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Abstract: FR-PO908

Mechanism of CLIC5A Action in Podocytes

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 a major podocyte protein essential for assembly of the Ezrin/NHERF2/Podocalyxin complex that shapes the apical domain of foot processes. CLIC5A does not function as an ion channel, but it stimulates apical plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) generation by PI4P5 kinases (PI4P5Ks) through localized Rac1 activation. Docking on PI(4,5)P2 then changes the Ezrin conformation causing Ezrin to link Podocalyxin to actin. CLIC5A deletion in vivo abrogates podocyte Ezrin activation and heightens hypertension-induced glomerular injury.

Methods

We defined the relationship between CLIC5A, Rac1 and Ezrin by pull-down of Rac-GTP with Pak Binding Domain (PBD) beads, GST-CLIC5A pull-down, and Yeast-two-Hybrid (Y2H) assay, in which direct protein-protein interactions generate blue colonies by α-gal LacZ activity and growth on "DDO media" (Figure).

Results

CLIC5A overexpression in cultured podocytes and HEK293 cells increased Rac1-GTP levels 5-10 fold, and CLIC5A, Ezrin and PI4P5Kiα, PI5P4Kα, and PI4P5Kγ isoforms were all co-precipitated from cell lysates with GTP-Rac1 by PBD beads. Notably, Pak-PBD pulled the PI4P5Kα3 isoform from kidney cortex lysates of CLIC5A+/+ but not CLIC5A-/- mice. GST-CLIC5A pulled Rac1-GTP and -GDP from cell lysates, but failed to interact directly with purified, recombinant Rac1-GDP or -GTP. Domain-based Y2H screening and mapping showed that CLIC5A interacts directly with the ezrin C-terminus (lys516-leu586; Figure) and the βPix/ARHGEF7 interacting protein Shank2. The CLIC5A/Ezrin interaction required the active conformation of Ezrin, and siRNA knockdown of Ezrin reduced CLIC5A-induced Rac1 activation in cultured podocytes.

Conclusion

Thus, CLIC5A, Rac1-GTP, Ezrin and PI4P5K exist in a protein complex. In which CLIC5A interacts directly with Ezrin. CLIC5A-dependent Rac1 activation requires the direct interaction with Ezrin, and probably Shank2, leading to the formation of the CLIC5A/Ezrin/ Rac1-GTP/PI4P5Kα3 complex that generates PI(4,5)P2 clusters at the apical plasma membrane.

Funding

  • Private Foundation Support