A Direct CLIC5A/Ezrin Interaction Enables CLIC5A‐Dependent Rac1 Activation
Bibliographic record
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 ( 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.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.004 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".