Phospho-Regulatory Network of the Right Open Reading Frame Kinase 1 (RIOK1), Its Functional Relevance, and Cancer Treatment Prospects

Right open reading frame kinase 1 (RIOK1) is an atypical kinase involved in ribosome biogenesis, cell cycle progression, and chromosome organization. Its overexpression is linked to tumor progression, metastasis, and chemoresistance, while its absence alters protein phosphorylation across various biological processes. Although the oncogenic role of RIOK1 is recognized, its phospho-regulatory network and the functional relevance of its phosphorylation sites remain unknown. Here, we present the first large-scale phosphoproteomic analysis of RIOK1. Through a systematic assembly of 671 mass spectrometry-based datasets and 157 datasets that quantified RIOK1 phosphopeptides in different experimental conditions, we compiled 13 RIOK1 phosphorylation sites. Notably, phosphorylation at S21 and S22 was predominant, observed in 83% of phosphoproteomics datasets, highlighting their functional significance, and these sites may have a role in RIOK1's interaction with the protein arginine methyltransferase 5 complex. We identified co-differentially regulated phosphorylation events in potential upstream kinases and experimentally validated interactors, providing insights into RIOK1's broader signaling context. The phosphorylation sites in five potential upstream kinases (eukaryotic translation initiation factor 2 alpha kinase 4, ataxia telangiectasia mutated protein kinase, B-Raf proto-oncogene, mitogen-activated protein kinase kinase kinase 3, and polo-like kinase 1) co-differentially regulated with RIOK1 were identified, which regulate their activity in concert with RIOK1 in cancers. Together, this study represents the first comprehensive map to date of RIOK1 phosphorylation and its regulatory associations, highlighting its potential as a therapeutic target in cancers.