Abstract
Background and Aims
Activation 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. However, the role of MAP kinases and their substrate proteins in the vasopressin signaling, including the regulation of AQP2 expression and phosphorylation, 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. Tripartite motif-containing protein 28 (TRIM28) was identified as a substrate of ERK1 as well as a vasopressin-responsive protein via bioinformatic tools. We further evaluated whether TRIM28 plays a role in vasopressin-mediated regulation of AQP2 in the kidney CD.
Method
To 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 high-throughput profiles of transcriptome and proteome (http://hpcwebapps.cit.nih.gov/ESBL/Database/index.html); and 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 protein (TRIM28) was examined by immunohistochemistry. The role of TRIM28 in vasopressin-mediated AQP2 regulation was examined by quantitative real-time PCR (qRT-PCR) and semiquantitative immunoblotting after RNA interference of TRIM28 in mpkCCDc11 cells.
Results
Immunoblotting 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 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.
Conclusion
We 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.
P0013MAP KINASES-REGULATED PROTEINS IN SIGNALING PATHWAYS OF VASOPRESSIN IN KIDNEY COLLECTING DUCT
