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

Abstract

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 (ezrin, radixin, moesin) 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.