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/.

Fig. 1.

Flow diagram of methodology. A: Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) labeling and isolation of nuclear fractions. B: sample preparation for LC-MS/MS. C: analysis of output from LC-MS/MS.

Fig. 2.

Comparison of proteins identified by MS from eluate and flow-through fractions. A: Venn diagrams show proteins that are common to nuclear extract and nuclear pellet as well as proteins that are unique to one of the two samples. B: the 10 most common Gene Ontology Biological Process terms assigned to the proteins in the eluate and flow-through subfractions of nuclear fractions.

Fig. 3.

Plots showing −log(p) vs. log₂(dDAVP/vehicle) for the eluate (A) and flow-through peptides (B). Peptides satisfying two statistical criteria [P < 0.05 by t-test and log₂(dDAVP/vehicle) outside of a 95% confidence interval for vehicle-versus-vehicle experiments] are found within the shaded areas.

Fig. 4.

Distribution of phosphorylation sites that changed significantly in response to dDAVP. A: Venn diagram of regulated sites identified in the nuclear fractions. B: plot of values for the 7 peptides quantified in both the nuclear extract and the nuclear pellet. C: consensus logos for the peptides that were decreased (left) or increased (right) in abundance in response to dDAVP.

Fig. 5.

Classification of proteins with peptides that were significantly changed in abundance in response to dDAVP. Proteins are identified by their official gene symbols and are classified according to the “[FUNCTION]” annotations in the respective Swiss-Prot records. Abundance changes for each phosphorylation site shown in Tables 1 and 2 are reported as log₂(dDAVP/vehicle). Positive abundance changes are labeled green, and negative abundance changes are labeled red. Values in bold refer to changes in the nuclear extract.

Fig. 6.

Vasopressin signaling network constructed using STRING 9.0. The core vasopressin signaling network (see text) is indicated by the gray nodes. The red nodes correspond to proteins identified in this study, while the blue nodes indicate proteins from Schenk et al. (49). The pink nodes indicate proteins common to both studies. Many of the proteins fell into one of two functional categories (“Cell-Cell Adhesion” and “Transcriptional Regulation”).

Fig. 7.

Immunoblotting of phospho-β-catenin and phospho-c-Jun (n = 3). A: immunoblotting of phospho-β-catenin (Ser552) showed a significant increase in abundance in response to dDAVP. B: immunblotting of phospho-c-Jun (Ser73) revealed a significant decrease in abundance in response to dDAVP.

Fig. 8.

β-Catenin structure and alignment. A: NHLBI-AbDesigner (http://helixweb.nih.gov/AbDesigner/) was employed to construct a visual representation of the domains, phosphorylation sites, and other structural information. B: alignment across 11 species of β-catenin sequences spanning the Ser552 phosphorylation site.

Fig. 9.

c-Jun structure and alignment. A: NHLBI-AbDesigner (http://helixweb.nih.gov/AbDesigner/) was used to construct a visual representation of the domains, phosphorylation sites, and other structural information. B: alignment across 11 species of c-Jun sequences spanning the Ser73 phosphorylation site.

Fig. 10.

Mylk structure. NHLBI-AbDesigner (http://helixweb.nih.gov/AbDesigner/) was employed to construct a visual representation of the domains, phosphorylation sites, and other structural information of Mylk.