Bayesian analysis of dynamic phosphoproteomic data identifies protein kinases mediating GPCR responses.

BACKGROUND: A major goal in the discovery of cellular signaling networks is to identify regulated phosphorylation sites ("phosphosites") and map them to the responsible protein kinases. The V2 vasopressin receptor is a G-protein coupled receptor (GPCR) that is responsible for regulation of renal water excretion through control of aquaporin-2-mediated osmotic water transport in kidney collecting duct cells. Genome editing experiments have demonstrated that virtually all vasopressin-triggered phosphorylation changes are dependent on protein kinase A (PKA), but events downstream from PKA are still obscure. METHODS: Here, we used: 1) Tandem mass tag-based quantitative phosphoproteomics to experimentally track phosphorylation changes over time in native collecting ducts isolated from rat kidneys; 2) a clustering algorithm to classify time course data based on abundance changes and the amino acid sequences surrounding the phosphosites; and 3) Bayes' Theorem to integrate the dynamic phosphorylation data with multiple prior "omic" data sets covering expression, subcellular location, known kinase activity, and characteristic surrounding sequences to identify a set of protein kinases that are regulated secondary to PKA activation. RESULTS: Phosphoproteomic studies revealed 185 phosphosites regulated by vasopressin over 15 min. The resulting groups from the cluster algorithm were integrated with Bayes' Theorem to produce corresponding ranked lists of kinases likely responsible for each group. The top kinases 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) mitogen-activated protein kinase and cyclin-dependent kinase pathways that control cell proliferation; and 3) calcium/calmodulin-dependent signaling. CONCLUSIONS: Our findings identify a novel set of downstream small GTPase effectors and calcium/calmodulin-dependent kinases with potential roles in the regulation of water permeability through actin cytoskeleton rearrangement and aquaporin-2 trafficking. The proposed signaling network provides a stronger hypothesis for the kinases mediating V2 vasopressin receptor responses, encouraging future targeted examination via reductionist approaches. Furthermore, the Bayesian analysis described here provides a template for investigating signaling via other biological systems and GPCRs. Video abstract.