Journal Article

Quantitative phosphoproteomic analysis reveals vasopressin V2-receptor-dependent signaling pathways in renal collecting duct cells

Rinschen, Markus M, Yu, Ming-Jiun, Pisitkun, Trairak, Wang, Guanghui, Boja, Emily S, Hoffert, Jason D, Knepper, Mark A

2010

Overview

Vasopressin's action in renal cells to regulate water transport depends on protein phosphorylation. Here we used mass spectrometry-based quantitative phosphoproteomics to identify signaling pathways involved in the short-term V2-receptor-mediated response in cultured collecting duct cells (mpkCCD) from mouse. Using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) with two treatment groups (0.1 nM dDAVP or vehicle for 30 min), we carried out quantification of 2884 phosphopeptides. The majority (82%) of quantified phosphopeptides did not change in abundance in response to dDAVP. Analysis of the 273 phosphopeptides increased by dDAVP showed a predominance of so-called \"basophilic\" motifs consistent with activation of kinases of the AGC family. Increases in phosphorylation of several known protein kinase A targets were found. In addition, increased phosphorylation of targets of the calmodulin-dependent kinase family was seen, including autophosphorylation of calmodulin-dependent kinase 2 at T286. Analysis of the 254 phosphopeptides decreased in abundance by dDAVP showed a predominance of so-called \"proline-directed\" motifs, consistent with down-regulation of mitogen-activated or cyclin-dependent kinases. dDAVP decreased phosphorylation of both JNK1/2 (T183/Y185) and ERK1/2 (T183/Y185; T203/Y205), consistent with a decrease in activation of these proline-directed kinases in response to dDAVP. Both ERK and JNK were able to phosphorylate residue S261of aquaporin-2 in vitro, a site showing a decrease in phosphorylation in response to dDAVP in vivo. The data support roles for multiple vasopressin V2-receptor-dependent signaling pathways in the vasopressin signaling network of collecting duct cells, involving several kinases not generally accepted to regulate collecting duct function.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Amino Acid Sequence / Amino acids / Amino Acids - chemistry / Animals / Biological Sciences / Cell culture / Cell Culture Techniques / Cell lines / Cells / Cells, Cultured / cyclin-dependent kinase / Deamino Arginine Vasopressin - pharmacology / Hormones / Immunoblotting / Isotope Labeling / Isotopic labeling / Kidney cells / Kidney Tubules, Collecting - metabolism / Kidneys / Kinases / MAP Kinase Kinase 4 - metabolism / Mass spectrometry / Mass spectroscopy / Mice / mitogen-activated protein kinase / Mitogen-Activated Protein Kinase 3 - metabolism / Phosphopeptides - chemistry / Phosphopeptides - metabolism / Phosphoproteins - chemistry / Phosphoproteins - metabolism / Phosphorylation / Phosphorylation - drug effects / Physiology / Protein isoforms / protein phosphorylation / Proteome / Proteomics / Quantification / Receptors, Vasopressin - drug effects / Receptors, Vasopressin - metabolism / Signal Transduction / Stable isotopes / vasopressin / Water transport