Clustered phosphatidylinositol 4,5 bisphosphate accumulation and ezrin phosphorylation in response to CLIC5A

CLIC5A is a component of the ezrin-NHERF2-podocalyxin complex in renal glomerular podocyte foot processes. We explored the mechanism(s) by which CLIC5A regulates ezrin function. In COS-7 cells, CLIC5A augmented ezrin phosphorylation without changing ezrin abundance, increased the association of ezrin with the cytoskeletal fraction, enhanced actin polymerization and the formation of cell surface projections. CLIC5A caused the PI(4,5)P2 reporter RFP-PH-PLC to translocate from cytosol to discrete plasma membrane clusters at the cell surface, where it co-localized with CLIC5A. Transiently expressed HA-PIP5Kα co-localized with GFP-CLIC5A and was pulled from cell lysates by GST-CLIC5A, and silencing of endogenous PIP5Kα abrogated CLIC5A-dependent ERM phosphorylation. N- and C-terminal deletion mutants of CLIC5A, which failed to associate with the plasma membrane failed to co-localize with PIP5Kα, did not alter the abundance of PI(4,5)P2 plasma membrane clusters and failed to enhance ezrin phosphorylation. Relative to wild-type mice, in CLIC5 deficient mice glomerular ezrin phosphorylation was diminished and the cytoskeletal association of both ezrin and NHERF2 was reduced. Therefore, the mechanism of CLIC5A action involves clustered plasma membrane PI(4,5)P2 accumulation through an interaction of CLIC5A with PI(4,5)P2 generating kinases, in turn facilitating ezrin activation, and actin-dependent cell surface remodeling.