Bayesian Analysis of Dynamic Phosphoproteomic Data Identifies Protein Kinases Mediating Responses to a Galphas-Coupled GPCR

A major goal in the discovery of signaling networks is to identify regulated phosphorylation sites and map them to the protein kinases responsible for their phosphorylation. The V2 vasopressin receptor is a Galphas-coupled GPCR that is responsible for regulation of renal water excretion through control of osmotic water transport in kidney collecting duct cells. Genome editing experiments have demonstrated that virtually all vasopressin-triggered phosphorylation changes are dependent on PKA, but events downstream from PKA are obscure. Here we used: 1) TMT-based quantitative phosphoproteomics to track phosphorylation changes over time in native collecting duct cells isolated from rats; 2) a clustering algorithm to classify time course data; and 3) Bayes’ Theorem to integrate the dynamic phosphorylation data with multiple prior “omic” data sets to identify a set of protein kinases that are regulated secondary to PKA activation. The data establish three PKA-dependent protein kinase modules whose regulation mediate the physiological effects of vasopressin at a cellular level. The three modules are 1) a pathway involving several Rho/Rac/Cdc42-dependent protein kinases that control actin cytoskeleton dynamics; 2) MAP kinase and cyclin-dependent kinase pathways that control cell proliferation; and 3) calcium/calmodulin-dependent signaling. The findings provide a template for investigating signaling via other Galphas-coupled GPCRs.