Functional phosphoproteomic analysis of SMG1 in nonsense-mediated mRNA decay and DNA damage repair in cancer.

Introduction: SMG1, a phosphatidylinositol 3-kinase-related kinase (PIKK) family serine/threonine kinase, is a key regulator of nonsense-mediated mRNA decay (NMD) through phosphorylation of UPF1 and plays important roles in genome stability, p53 activation, and tumor suppression. Although 77 phosphorylation sites have been reported on SMG1, their regulatory significance has not been systematically characterized. Methods: A comprehensive meta-analysis of more than 3,800 PubMed-indexed human phosphoproteomic studies was performed to curate Class I SMG1 phosphosites. A total of 678 qualitative and 173 quantitative phosphoproteomic datasets were analyzed to identify predominant phosphorylation sites. Co-regulation, co-occurrence, motif enrichment, kinase prediction, and cancer phosphoproteomic analyses were conducted to investigate site-specific regulatory networks and biological functions. Results: Three C-terminal phosphosites (T3573, S3570, and S3556) emerged as the most frequently detected and dynamically regulated sites across datasets. Co-regulation network analysis indicated distinct functional associations for each site, with T3573 linked to cytoskeletal regulators, S3570 to RNA-processing factors, and S3556 to chromatin- and DNA damage-associated proteins. Co-occurrence analysis suggested that these phosphosites can be phosphorylated simultaneously, indicating coordinated regulation. Multiple core NMD components, including SMG7, UPF1, SMG9, CASC3, and CTIF, exhibited strong positive co-regulation with S3570 and/or S3556. In silico and peptide-array-based kinase predictions identified ATM, PRKDC (DNA-PKcs), and BRAF as candidate upstream kinases for S3556, linking SMG1 phosphorylation to DNA damage signaling. Motif and co-regulation analyses expanded the putative SMG1 substrate repertoire by identifying numerous [S/T]-Q-containing DNA damage response proteins, including BRCA1, RAD51AP1, NBN, PNKP, TP53BP1, and PBRM1. UALCAN analysis further revealed differential regulation of SMG1 phosphosites across multiple cancer types. Discussion: These findings identify SMG1 as a central phosphorylation hub integrating nonsense-mediated mRNA decay with genome maintenance pathways and provide the first systematic phosphosite-centric framework for understanding its regulation. The identified regulatory phosphosites, candidate upstream kinases, and phosphorylation networks establish a foundation for future mechanistic studies and support the potential of SMG1 phosphorylation as a diagnostic and therapeutic target in cancer.