ABSTRACT
CLIC5A (encoded by CLIC5) 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 and enhanced actin polymerization and the formation of cell surface projections. CLIC5A caused the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] reporter RFP–PH-PLC to translocate from the cytosol to discrete plasma membrane clusters at the cell surface, where it colocalized with CLIC5A. Transiently expressed HA–PIP5Kα colocalized 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 colocalize 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, the phosphorylation of glomerular ezrin 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.
Footnotes
Competing interests
The authors declare no competing interests.
Author contributions
A.A.-M. and L.L. performed essentially all of the experiments and prepared a near-final draft of the manuscript. R.T.A. helped to design and interpret PI(4,5)P2 and negative-charge reporter assays. As principal investigator, B.J.B. designed the overall experimental approach, supervised the studies and prepared the final draft of the paper. All authors approved the final version of the manuscript.
Funding
B.J.B. was supported by operating funds from Canadian Institutes of Health Research (CIHR) [grant number MOP 641814]; and the Kidney Foundation of Canada (KFOC). R.T.A. is supported by operating funds from KFOC and by a Clinician Scientist Award from CIHR and a Clinical Investigator Award from Alberta Innovates-Health Solutions. The Division of Nephrology provided support for tuition and core infrastructure.
- Received December 7, 2013.
- Accepted September 3, 2014.
- © 2014. Published by The Company of Biologists Ltd