Hoffert et al.
were the first to utilize a quantitative phosphoproteomic approach to analyze the response of the collecting duct to AVP [19
]. Isolated inner medullary collecting ducts from rats were incubated in the presence or absence of 1 nM (deamino-Cys1, D-Arg8)-vasopressin (dDAVP; a V2-receptor-specific analog of vasopressin) for 10 min and then processed for LC-MS/MS analysis utilizing a strategy similar to that shown in . A total of 714 phosphorylation sites on 223 proteins were identified (data available at [101
]), including four phosphorylation sites in the C-terminus of the water channel AQP2, one of the main molecular targets of vasopressin. One site, Ser-256, had been previously identified as being critical for AQP2 trafficking to the apical cell membrane [35
], while the other three sites (Ser-261, Ser-264 and Ser-269) were previously unidentified. Label-free LC-MS/MS quantification of reconstructed MS1 precursor ion intensities demonstrated that two of these sites are reciprocally regulated: phosphorylation at Ser-256 increases while phosphorylation at Ser-261 decreases in the presence of AVP. Subsequent studies have examined the phosphorylation kinetics and physiological roles of these newly identified phosphosites. Immunoblotting with newly generated phospho-specific antibodies against each site demonstrated that phosphorylation at Ser-264 and Ser-269 also increase with dDAVP and that prior phosphorylation at Ser-256 is a requirement for phosphorylation at these downstream residues [4
]. Phosphorylation at Ser-269 is believed to play a role in retention at the apical membrane [4
] through a reduction in the rate of endocytosis and decreased interaction with a number of proendocytotic proteins including heat-shock protein 70, heat-shock cognate 70, dynamin and clathrin [36
]. Ser-261 phosphorylation may be involved in regulating polyubiquitylation and proteasomal degradation of AQP2 [37
]. The kinase(s) responsible for phosphorylating AQP2 at these sites remain unknown.
A subsequent large-scale phosphoproteomic study by Bansal et al.
also analyzed the AVP response in isolated rat inner medullary collecting ducts [38
], but with a number of important modifications designed to increase the sensitivity of the analysis: an upstream strong cation-exchange chromatography step was added, which stratified the peptide sample and increased the recovery of phosphopeptides after IMAC; samples were run on a higher sensitivity mass spectrometer, the Orbitrap XL™ (Thermo Scientific); and spectra were searched with multiple algorithms (Sequest, Inspect and OMSSA). As a result of these advances, over 2700 unique phosphopeptides were identified (data available at [102
]). The authors also performed label-free MS quantitation, which identified 29 phosphopeptides that changed significantly with AVP treatment (p < 0.05; n = 3 unfractionated biological replicates). A major finding from this study was the identification of three phosphorylation sites on urea transporters UT-A1 and UT-A3, all of which were shown by MS to increase in abundance in response to AVP. AVP-mediated phosphorylation of UT-A1 is believed to increase the permeability of the collecting duct to urea, possibly by affecting membrane trafficking [39
]. Immunoblotting with phospho-specific antibodies to Ser-84 and Ser-486 of rat UT-A1 confirmed responsiveness to AVP and involvement of PKA. Another phosphoprotein that was increased with AVP was β-catenin (Ctnnb1
), a protein with both structural and signaling roles that has been proposed to play a role in regulation of AQP2 [40
]. Other phosphorylation sites that increased with AVP that have potential roles in the regulation of water and/or urea transporter activity included sites in protein kinase PCTAIRE-3 (Pctk3
) and plexin domain containing-2 (Plxdc2
). Phosphorylation sites decreased with AVP were present in septin-9 (Sept9
), ArfGAP with FG repeats 1 (Agfg1
) and epsin-3 (Epn3
A recent study by Rinschen et al.
measured AVP-responsive phosphoproteins in a SILAC-labeled mouse cortical collecting duct cell line (mpkCCD) [41
]. Cells labeled with heavy or light amino acids were exposed to either dDAVP (0.1 nM) or vehicle control for 30 min and processed for LC-MS/MS analysis. Of the 2884 phosphopeptides that were quantified, 273 increased and 254 decreased with AVP. The threshold for change was based on the results of preliminary experiments in which both heavy and light samples were vehicle treated (data are available at [103
]) Identification of upregulated phosphorylation sites on calmodulin-dependent kinase II pointed to a potential role for the Ca+2
-calmodulin pathway in the response of the collecting duct to AVP. This finding was consistent with prior studies demonstrating that vasopressin triggers both intracellular calcium mobilization [42
] and calmodulin-dependent phosphorylation of myosin regulatory light chain protein by myosin light chain kinase [43
], which are both critical for AQP2 trafficking. In order to assess which kinase pathways may be regulated by vasopressin, the authors performed a global analysis of phosphorylation motifs (i.e., the amino acid sequences surrounding the phosphorylation site) for phosphopeptides regulated by AVP. This analysis was performed using the motif-x algorithm [44
], which generates sequence logos representing amino acid sequences that are significantly over-represented in input datasets compared with background. For phosphopeptides increased by AVP, there was an over-representation of so-called ‘basophilic’ phosphorylation motifs, which contained positively charged amino acids arginine and lysine prior to the site of phosphorylation (i.e., R- [R/K]-X- [S/T]*, where X is any amino acid and * indicates the site of phosphorylation). These basophilic motifs are associated with phosphorylation by the AGC class of kinases, which includes members such as PKA, PKC, PKG and some calmodulin-dependent kinases. For phosphopeptides decreased by AVP, there was an over-representation of ‘proline-directed’ motifs, which contain a single proline residue at position +1 (i.e., X-X- [S/T]*-P-X) and are associated with phosphorylation by MAP kinases and cyclin-dependent kinases. Thus, these results reveal that AVP signaling is associated with inactivation of one or more proline-directed kinases. A similar profile was demonstrated in a recent analysis of the AVP response in the medullary thick ascending limb of rat kidney [45