EGFR inhibition with erlotinib induces membrane accumulation of aquaporin-2 via PDK1 and RSK activation
Authors: Babicz Richard , Bouley Richard , Cheung Pui , Baylor Noah , Faber Daphne , Brown Dennis 
Published: May 2024

In the canonical aquaporin-2 (AQP2) signaling pathway, vasopressin (VP) binds to its V2 receptor, which in turn activates adenylate cyclase and protein kinase A (PKA), resulting in AQP2 S256 phosphorylation and accumulation of AQP2 in the membrane. The epidermal growth factor receptor (EGFR) inhibitor erlotinib also increases AQP2 S256 phosphorylation and membrane accumulation, yet it does not increase cAMP or PKA activity. We hypothesize that the ribosomal s6 kinase (RSK), a downstream effector in the EGFR-MAPK/ERK pathway, is the terminal kinase phosphorylating AQP2 in this novel PKA-independent pathway activated by erlotinib, and that the phosphoinositide dependent kinase-1 (PDK1), a well-established activator of RSK, is indispensable for erlotinib-induced AQP2 activation. Using our LLC-AQP2 cell model, we show that erlotinib-induced AQP2 membrane accumulation and S256 phosphorylation are abolished by the specific RSK inhibitor BI-D1870. RSK knockdown with siRNA also blocked AQP2 S256 phosphorylation and membrane accumulation, as did RSK knockout with CRISPR/cas9 editing. Next, rat kidney slices were incubated in Hank’s buffer with or without BI-D1870 and treated with erlotinib. BI-D1870 inhibited AQP2 membrane accumulation in collecting duct principal cells, supporting the relevance of this pathway in kidney cells in situ. An in-vitro kinase assay using purified proteins demonstrated that RSK directly phosphorylates AQP2 at the S256 residue. Additionally, erlotinib consistently increased phosphorylation of RSK T359, a residue associated with RSK’s active state. In canonical RSK activation, a phosphorylation cascade results in PDK1 binding to and activating RSK. We found that PDK1 inhibition with an experimental compound PS-222 also blocked erlotinib-induced AQP2 membrane accumulation and S256 phosphorylation. This implicates PDK1 along with RSK in the novel erlotinib-induced pathway of AQP2 traffcking. Our data show that RSK and PDK1 are terminal effectors in the novel, PKA-independent pathway of AQP2 activation induced by erlotinib. Further elaboration of this new non-canonical pathway promises to uncover potential pharmacological targets for the treatment of water balance disorders. This work was supported by the National Institutes of Health (NIH) grant DK096586 (D. Brown). P. W. Cheung was supported was supported by NIH K-award DK115901. Richard Babicz is the recipient of the Ben J Lipps Research Fellowship (American Society of Nephrology). Additional support for the Program in Membrane Biology Microscopy Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (DK043351). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.