Vasopressin (VP) regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the C-terminus, and among them, serine-256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of the serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells, and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and western blotting with phospho-specific antibodies. Using methyl-b-cyclodextrin (MBCD), cold block or bafilomycin, and Taxol, we blocked AQP2 at the plasma membrane, in the peri-nuclear trans-Golgi network (TGN), and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared to baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the TGN, or in cytoplasmic vesicles, and that this event is dependent on the expression of PKA in these cells.

Vasopressin (VP) activates PKA, resulting in phosphorylation and membrane accumulation of aquaporin-2 (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 identify the ribosomal s6 kinase (RSK) as the final mediator phosphorylating AQP2 in this novel, erlotinib-induced pathway. We found that RSK was expressed in medullary principal cells in rat kidneys. RSK inhibition with BI-D1870 or siRNA blocked erlotinib-induced AQP2 S256 phosphorylation and membrane trafficking. CRISPR-generated RSK knockout cells failed to show increased S256 phosphorylation in response to erlotinib. Like PKA, RSK was able to phosphorylate AQP2 S256 in vitro. Stimulation of PDK1, a known activator of RSK, caused AQP2 S256 phosphorylation and membrane accumulation similar to erlotinib and VP. We conclude that RSK is the terminal kinase phosphorylating AQP2 at S256 upon EGFR inhibition by erlotinib.