Re‐evaluation of the effect of the phosphatase inhibitor okadaic acid on aquaporin 2 phosphorylation and trafficking

Abstract

Aquaporin‐2 (AQP2), the vasopressin (VP)‐sensitive water channel, plays an important role in water reabsorption. Accumulation of AQP2 on the plasma membrane in kidney collecting duct principal cells increases water permeability and is necessary for urine concentration. AQP2 trafficking is tightly regulated by phosphorylation and dephosphorylation, and the role of each of four phosphoserine residues (pS256, pS261, pS264 and pS269) has been intensively studied. Recent work in our laboratory has shown that erlotinib (Erl), an epidermal growth factor receptor (EGFR) inhibitor, induces AQP2 membrane accumulation in the absence of VP. Western blot analysis using selective phospho anti‐AQP2 antibodies showed a VP‐like effect. Here, we investigate how phosphatase inhibition affects trafficking and intracellular location of AQP2 in the presence of VP and erlotinib. We performed immunohistochemistry and western blotting on LLC‐PK1 cells stably expressing WT AQP2. Cells were treated with okadaic acid (OA, 1 μM), a phosphatase inhibitor specific for PP1 and PP2A, for 10 minutes before treatment with either VP (10 nM) or Erl (2 μM). Interestingly, OA treatment alone did not significantly increase AQP2 membrane accumulation in our cell model, in contrast to a previous report in CD 8 cells (Valenti et al 2000), but did modify its intracellular distribution into a perinuclear position. Despite the lack of AQP2 membrane accumulation, western blot analysis showed that OA alone increases phosphorylation at all four phosphorylation sites. The VP or Erl induced AQP2 membrane accumulation is not affected by OA, but unexpectedly, pretreatment with OA did not inhibit VP or Erl induced S261 dephosphorylation. This suggests that phosphoserines 256 and 269 are the main substrates of phosphatases inhibited by OA (i.e., PP1, PP2a) and that serine 261 is the substrate for an OA insensitive phosphatase activated by VP and Erl.Support or Funding InformationThe American Physiological Society supported this work