Co-Regulation Analysis of Domain-Specific Phosphosites Reveals Insights into the Phosphoregulation-Directed Functional Diversity of Nucleolin

Nucleolin (NCL) is a highly phosphorylated, multilocalized, pleiotropic nucleolar protein involved in chromatin organization, DNA damage response, the cell cycle, RNA metabolism, and ribosome biogenesis. A large-scale integration of publicly available human cellular phosphoproteome datasets led to the identification of 18 frequently detected/reported phosphosites across 1266 profiling and 507 differential datasets. Amidst various strategies currently being considered to target NCL in cancers, multi-domain distribution of these phosphosites differentially regulated across distinct cancers highlighted its plausible involvement in tumor-associated regulatory mechanisms. This demands the exploration of the potential kinases and interactors associated with these phosphorylation events. We performed a co-occurrence analysis to explore potential coordinated phosphorylation events among them and across other phosphoproteins. An integrative analysis of phosphosite-specific co-regulation, upstream kinases, and interaction networks was employed to explore potential phosphosite-specific signaling associations. Intriguingly, our analysis inferred the association of the predominant phosphosites within the acidic tract with chromatin organization and histone-related functions, whereas TPxKK motif phosphosites were linked to NCL localization, chromosome segregation, and nucleolar reorganization. Phosphosites in the RRM domain showed a potential association with RNA-binding proteins and metabolic pathways. Collectively, these findings suggest a domain-specific phosphosite-centric regulatory network of NCL, providing a valuable reference framework for future experimental studies and phosphosite-directed therapeutic strategies in cancers.