression coregulation analysis, aimed at interpreting phosphosites that consistently show either similar or opposite patterns of expression, was performed by computing the expression patterns of phosphosites in parallel with the predominant MAST2 phosphosite. Their frequencies were then ranked across differential datasets to ensure consistency, and Fisher's exact test was performed to assess the likelihood of the coregulation pattern. Potential biases within the datasets were mitigated using additional cutoffs. Interpreting these datasets, we identified the majority of high-confidence coregulated protein phosphosites of both predominant phosphosites to be involved in transcriptional regulation. This is consistent with reports that a nearby Arg89Gln mutation in MAST2 disrupts its transcriptional regulatory activity. Co-occurrence analysis of phosphosites within MAST2 revealed that these predominant sites tend to co-occur positively and share similarity in expression coregulation patterns with phosphosites in other proteins. Finally, novel upstream kinases that potentially phosphorylate the predominant phosphosites of MAST2, as well as potential downstream substrates that are phosphorylated by MAST2, were also identified from the high-confidence coregulation dataset. We propose that phosphorylations at S74 and S148 of MAST2 are functionally similar, and that these phosphosites are candidate regulatory sites influencing the transcriptional regulatory activity of MAST2.

High-density lipoprotein-binding protein (HDLBP), also called Vigilin, is a multifunctional RNA-binding protein with established roles in RNA transport and regulation, chromosome segregation, lipid homeostasis, and translational regulation. Frequently detected to be perturbed in phosphoproteome analysis, phosphorylation is indicated as a major mechanism in the regulation of HDLBP functions; however, its phosphorylation landscape remains unexplored. We performed a meta-phosphoproteome analysis of HDLBP to map site-specific functional and regulatory roles of its two most frequently detected phosphosites, S31 and S944. Co-occurrence analysis across multiple datasets indicated that they can be phosphorylated together, suggesting potential co-ordinated regulation. Site-specific co-regulation analysis revealed distinct phospho-regulatory networks, with upstream kinases identified exclusively for S944. Functional enrichment of co-regulated protein phosphosites (CPPs) highlighted its role in RNA metabolism, chromosome organization, and nucleoplasmic transport, while functional annotation of site-specific phosphorylation of CPPs indicates its involvement in cell cycle regulation, apoptosis, and carcinogenesis. Additionally, the potential role of CPPs in the lipid homeostasis network was explored. Furthermore, the differential expression of HDLBP phosphosites across multiple cancers was observed using UALCAN, suggesting a potential role for phospho-regulation of HDLBP in tumor-associated pathways. Together, these findings provide the first integrated view of HDLBP phosphorylation and could serve as a valuable framework for future targeted studies to elucidate the mechanistic roles of site-specific HDLBP phosphorylation in cellular and pathophysiological processes.

Drug resistance poses a significant obstacle to the success of anti-cancer therapy in head and neck cancers (HNCs). We aim to develop a platform for visualizing and analyzing molecular expression alterations associated with HNC drug resistance. Through data mining, we convened differentially expressed molecules and context-specific signaling events involved in drug resistance. The driver genes, interaction networks and transcriptional regulations were explored using bioinformatics approaches. A total of 2364 differentially expressed molecules were identified in 78 distinct drug-resistant cells against 14 anti-cancer drugs, comprising 1131 mRNAs, 746 proteins, 62 lncRNAs, 257 miRNAs, 1 circRNA, and 166 post-translational modifications. Among these, 255 molecules were considerably, the signature driver genes of HNC drug resistance. Further, we also developed a landscape of signaling pathways and their cross-talk with diverse signaling modules involved in drug resistance. Additionally, a publicly-accessible database named “HNCDrugResDb” was designed with browse, query, and pathway explorer options to fetch and enrich molecular alterations and signaling pathways altered in drug resistance. HNCDrugResDb is also enabled with a Drug Resistance Analysis tool as an initial platform to infer the likelihood of resistance based on the expression pattern of driver genes. HNCDrugResDb is anticipated to have substantial implications for future advancements in drug discovery and optimization of personalized medicine approaches.