Revealing RELL1 signaling relying on the phosphoregulatory network analysis.

Introduction: RELL1 (Receptor Expressed in Lymphoid Tissues-Like 1) is a member of the TNF receptor superfamily. Although the physiological ligands and mechanistic insights into cell signaling still remain to be elucidated, its over-expression is associated with poor prognosis and enhanced immune cell infiltration in cancers, marking it a potential oncogenic driver and immunotherapeutic target. Despite extensive phosphoproteomic evidence indicating that RELL1 undergoes phosphorylation, the functional significance of its phosphosites has not been systematically investigated. Methods: Global human phosphoproteomic datasets were systematically analyzed to identify predominant RELL1 phosphosites. The two most frequently detected phosphosites were selected as predominant sites for phosphosite-centric analysis. To explore their functional significance, their highly co-phosphoregulated sites in other proteins across diverse conditions were examined. A stringent criteria was applied to determine protein phosphosites that showed either positive or negative co-regulation with predominant sites. These co-regulated sites were further analyzed within known interacting proteins, and predicted the upstream regulators of RELL1, followed by enrichment analysis. Results: Exemplified by the detection of its phosphorylated forms in 474 qualitative and 150 quantitative phosphoproteomic datasets across multiple experimental conditions, we propose phosphorylation as a potential regulatory mechanism associated with RELL1 function. However, the functional role of any of the phosphosites in RELL1 phosphosites remains unknown. Among the detected phosphosites, S244 and S161 were the most frequently observed sites perturbed across datasets, and hence, considerably the predominant sites. Co-phosphoregulation analysis revealed plausible regulatory networks comprising interacting proteins and predicted upstream kinases. Bioinformatics analyses revealed strong associations between these phosphosites and biological processes such as nuclear transport and cell cycle regulation. Conclusion: This systems-level phosphoproteomic investigation provides the potential phosphoregulatory map for RELL1 phosphosites, which could lay a foundation for further experimental investigations as well as in establishing its functional roles contributing to advancements in RELL1 signaling events relevant to immuno-oncotherapy.