Phosphoproteomic identification of vasopressin‐regulated protein kinases in collecting duct cells

Abstract

Background and Purpose The peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP‐mediated activation of a PKA‐dependent signalling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins. Experimental Approach We carried out systems‐level analysis of large‐scale phosphoproteomic data quantifying vasopressin‐induced changes in phosphorylation in aquaporin‐2‐expressing cultured collecting duct (mpkCCD) cells. Quantification was done using stable isotope labelling (SILAC method). Key Results Six hundred forty phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin‐regulated cellular processes. The vasopressin‐regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups. Among the vasopressin‐regulated phosphoproteins were 25 distinct protein kinases. Among these, six plus PKA appeared to account for phosphorylation of about 81% of the 313 vasopressin‐regulated phosphorylation sites. The six downstream kinases were salt‐inducible kinase 2 (Sik2), cyclin‐dependent kinase 18 (Cdk18), calmodulin‐dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen‐activated kinase 3 (Mapk3) and myosin light chain kinase (Mylk). Conclusion and Implications In V2 receptor‐mediated signalling, PKA is at the head of a complex network that includes at least six downstream vasopressin‐regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data reported in this study are provided as a web‐based data resource for future studies of GPCRs.

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

“…2 ). Seven of the 66 decreasing phosphosites had basic amino acids in positions + 5 and + 6 (as previously seen [ 21 ]) (Group I.A.1). The remaining 59 decreasing phosphosites (Group I.A.2) were partitioned into three groups based on their time-course pattern.…”

“…The V2R is a G-protein coupled receptor (GPCR) that signals through the heterotrimeric G-protein alpha subunit (G α s) to activate adenylyl cyclase type 6 [ 17 , 18 ] and increase cyclic adenosine monophosphate (cAMP) levels [ 19 ] in collecting duct cells. Protein kinase A (PKA) appears to be the main cAMP effector in the collecting duct, mediating altered phosphorylation of multiple proteins [ 20 ], many of which are themselves protein kinases that phosphorylate their own sets of substrates [ 21 ]. One major target of PKA is AQP2 [ 20 ].…”

“…through PKA mediated phosphorylation of ARPP19/ENSA [ 22 ]. Most of the data gathered thus far represent steady-state responses to vasopressin after 15 min or more of vasopressin exposure [ 21 – 25 ]. Additional information needed for kinase/substrate mapping can, in principle, be obtained through observation of dynamic responses to vasopressin since the various kinases involved are likely to be activated at different time points after initiation of vasopressin exposure.…”

Aurora Kinase A Is Involved in Controlling the Localization of Aquaporin-2 in Renal Principal Cells

“…In quantitative phosphoproteomic analyses, AAK1 was shown to be phosphorylated upon V2R activation in renal collecting duct cells [ 48 , 49 ]. However, its inhibition by our AURKA inhibitors is unlikely because inhibition of clathrin-mediated endocytosis in renal principal cells causes an accumulation of AQP2 in the plasma membrane [ 50 ].…”

“…They could potentially constitute additional components of the machinery controlling AQP2 in renal principal cells. Phosphoproteomic studies revealed an AVP/V2R-dependent signaling network in renal-collecting duct cells that involves the regulation of a plethora of kinases, partly in a PKA-independent manner [ 48 , 49 , 81 ]. We had previously identified 19 potential regulators of the AQP2 redistribution via siRNA-mediated knockdown of 719 kinase-related genes, one of which was AURKA [ 34 ].…”