Discov Oncol. 2026 Apr 10. doi: 10.1007/s12672-026-04982-6. Online ahead of print.

ABSTRACT

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.

PMID:41963591 | DOI:10.1007/s12672-026-04982-6

Int J Mol Sci. 2026 Feb 25;27(5):2137. doi: 10.3390/ijms27052137.

ABSTRACT

Protein Kinase N1 (PKN1) is a PKC-related serine/threonine kinase of the AGC group within the eukaryotic protein kinase superfamily (ePK) that orchestrates oncogenic, metabolic, and cytoskeletal signaling. Despite these critical roles, the phosphorylation-dependent regulatory network of PKN1 remains largely undefined. We performed a large-scale phosphoproteomic data integration of publicly available human datasets (892 profiling datasets and 191 differential datasets) to identify recurrent PKN1 phosphorylation sites. This analysis identified two predominant PKN1 phosphosites, S562 and S916, that were frequently observed and differentially regulated across studies. The S916 maps to a turn motif (TM) in the AGC group of kinases, which is evolutionarily conserved among PKN paralogs, while S562 is non-conserved and appears to be PKN1-specific. Co-regulation and enrichment analyses suggest that S916 is associated with insulin/AMPK signaling and metabolic pathways, whereas S562 co-occurs with phosphosites involved in cell division, cytoskeletal regulation, and microtubule cytoskeleton organization. Integrating predicted and experimentally validated kinases, substrates, and interactors, we reconstructed a phospho-regulatory network that positions PKN1 at the crossroads of cytoskeleton organization and metabolic signaling. To assess the disease relevance of these phosphorylation events, we integrated transcriptomic and phosphoproteomic data from the hepatocellular carcinoma database (HCCDB). PKN1 was markedly up-regulated in HCC, and its phosphorylation at S916 was positively co-regulated with multiple oncogenic and proliferation-associated protein phosphosites. These results predict S562 and S916 as potential sites for targeted biochemical validation and functional experiments. The identification of S562 and S916 as key regulatory sites provides new mechanistic insight into PKN1 activation and highlights potential avenues for therapeutic targeting.

PMID:41828364 | PMC:PMC12984926 | DOI:10.3390/ijms27052137