MAP kinases‐Regulated Proteins in Downstream Signaling Pathways of Vasopressin V2 Receptor in Kidney Collecting Duct

Abstract

BackgroundActivation of G protein‐coupled vasopressin V2 receptor (V2R) is critical in water and electrolyte transport in the kidney collecting duct (CD) cells. Stimulation of V2R affects several downstream pathways, including PKA, PI3K/AKT, Wnt, and Ca2+/calmodulin. Previous studies have shown that MAP kinases are also involved as an apparent downstream signaling pathway of V2R, which affects AQP2 phosphorylation (Ser261). However, the role of MAP kinases and their substrate proteins in the vasopressin signaling, including the regulation of AQP2, are unclear. In the present study, substrates of MAP kinases were identified using bioinformatic analyses, and they were mapped on the downstream signaling pathways of V2R.MethodsTo identify comprehensive substrates of MAP kinases in the kidney CD, we investigated 1) the expression of MAP kinases in CD cells by use of databases based on highthroughput profiles of transcriptome and proteome ( http://hpcwebapps.cit.nih.gov/ESBL/Database/index.html); 2) MAP kinases substrates expressed in the CD cells by use of protein phosphorylation databases (PhosphoNetworks and RegPhos 2.0). The identified substrates were mapped on the downstream signaling of V2R. Cellular and subcellular localization of selected substrate proteins was examined by immunohistochemistry. Substrate protein‐mediated AQP2 regulation was examined quantitative real‐time PCR (qRT‐PCR) and semiquantitative immunoblotting.ResultsImmunoblotting of mpkCCDc11 cells revealed that both p‐ERK1/2 and pS261‐AQP2 expression was decreased in response to dDAVP (10−9 M) stimulation. In silico analyses demonstrated that five MAP kinases (ERK1, ERK2, ERK3, JNK2, and MAPK p38 alpha) were identified as the MAP kinases expressed in kidney CD cells. Based on several protein kinase‐substrates databases, 189 proteins were identified as the substrates of the five MAP kinases. In particular, sequential data mining revealed that tripartite motif‐containing 28 (TRIM28), as the substrate of ERK1, has the only one phosphorylation site which was down‐regulated by vasopressin stimulation. Since TRIM28 is a transcription cofactor and also a ubiquitin‐protein E3 ligase, we examined whether TRIM28 is involved in the regulation of AQP2 expression as a mediator of MAP kinases action. Immunofluorescence labeling of mouse and rat kidneys revealed that TRIM28 was exclusively expressed in the nuclei of the tubular epithelial cells, including CD cells. dDAVP‐induced AQP2 mRNA and protein up‐regulation was significantly attenuated in mpkCCDc11 cells with siRNA‐mediated knockdown of TRIM28.ConclusionsWe identify MAP kinase substrates in the kidney CD, which are mapped on the downstream signaling pathways of V2R. TRIM28 is identified as a substrate of MAP kinases that involves in vasopressin signaling pathways. TRIM28 is likely to play a role in the regulation of AQP2 expression, particularly in the AQP2 transcription.Support or Funding InformationThis work was supported by the National Research Foundation of Korea funded by the Ministry of Science, Information and Communication Technology, Korea (2019R1A2C1007753).