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Abstract: TH-PO335

Activation of PDK1/RSK Results in Aquaporin-2 S256 Phosphorylation and Membrane Accumulation After Downregulation of EGFR Signaling

Session Information

Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders

  • 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic


  • Babicz, Richard Samuel, Massachusetts General Hospital, Boston, Massachusetts, United States
  • Cheung, Pui Susan Wen, Massachusetts General Hospital, Boston, Massachusetts, United States
  • Bouley, Richard, Massachusetts General Hospital, Boston, Massachusetts, United States
  • Baylor, Noah, Massachusetts General Hospital, Boston, Massachusetts, United States
  • Brown, Dennis, Massachusetts General Hospital, Boston, Massachusetts, United States

Group or Team Name

  • Program in Membrane Biology

Vasopressin (VP) activates PKA, resulting in phosphorylation and membrane accumulation of AQP2. Epidermal growth factor receptor (EGFR) inhibition with erlotinib also induces AQP2 membrane trafficking with a phosphorylation pattern similar to VP, but without increasing PKA activity. Here, we identified the novel kinase pathway(s) that are activated when EGFR signaling is inhibited.


We used several inhibitors that block EGFR signaling to identify which pathway inhibited erlotinib-induced AQP2 membrane accumulation in LLC-PK1 cells, and rat kidney slices. We incubated purified GST-AQP2 c-terminus with recombinant kinase and 10uCi (Y32P) ATP using PKA as a positive control and our purified kinase target (RSK). Inhibitors of PKA and RSK and purified GST-AQP2 S256A c-terminus were negative controls. RSK phosphorylated AQP2 at S256, and we asked if the intermediate kinase PDK-1 stimulated RSK activity and AQP2 phosphorylation by western blotting, and membrane accumulation by immunocytochemsitry.


Inhibiting 90 kDa ribosomal S6 kinase (RSK) significantly reduced erlotinib-induced S256 phosphorylation. Upon blocking or knocking down RSK, erlotinib no longer induced AQP2 membrane accumulation. In rat kidneys, RSK was expressed in medullary principal cells. Erlotinib did not induce AQP2 membrane accumulation when cells/tissues were pre-treated with the RSK inhibitor, BI-D1870. Using purified proteins, RSK directly phosphorylated WT- but not S256A-AQP2, similar to PKA. We conclude that RSK phosphorylates AQP2 at S256 upon EGFR inhibition by erlotinib. RSK is activated by phosphorylation of a critical S221 residue by the intermediate kinase PDK-1. Incubation of cells/tissues with PS210, a direct activator of PDK-1, caused a significant increase in both RSK S221 and AQP2 S256 phosphorylation, and plasma membrane accumulation of AQP2.


Activation of PDK1/RSK upon erlotinib exposure is a key step in stimulating AQP2 membrane accumulation via direct RSK-mediated phosphorylation of the critical S256 C-terminal residue. This represents a novel, VP-independent pathway by which collecting duct water permeability could be modulated.


  • NIDDK Support