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

Abstract

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 socalled "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.aquaporin-2 | MAP kinase | mass spectrometry | protein kinase A | SILAC R egulation of osmotic water transport across the renal collecting duct epithelium is responsible for precise control of renal water excretion and regulation of the osmolality of body fluids. This precise control is achieved largely by actions of the peptide hormone vasopressin to regulate the water channel aquaporin-2 (AQP2) in collecting duct cells. Vasopressin binds to a G protein-coupled receptor (the type 2 vasopressin receptor or V2R) in the basolateral plasma membrane of collecting duct (CD) principal cells, triggering a complex signaling response that involves an increase in intracellular cAMP and spike-like, aperiodic increases in intracellular calcium (1, 2).The advent of methodologies for large-scale, mass spectrometry (MS)-based phosphoproteomics offers the opportunity for comprehensive discovery of signaling pathways in mammalian cells. In particular, the Stable Isotope Labeling by Amino acids in Cell culture (SILAC) methodology is a metabolic-labeling approach that allows high-precision quantification of individual peptides in cultured cells using tandem MS (3), leading to powerful quantitative approaches to discovery of signaling pathways (4-6...

Quantitative phosphoproteomic analysis reveals cAMP/vasopressin-dependent signaling pathways in native renal thick ascending limb cells

“…Phosphorylation at this site was increased by approximately fourfold in response to the cAMPgenerating hormone mixture. This response is similar to what was found previously in inner medullary collecting duct suspensions (26) and cultured collecting duct cells in response to vasopressin (12). β-Catenin is a structural protein that binds to cadherins at adherens junctions and plays an additional role as a transcriptional coregulator in the Wnt signaling pathway.…”

“…The basophilic pattern for up-regulated sites suggests that cAMP-mediated signaling in the mTAL involves regulation of kinases in the AGC and/or CAMK families. A similar pattern was recently reported in cultured renal collecting duct cells in response to vasopressin (12). Previous studies in mTAL have concluded that at least one AGC kinase, namely PKA, plays a critical role in vasopressin-mediated signaling (13).…”

“…This pattern suggests that cAMP-mediated signaling in the mTAL involves down-regulation of kinases in the MAPK (CMGC-II) and/ or cyclin-dependent (CMGC-I) kinase families (7). A similar reduction in phosphorylation at proline-directed sites was recently reported in response to vasopressin in cultured renal collecting duct cells (12). Several publications have implicated MAP kinases in the regulation of various processes in TAL cells (15)(16)(17).…”

“…MS spectra were searched using three different search algorithms: InsPecT, SEQUEST, and OMSSA (SI Materials and Methods). Searches were conducted against the most recent Rattus norvegicus RefSeq Database (National Center for Biotechnology Information) using the targetdecoy approach with filters adjusted to limit the false discovery rate to <2% as described in previous work (12). Quantification of relative phosphopeptide abundance (area under MS 1 time-course curve or extracted ion chromatogram elution profile) was implemented using QUOIL, an in-house software program designed for quantification of label-free peptides by LC-MS (30).…”

Angiotensin II regulates phosphorylation of actin‐associated proteins in human podocytes

“…For relative quantification of the phosphoproteome, AB8 3F-AT1R podocytes were grown in stable isotope labeling with amino acids in cell culture (SILAC) (16) 4 arginine, [ 12 C] 6 lysine) for 3 passages. This labeling period is sufficient to achieve .98% saturation of labeling (17). On the day of the experiment, 1 set of cells was treated with AngII (100 nM for 15 min; Sigma-Aldrich, St. Louis, MO, USA), another with vehicle, and then samples were pooled 1:1 (AngII/vehicle).…”