Quantitative Proteomics Identifies Vasopressin-Responsive Nuclear Proteins in Collecting Duct Cells

Abstract

Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (b-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 59-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCb), and Scnn1g (ENaCg), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in b-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct.

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.…”

“…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. This finding potentially suggested that transcriptional regulator ␤-catenin could be involved in the transcriptional regulation in response to vasopressin stimulation.…”

“…The AVP-induced cAMP/PKA signaling pathway interacts with other signals, such as phosphoinositide pathways (19,31) and Wnt/␤-catenin signaling (34). However, cross talk between AVP and Wnt/␤-catenin signaling in the kidney collecting ducts, particularly for the regulation of AVP-induced AQP2 expression, is unknown.…”

“…However, cross talk between AVP and Wnt/␤-catenin signaling in the kidney collecting ducts, particularly for the regulation of AVP-induced AQP2 expression, is unknown. Previous studies demonstrated that AVP-mediated signaling induces phosphorylation (Ser 552) and nuclear translocation of ␤-catenin (34). Moreover, the binding sites of DNA-bound T cell factor/lymphoid enhancer factor (TCF/LEF), the main partner for ␤-catenin in transcriptional regulation of target genes, were identified at the promoter region of the AQP2 gene (28).…”

Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells

“…These proteomic and transcriptomic data have been made publicly available in databases (3). Many of these studies have focused on whole cells (4-6), whereas several analyzed subcellular fractions (7)(8)(9)(10)(11)(12). Analysis of subcellular fractions is technically demanding, but it has the benefit of identifying low abundance proteins with important functions.…”