Systems level phosphoproteomics reveals CAMKK2 driven kinase signaling underlying malignant phenotypes in gastric cancer

Abstract Gastric cancer is driven by aberrant kinase signaling that promotes uncontrolled proliferation and malignant progression. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is overexpressed in gastric cancer; however, the global phosphorylation networks downstream of CAMKK2 remain incompletely defined. In this study, we investigated the functional and signaling consequences of CAMKK2 inhibition in gastric cancer cells using an integrated phenotypic and quantitative phosphoproteomics approach. Pharmacological inhibition of CAMKK2 using STO-609 in AGS cells significantly suppressed proliferation, clonogenic growth, migration, and invasion, and induced defects in nuclear morphology indicative of impaired cell cycle progression. Tandem mass tag (TMT) based phosphoproteomic profiling identified over 10,500 phosphopeptides and revealed extensive phosphoproteome remodeling following CAMKK2 inhibition, characterized predominantly by hypophosphorylation of proteins involved in nuclear signaling, RNA processing, and cell cycle regulation. Kinase substrate enrichment and motif analyses demonstrated coordinated attenuation of CDK, MAPK, and mitotic kinase-associated signaling pathways, with convergence on E2F regulated transcriptional programs. Collectively, these findings establish CAMKK2 as a central regulator of kinase signaling networks that sustain proliferative and malignant phenotypes in gastric cancer and highlight CAMKK2 inhibition as a potential therapeutic strategy. Competing Interest Statement The authors have declared no competing interest. Footnotes mohammadanajar{at}yenepoya.edu.in, Keshava.prasad{at}nitte.edu.in, prashantmodi{at}yenepoya.edu.in Data Availability Statement Data related to the study are provided in the manuscript and supplementary rest of the data and can be provided request. Abbreviations - CAMKK2 - Calcium/calmodulin-dependent protein kinase kinase - GC - Gastric cancer - TMT - Tandem mass tag - LC-MS/MS - Liquid chromatography tandem mass spectrometry - GO - Gene Ontology - KSEA - Kinase substrate enrichment analysis - MAPK - Mitogen activated protein kinase - CDK - Cyclin-dependent kinase - ERK - Extracellular signal-regulated kinase - AKT - Protein kinase B - ATM - Ataxia telangiectasia mutated - ATR - Ataxia telangiectasia and Rad3-related - PRKDC - Protein kinase DNA-activated catalytic subunit - E2F - E2 factor transcription factor - PCA - Principal component analysis - FDR - False discovery rate - PTM - Post-translational modification - Fe-NTA - Ferric nitrilotriacetic acid - TiO₂ - Titanium dioxide - ACN - Acetonitrile - TFA - Trifluoroacetic acid - TEABC - Triethylammonium bicarbonate - SEM - Standard error of the mean - SDS-PAGE - Sodium dodecyl sulfate–polyacrylamide gel electrophoresis - DHB - 2,5-Dihydroxybenzoic acid - X2K - eXpression2Kinases.