Integrative phosphoproteomic analysis reveals co-regulatory phosphorylation networks of rhotekin in cancer progression.

Rhotekin (RTKN), a Rho GTPase effector, promotes the development of malignancies, including breast, gastric, and colon cancers, by enhancing cell proliferation and migration while inhibiting apoptosis. Despite its oncogenic role, the phosphoregulatory network of RTKN remains largely unexplored, with no experimental evidence on its upstream kinases and functional phosphosites. To characterize RTKN-associated phosphorylation dynamics, PubMed-indexed studies were systematically retrieved using predefined MeSH terms to compile large-scale cellular phosphoproteomics datasets. Analysis of 618 quantitative profiling and 179 differential abundance datasets identified 27 Class-I phosphosites in RTKN. Among these, five sites-Ser106, Ser220, Ser520, Ser529, and Ser543 were consistently observed across multiple datasets, suggesting them as predominant sites with potential functional significance. Structural mapping of predominant sites onto the AlphaFold2-predicted model indicated that these sites are located in accessible regions, highlighting their potential susceptibility to kinase-mediated regulation. As these sites represent understudied phosphosites, a robust strategy was employed to identify their functional role by assessing co-regulated phosphosites on other proteins (PsOPs). ACIN1_Ser243, CTNNA1_Ser641, and SHROOM2_Ser1036 were among the top positively co-regulated PsOPs, whereas MICALL1_Ser644, PRP4K_Ser366, and MYO18A_Ser1970 were negatively co-regulated. PsOPs were mainly involved in apoptosis, cell growth, motility, and cytoskeletal reorganization, suggesting potential functional convergence with RTKN. Additionally, phosphorylation at RTKN Ser520 and Ser543 co-occurred across multiple datasets. Moreover, TRPM7 and PAK4 were identified as predicted upstream kinases phosphorylating RTKN at Ser220 and Ser520. Pathway analysis showed involvement of co-regulated proteins in cancer-associated signaling pathways. These findings provide a foundation for future research to elucidate the phosphosite-specific role of RTKN in cancer.