Quantitative phosphoproteomics implicates clusters of proteins involved in cell‐cell adhesion and transcriptional regulation in the vasopressin signaling network

Abstract

Vasopressin regulates transport in the renal collecting duct in part by modifying the phosphorylation of transcriptional regulators present in the nucleus. To assess changes in the nuclear phosphoproteome of vasopressin‐sensitive mpkCCD cells in response to dDAVP, a vasopressin V2 receptor analog, we employed stable isotope labeling and mass spectrometry to quantify changes in the abundance of phosphorylated peptides in nuclear fractions. Of the 1,251 phosphorylation sites identified, 39 sites changed significantly in response to dDAVP. Network analysis showed that the regulated proteins fell into two major clusters: cell‐cell adhesion and transcriptional regulation. The hubs of these two clusters were the transcriptional coactivator beta‐catenin and the transcription factor c‐Jun respectively. Phosphorylation of beta‐catenin at Ser552, a known target of protein kinase A or Akt in the collecting duct, was increased in response to dDAVP (log2(dDAVP/vehicle) = 1.79). Phosphorylation of c‐Jun at Ser73, a known canonical target for the MAP kinase Jnk2 in the collecting duct, was decreased (log2(dDAVP/vehicle) = −0.53). The data suggest that vasopressin‐mediated control of transcription in the renal collecting duct involves selective changes in the nuclear phosphoproteome.