CIT kinase phosphorylation as significant regulatory node for cellular checkpoints.

Introduction: Citron Rho-interacting serine/threonine kinase (CIT) is a major cytosolic protein kinase essential for midbody organisation, abscission, and cytokinesis. Dysregulation and mutations in CIT are associated with multiple cancers and neurodevelopmental disorders, including microcephaly. Although global phosphoproteomic studies have identified more than 50 phosphosites in CIT, their functional relevance and the kinases regulating them remain largely unexplored. Methods: To systematically investigate the phosphoregulation of CIT, we curated and integrated global phosphoproteomic datasets, along with their associated experimental conditions, to comprehensively catalogue phosphorylation events reported for CIT. To assess the functional significance of CIT, we examined proteins that were differentially co-regulated with its predominant phosphosite. Results: Serine 440 (S440), located outside the kinase domain (representing over 55% of CIT-associated phospho-signalling events across 100 experimental conditions, including Enterovirus A71 infection, metformin, and interleukin-33), was identified as its predominant phosphosite. Motif analysis revealed the presence of a D(S/T)P/P(S/T)D motif recognised by the CIT kinase domain, suggesting S440 as a predicted autophosphorylation site. Co-phosphoregulation analysis identified 136 interacting proteins and 82 predicted substrates that were positively co-regulated with CIT_S440. The resulting phospho-regulatory network comprised essential cell cycle and DNA repair regulators, including MDC1 and TRIP12. Significantly, over 120 co-regulated phosphosites were functionally linked to DNA repair and cell cycle regulation. Aberrant phosphorylation of CIT_S440 observed across cancers of the breast, colon, and bladder suggests CIT_S440 as a potential onco-phosphosite critically involved in cellular checkpoint signalling. Discussion: These findings suggest that CIT_S440 functions as a promising therapeutic target, and the phosphosite-centric regulatory network derived in this study could serve as a platform to evaluate its phosphosite-specific therapeutic interventions.