Integrative phosphoproteomic analysis identifies functional roles of TRPM7 phosphosites in oncogenesis.

Introduction: Transient Receptor Potential Melastatin 7 (TRPM7) is a 'chanzyme' with dual functions, acting both as a channel for divalent ions and as a serine/threonine kinase. Overexpression of TRPM7 has been linked to the development of various diseases, particularly cancers, making it a promising molecular target. Despite its relevance in oncogenesis, the phospho-regulatory network of TRPM7 remains largely unexplored, with limited evidence on its upstream kinases, downstream substrates, and site-specific phospho-regulated functions. Methods: To address this knowledge gap, we employed a co-differential detection-based strategy to analyse publicly available phosphoproteomics datasets. Results: Through the analysis of 569 phosphoproteomics profiling datasets and 116 differential abundance datasets, we identified 55 and 38 Class I phosphosites in TRPM7, of which 13 have not been previously reported. Among the Class I phosphosites, S1504, S1255, S1513, S1477, and S1387 emerged as the predominant phosphosites in TRPM7. Furthermore, all known interactors and substrates of TRPM7 were associated with broad cellular functions such as protein phosphorylation, chromatin remodeling, transcriptional regulation, intracellular signal transduction, DNA damage response, and apoptosis, whereas the co-differentially regulated interactors and substrates of TRPM7 were associated with more specialized functions, including positive regulation of stem cell population maintenance, regulation of mRNA splicing via the spliceosome, and regulation of the G2/M phase transition of the cell cycle. Finally, we identified potential upstream kinases for TRPM7, including PRKCD, CLK2, STK39, PKN2, MAST3, PRKD3, and MAP4K4. Discussion: These findings provide a comprehensive resource of TRPM7 phosphosites, their potential regulatory kinases, and associated biological functions, laying the groundwork for future mechanistic and therapeutic studies.