Phosphoproteomic identification of vasopressin-regulated protein kinases in collecting duct cells

Br J Pharmacol. 2021 Mar;178(6):1426-1444. doi: 10.1111/bph.15352. Epub 2021 Feb 14.


Background and purpose: The peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP-mediated activation of a PKA-dependent signalling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins.

Experimental approach: We carried out systems-level analysis of large-scale phosphoproteomic data quantifying vasopressin-induced changes in phosphorylation in aquaporin-2-expressing cultured collecting duct (mpkCCD) cells. Quantification was done using stable isotope labelling (SILAC method).

Key results: Six hundred forty phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin-regulated cellular processes. The vasopressin-regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups. Among the vasopressin-regulated phosphoproteins were 25 distinct protein kinases. Among these, six plus PKA appeared to account for phosphorylation of about 81% of the 313 vasopressin-regulated phosphorylation sites. The six downstream kinases were salt-inducible kinase 2 (Sik2), cyclin-dependent kinase 18 (Cdk18), calmodulin-dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen-activated kinase 3 (Mapk3) and myosin light chain kinase (Mylk).

Conclusion and implications: In V2 receptor-mediated signalling, PKA is at the head of a complex network that includes at least six downstream vasopressin-regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data reported in this study are provided as a web-based data resource for future studies of GPCRs.

Keywords: Camkk2; Cdk18; GPCR signalling; Prkd2; Sik2; V2 receptor signalling; aquaporin-2-expressing cultured collecting duct cells.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Aquaporin 2 / metabolism
  • Kidney Tubules, Collecting* / metabolism
  • Mice
  • Phosphorylation
  • Protein Kinases* / metabolism
  • Proteome
  • Vasopressins* / metabolism


  • Aquaporin 2
  • Proteome
  • Vasopressins
  • Protein Kinases