Mechanism of CLIC5A-Dependent Rac1 Activation and ERM Phosphorylation
Creators: Rahman, Md M
Publisher: name: University of Alberta Library
Year: 2024

<p>The chloride intracellular channel (CLIC) isoform 5A was discovered in a complex with ezrin and actin in placenta microvilli. CLIC5A is also greatly enriched in kidney glomeruli and is essential for assembly of the ezrin/NHERF2/podocalyxin complex at the apical domain of glomerular podocyte foot processes. CLIC proteins are soluble members of the GST family whose crystal structure is inconsistent with the theory that they are membrane-spanning chloride channels. CLIC5A stimulates Rac1 activity and ezrin, radixin, moesin (ERM) protein phosphorylation. The goals of this thesis are to better understand direct and functional relationships of CLIC5A with Rac1, ERM proteins and taperin in the organization of the cortical actin cytoskeleton of cells.<br>
Though CLIC5A expression increased Rac1-GTP levels, and CLIC5A was part of the Rac1-GTP complex, I found the CLIC5A interaction with Rac1 to be indirect. Unbiased yeast two-hybrid screening revealed that radixin and taperin bind CLIC5A directly. I observed that CLIC5A interacts directly with the carboxyterminal domain of all ERM proteins, but its affinity for ezrin is the highest.  Furthermore, the last 16 aa of ezrin are necessary for CLIC5A binding, and phosphorylation of ezrin at T567 markedly enhances its interaction with CLIC5A. <br>
In renal glomeruli, endogenous CLIC5A was predominantly soluble, and Staurosporine and phospholipase C activation reduced the small proportion of CLIC5A associated with the membrane fraction. In cultured cells, triple ezrin, radixin, and moesin silencing reduced CLIC5A localization to the apical cell periphery, suggesting that CLIC5A localizes to the cortical cytoskeleton by interacting with ERM proteins.<br><br>
The ezrin 432-586(T567D) mutant, which has a high affinity for CLIC5A, competitively inhibited CLIC5A-dependent phosphorylation of endogenous ERM proteins and Rac1 activation, but ezrin 432-570, which does not bind CLIC5A, was without effect. Knockdown of endogenous ezrin also reduced CLIC5A-dependent Rac1 activation, and CLIC5A expression enhanced Rho GDI/ezrin co-immunoprecipitation. These findings indicate that CLIC5A-stimulated Rac1 activation is ezrin dependent, and that CLIC5A enhances Rho GDI/ezrin binding promoting Rac1 activation.<br><br>
CLIC5A-stimulated Rac1-GTP activation increased PI4P5KIα and PI5P4KIα lipid kinase precipitation with the Rac1-GTP complex, and in kidneys in vivo, precipitation of the PI4P5Kα3 kinase isoform with Rac1-GTP required CLIC5A.  I therefore postulate that CLIC5A promotes Rac1 activation in ERM complexes, stimulating localized PI4,5P2 generation by PI4P5Kα3.<br><br>
Taperin. a protein phosphatase 1 (PP1) regulatory subunit, exists as isoform 1 (taperin 1, 1-711 aa) and as splice variants 2, 3 and 4, all starting at aa 307. I found that taperin isoforms 1 and 2 interact directly with CLIC5A and that residues 307-385 of taperin are sufficient for the interaction. The 45KGVVF49 motif of CLIC5A, conserved among all CLICs, is required for its interaction with taperin.  As expected, mutation of the 577KISF580 motif of taperin 1 (1-711 aa) or 2 (307-711 aa) to 577KASA580 abolished binding of the PP1 catalytic subunit (PP1c). In the presence of wild-type CLIC5A, immobilized GST-ezrin 432-586 captured taperin 1 and PP1c, but neither taperin nor PP1c were captured by GST-ezrin in the presence of mutant CLIC5A 45KGAVY49. GFP-taperin 1 localized predominantly to the nucleus and the cell periphery.  By contrast, GFP-taperin 2 localized exclusively to the nucleus. In the presence of wild-type CLIC5A, but not CLIC5A 45KGAVY49, there was a dramatic reduction in the taperin 1 nuclear localization, without effect on taperin 2, and RFP-CLIC5A co-localized with GFP-taperin 1 at the cell periphery. Interestingly, PP1cα and PP1cβ, but not PP1cγ, were captured by GST-CLIC5A even when taperin was silenced, and purified GST-CLIC5A precipitated all purified PP1c isoforms in vitro, independent of taperin. Taperin knockdown did not change CLIC5A localization at the cell periphery, but reduced ERM phosphorylation. Therefore, CLIC5A brings taperin 1/PP1c into the CLIC5A/ezrin complex, and it profoundly reduces the nuclear localization of taperin 1.<br>
To sum up, CLIC5A interacts directly with ERM proteins, and the interaction is enhanced by ERM phosphorylation. The ezrin/CLIC5A interaction enhances the sequestration of Rho GDI by ezrin to facilitate Rac1 activation. CLIC5A also brings taperin 1/PP1c into the ezrin complex and reduces the nuclear localization of taperin 1.    </p>