Hyperactivation of MEK/ERK pathway by Ca²⁺/calmodulin-dependent protein kinase kinase 2 promotes cellular proliferation by activating cyclin-dependent kinases and minichromosome maintenance protein in gastric cancer cells

Although CAMKK2 is overexpressed in several cancers, its role and relevant downstream signaling pathways in gastric cancer (GC) are poorly understood. Treatment of AGS GC cells with a CAMKK2 inhibitor, STO-609, resulted in decreased cell proliferation, cell migration, invasion, colony-forming ability, and G1/S-phase arrest. Quantitative phosphoproteomics in AGS cells with the CAMKK2 inhibitor led to the identification of 9603 unique phosphosites mapping to 3120 proteins. We observed decreased phosphorylation of 1101 phosphopeptides (1.5-fold) corresponding to 752 proteins upon CAMKK2 inhibition. Bioinformatics analysis of hypo-phosphorylated proteins revealed enrichment of MAPK1/MAPK3 signaling. Kinase enrichment analysis of hypo-phosphorylated proteins using the X2K Web tool identified ERK1, cyclin-dependant kinase 1 (CDK1), and CDK2 as downstream substrates of CAMKK2. Moreover, inhibition of CAMKK2 and MEK1 resulted in decreased phosphorylation of ERK1, CDK1, MCM2, and MCM3. Immunofluorescence results were in concordance with our mass spectroscopy data and Western blot analysis results. Taken together, our data reveal the essential role of CAMKK2 in the pathobiology of GC through the activation of the MEK/ERK1 signaling cascade.