Quantitative phosphoproteomics in nuclei of vasopressin-sensitive renal collecting duct cells

Abstract

Vasopressin regulates transport across the collecting duct epithelium in part via effects on gene transcription. Transcriptional regulation occurs partially via changes in phosphorylation of transcription factors, transcriptional coactivators, and protein kinases in the nucleus. To test whether vasopressin alters the nuclear phosphoproteome of vasopressin-sensitive cultured mouse mpkCCD cells, we used stable isotope labeling and mass spectrometry to quantify thousands of phosphorylation sites in nuclear extracts and nuclear pellet fractions. Measurements were made in the presence and absence of the vasopressin analog dDAVP. Of the 1,251 sites quantified, 39 changed significantly in response to dDAVP. Network analysis of the regulated proteins revealed two major clusters ("cell-cell adhesion" and "transcriptional regulation") that were connected to known elements of the vasopressin signaling pathway. The hub proteins for these two clusters were the transcriptional coactivator β-catenin and the transcription factor c-Jun. Phosphorylation of β-catenin at Ser552 was increased by dDAVP [log(2)(dDAVP/vehicle) = 1.79], and phosphorylation of c-Jun at Ser73 was decreased [log(2)(dDAVP/vehicle) = -0.53]. The β-catenin site is known to be targeted by either protein kinase A or Akt, both of which are activated in response to vasopressin. The c-Jun site is a canonical target for the MAP kinase Jnk2, which is downregulated in response to vasopressin in the collecting duct. The data support the idea that vasopressin-mediated control of transcription in collecting duct cells involves selective changes in the nuclear phosphoproteome. All data are available to users at http://helixweb.nih.gov/ESBL/Database/mNPPD/.

Tankyrase-mediated β-catenin activity regulates vasopressin-induced AQP2 expression in kidney collecting duct mpkCCDc14 cells

“…Previous proteomic studies on vasopressin signaling in kidney collecting duct cells have suggested that the Wnt signaling pathway could also be involved in vasopressin-mediated AQP2 regulation (4,33,34). Recently, Schenk et al (34) demonstrated that vasopressin induced a change in the abundance of the transcriptional regulator ␤-catenin in the nuclear proteome of cortical collecting duct mpkCCD (clone 11) cells.…”

Vasopressin and the regulation of aquaporin-2

“…Recently, our laboratory has conducted a series of phosphoproteomic studies to identify proteins whose phosphorylation is regulated by vasopressin in collecting duct cells [22],[55],[18], [19],[56] and in renal thick ascending limbs [57]. An in depth presentation of these results is beyond the scope of the current review.…”

“…Among the most consistent phosphoproteomic responses to vasopressin has been a marked increase in β-catenin ( Ctnnb1 ) phosphorylation at Ser552, seen in native IMCD cells [55],[58], in cultured mpkCCD cells [18],[59],[56], and in native thick ascending limb cells [57]. In mpkCCD cells, vasopressin the increase in Ser552 phosphorylation was associated with translocation of β-catenin from the cytoplasm to nucleus, consistent with prior observations in A431 tumor cells [60].…”

Vasopressin-2 Receptor Signaling and Autosomal Dominant Polycystic Kidney Disease

“…56 Also, phosphorylation of c-Jun in the nucleus is decreased in response to dDAVP. 57 In conclusion, all of these studies suggest an important but ill-defined role for V2R-dependent modulation of MAPK signaling in the renal epithelium.…”