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Abstract: PO1684

A Direct CLIC5A/Ezrin Interaction Accounts for CLIC5A Plasma Membrane Localization and CLIC5A-Stimulated Rac1 Activation in Renal Glomeruli

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Rahman, Md. Mizanur, University of Alberta, Edmonton, Alberta, Canada
  • Li, Laiji, University of Alberta, Edmonton, Alberta, Canada
  • Ballermann, Barbara J., University of Alberta, Edmonton, Alberta, Canada
Background

CLIC5A is part of the Podocalyxin/Ezrin complex at the apical domain of podocyte foot processes. Ezrin binds membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) inducing its activation and C-terminal (T567) phosphorylation. Activated Ezrin bridges membrane-spanning proteins to cortical actin, shaping the cellular architecture. We reported that CLIC5A stimulates Rac1-GTP-dependent PI(4,5)P2 generation and Ezrin activation and that CLIC5A deletion disrupts the foot process architecture (J. Cell Sci. 127:5164, 2014 & Kidney Int. 89:833, 2016). Here, we investigated the mechanism by which CLIC5A interacts with Ezrin.

Methods

Direct protein-protein interactions were established by Yeast two-hybrid (Y2H) assay and confirmed by GST pull-down and reciprocal co-immunoprecipitation. Subcellular localization of endogenous proteins was determined by differential detergent fractionation of mouse glomeruli. Ezrin was released from glomerular membranes by stimulating phospholipase C-mediated PI(4,5)P2 hydrolysis with m-3M3FBS.

Results

We found that CLIC5A interacts directly with the Ezrin C-terminal domain, but only if Ezrin was in the activated state. The extreme 16 C-terminal amino acids of Ezrin were necessary for the interaction and Ezrin phosphorylation at T567 increased CLIC5A binding. Expression of a C-terminal ezrin fragment (Ezrin 432-586, phosphomimic T567D), but not C-terminally truncated Ezrin 432-570 effectively blocked CLIC5A-stimulated Ezrin and Rac1 activation. In glomeruli 49 +/- 4 % of endogenous CLIC5A was in the cytoplasmic, 33 +/- 3% in membrane and 18 +/- 1 % in the cytoskeleton-associated fraction. As expected, PI(4,5)P2 hydrolysis reduced Ezrin phosphorylation and shifted Ezrin from membrane and cytoskeletal fractions into the soluble pool. Similarly, PI(4,5)P2 hydrolysis shifted CLIC5A from membrane- and cytoskeletal fractions, increasing the soluble component to 72 +/- 13% (p = 0.041, n= 3, mean +/- STD).

Conclusion

CLIC5A interacts directly with active Ezrin, and this interaction is required for CLIC5A-dependent Rac1 activation. The release of CLIC5A together with Ezrin from the membrane fraction in response to PI(4,5)P2 hydrolysis suggests that CLIC5A localizes to the plasma membrane because of its direct interaction with Ezrin.

Funding

  • Government Support – Non-U.S.