Kidney Week

Abstract: SA-PO294

Identification of MAP Kinases-Regulated Proteins in Downstream Signaling Pathways of Vasopressin V2 Receptor in Kidney Collecting Duct

Session Information

• Fluid and Electrolytes: Basic - II
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Jang, Hyo-Ju, Kyungpook National University, Daegu, Korea (the Republic of)

Hyo-Ju Jang,

• Jung, Hyun Jun, University of Maryland School of Medicine, Baltimore, Maryland, United States

Hyun Jun Jung,

• Choi, Hyo-Jung, Kyungpook National University, Daegu, Korea (the Republic of)

Hyo-Jung Choi,

• Park, Eui-Jung, Kyungpook National University, Daegu, Korea (the Republic of)

Eui-Jung Park,

• Park, Hye-Jeong, Kyungpook National University, Daegu, Korea (the Republic of)

Hye-Jeong Park,

• Kwon, Tae-Hwan, Kyungpook National University, Daegu, Korea (the Republic of)

Tae-Hwan Kwon,

Background

Vasopressin signaling mediated by G protein-coupled V2 receptor (V2R) is critical in water and electrolyte transport in the kidney collecting duct (CD) cells. Stimulation of V2R affects several downstream signaling pathways, including PKA, PI3K/AKT, Wnt, and Ca²⁺/calmodulin. MAP kinases are also involved as an apparent downstream signaling pathway of V2R, however, the roles and their substrates of MAP kinases in the vasopressin signaling are unclear.

Methods

Comprehensive substrates of MAP kinases were identified using bioinformatic analyses: 1) expression of MAP kinases were studied using database based on high-throughput profiles of transcriptome and proteome (https://hpcwebapps.cit.nih.gov/ESBL/Database/index.html); 2) MAP kinase substrates expressed in the CD were identified using multiple protein phosphorylation databases. The identified substrates were mapped on the downstream signaling of V2R. Trim28-mediated AQP2 regulation was examined using immunoblotting and immunohistochemistry.

Results

Five MAP kinases (ERK1, ERK2, ERK3, JNK2, and MAPK p38 alpha) were identified as the MAP kinases expressed in kidney CD cells. From multiple protein kinase-substrates databases, 189 proteins were identified as the substrates of five MAP kinases. Among them, 51 transcription factors, 15 transcription co-regulators, 30 kinases, 4 E3 ligases and 1 deubiquitinating enzyme were classified. In particular, sequential data mining revealed that serine 595 in the tripartite motif-containing 28 (TRIM28), as the substrate of MAP kinases, was the only one phosphorylation site downregulated by vasopressin. Since TRIM28 is a transcription cofactor and also E3 ligase, we examined whether TRIM28 is a mediator of MAP kinases action on AQP2 expression. Immunofluorescent labeling of mouse and rat kidneys revealed that TRIM28 was exclusively localized in the nuclei of the tubular epithelial cells, including CD. dDAVP-induced AQP2 up-regulation was significantly attenuated in mpkCCD cells with TRIM28 knockdown.

Conclusion

We identified MAP kinase substrates of the kidney CD mapped on the downstream signaling pathways of V2R. TRIM28 was identified as a substrate of MAP kinases that involves in vasopressin-mediated signaling pathways, including regulation of AQP2.

Funding

• Government Support - Non-U.S.