A Direct CLIC5A/Ezrin Interaction Enables CLIC5A‐Dependent Rac1 Activation

Introduction The CLIC (Chloride Intracellular Channel) protein CLIC5A is a component of the ezrin/podocalyxin/actin complex in kidney podocytes, where it stabilizes actin‐based projections (foot processes). Similarly, in inner ear hair cells CLIC5A in the radixin complex stabilizes sensory stereocilia, also actin‐based projections. ERM ( e zrin, r adixin, m oesin) proteins help organize the cortical actin cytoskeleton, linking membrane‐spanning proteins to actin. We have reported that CLIC5A stimulates Rac1‐GTP‐dependent phosphatidylinositol‐4,5‐bisphosphate [PI(4,5)P2] generation, causing ezrin activation and phosphorylation, thus coupling podocalyxin, via ezrin, to the cytoskeleton ( J. Cell Sci. 127:5164, 2014 & Kidney Int. 89:833, 2016 ). Hypothesis Direct interaction(s) of CLIC5A with Rac1 and/or ezrin enable CLIC5A‐dependent Rac1 activation. Methods Rac1 activity was determined by Rac1‐GTP specific PAK‐PBD pull‐down from CLIC5A or vector transfected cells and by Rac1‐GTP G‐LISA ® (Cytoskeleton Inc). Association of CLIC5A with Rac1 was determined by GST‐CLIC5A pull‐down of exogenously expressed wild‐type GFP‐Rac1(WT), constitutively active GFP‐Rac1 (Q61L) and dominant negative GFP‐Rac1 (D17N) from COS7 cell lysates and by in vitro GST‐CLIC5A pull‐down of GTPγS‐ or GDP‐loaded recombinant His‐Rac1. Direct protein‐protein interactions were evaluated by Yeast two‐Hybrid (Y2H) screening of a mouse kidney protein domain library, and Y2H mapping of interacting domains in the related ERM protein ezrin, using CLIC5A as the bait (Fig 1A). The CLIC5A/ezrin association was studied by co‐immunoprecipitation and GST‐CLIC5A pull‐down. Results Exogenous CLIC5A expression activated Rac1 and CLIC5A was co‐precipitated with Rac1‐GTP and ezrin by PAK‐PBD affinity beads. GST‐CLIC5A pulled GFP‐Rac1 WT, ‐Q61L, and ‐D17N equally from cell lysates, but recombinant GTPγS‐ or GDP‐loaded His‐Rac1 did not directly bind GST‐CLIC5A, suggesting that the CLIC5A/Rac1 interaction is indirect. The Y2H screen revealed that CLIC5A interacts directly with the C‐terminal domain of radixin, and Y2H mapping (Fig. 1A) showed that CLIC5A similarly interacts directly with the ezrin C‐terminal domain (432‐586). Direct interactions with full‐length ezrin (1‐586) and the C‐terminally deleted ezrin (432‐570) domain were not detected. Furthermore, ezrin (432‐586), but not full‐length ezrin (1‐586) or ezrin (432‐570) was pulled down by GST‐CLIC5A and co‐immunoprecipitated with CLIC5A. Finally, siRNA silencing of endogenous ezrin significantly reduced CLIC5A‐stimulated Rac1 activation (Fig. 1B). Conclusion Although CLIC5A activates Rac1 and is part of the Rac1‐GTP complex, the CLIC5A/Rac1 interaction appears to be indirect. CLIC5A interacts directly with ezrin (and radixin), and Rac1 activation by CLIC5A is ezrin‐dependent. The finding that a direct interaction of CLIC5A with full‐length ezrin was not detected suggests that only the active, open ezrin conformation, freeing the C‐terminus, has a high affinity for CLIC5A. The data suggest that the direct CLIC5A/ezrin interaction may enhance localized Rac1 activation and thus Rac1‐GTP‐dependent PI(4,5)P2 generation.