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.

Microtubule-associated serine/threonine-protein kinase 3 (MAST3) is a member of the MAST kinase family implicated in neuronal and immune pathways and is predicted to associate with cytoskeletal regulation. However, insights into its functional role in cytoskeletal organization remain unexplored. In this study, we performed a large-scale phosphoproteomic analysis of MAST3 using 562 datasets to delineate its functional network. We identified four predominant phosphosites, S134, S146, S792, and S793, based on the frequency of detection and differential regulation, with S134 and S146 localized within the Domain of Unknown Function domain, a noncatalytic region. These phosphosites exhibited distinct coregulatory profiles, suggesting regulation through noncatalytic domains. Coregulated phosphosites were enriched for cytoskeleton-associated functions, including actin filament organization, microtubule organization, and spindle assembly. Additionally, predicted downstream substrates such as KIF15, EPB41L1, CP110, and HNRNPU, and binary interactors including LMNA, CKAP4, and CAMSAP2, further support the involvement of MAST3 in cytoskeletal regulation. The convergence of these cytoskeletal partners across phosphosites, substrates, and interactors suggests that MAST3 may act as a key modulator of cytoskeletal organization through phosphorylation-dependent protein–protein interactions. Notably, frequent phosphorylation of S146 across cancer types points to a potential tumor-specific regulatory role. Together, these findings provide the first systems-level insight into the role of MAST3 in cytoskeletal regulation and disease relevance.

Introduction: TNIK (Traf2- and Nck-interacting kinase) is a serine/threonine kinase that plays a crucial role in cytoskeletal organization, Wnt pathway activation, and cancer progression. Recent studies have implicated the role of TNIK in oncogenic signaling pathways and neuropsychiatric regulation. However, the phosphosignaling dynamics of TNIK remain largely unknown. Methods: To explore TNIK phosphoregulation, we systematically assembled and integrated global human phosphoproteomic datasets. We identified the predominant phosphosites based on the frequency. Relative solvent accessibility (RSA) and Phosphosite accessibility index (PAI) were calculated to determine the solvent exposure and structural flexibility of TNIK predominant phosphosites. To assess the functional significance of TNIK, we examined proteins that were differentially co-regulated with its predominant phosphosite, along with the corresponding upstream kinases, downstream substrates, and interacting proteins. Results: Analysis of the global human cellular phosphoproteome datasets revealed phosphosites S640, S680, S707, and S769 of TNIK to be the most frequently perturbed phosphosites across diverse experimental conditions. The results of the RSA and PAI analysis revealed that the predominant sites are located within highly solvent-exposed and structurally flexible regions. Notably, we obtained a large number of co-regulated proteins that were associated with cell growth, carcinogenesis, and apoptosis. The interactors identified were primarily enriched towards carcinogenesis. Our analysis revealed PRKAA1 and RPS6KB2 as robust upstream kinases of TNIK_S640 and TNIK_S707. We also identified many proteins involved in RNA splicing, cytoskeletal organisation, and cell migration as potential downstream substrates of TNIK. Discussion: Considering the challenges in targeted experimental analysis of these sites, a global co-regulation analysis approach was employed. Our results show that these phosphorylation sites in TNIK can influence carcinogenesis and related biological functions. It offers new insights into TNIK-mediated cellular functions, deepening our comprehension of its involvement in carcinogenesis and RNA splicing.

Dengue virus (DENV) is a mosquito-borne RNA virus that causes serious illness in humans, ranging from mild fever to severe clinical manifestations, with dengue virus type 2 (DENV-2) being the most virulent among its four serotypes. Despite extensive research, no specific antiviral therapy is currently available, making the host-directed method an appealing therapeutic approach. Evidence shows that DENV manipulates host kinase-driven phosphorylation pathways to control viral pathogenesis. Using the kinase–substrate phosphomotif approach, we predicted phosphorylation sites across the DENV proteome and their potential human kinases. The predicted kinase–substrate interactions were systematically integrated with DENV-2-induced human phosphoproteome datasets, protein–protein interactions, and experimentally-validated viral phosphosites. The therapeutic relevance of the identified host kinases was corroborated by the impact of their inhibitors on DENV-2 infection. Among the 359 potential human kinases predicted to phosphorylate DENV-2 proteins, based on human phosphoproteome and kinase–viral protein interaction analyses, CDK9 emerged as a central hub kinase. Molecular docking analyses further revealed that the host kinases CDK9, EEF2K, HASPIN, and TNNI3K form stable interactions with the viral capsid and NS5 proteins. Additionally, a conservation analysis suggested that the predicted phosphorylation sites are evolutionarily conserved across DENV-2 strains. Computational prediction tools supported the predicted kinase–substrate interactions, underscoring the role of host kinases as key regulators of DENV infection, which may act as potential therapeutic targets. This study highlights the interplay between dengue viral and host proteins, providing insights into host-directed therapeutic strategies for DENV-2 infection and their potential to address the current lack of effective antiviral interventions.