Rac1 Suppression by the Focal Adhesion Protein GIT2 and Podocyte Protection

Background: Podocytes have an intricate structure featured by numerous actin-based projections called foot processes. Rho-family of small GTPases, including Ras-related C3 botulinum toxin substrate 1 (Rac1), play important roles in actin cytoskeletal remodeling required for cell morphology and adhesion. We previously showed that Rac1 activation in podocytes causes foot process effacement and proteinuria, but the upstream and spatiotemporal regulatory mechanism directing Rac1 is largely unknown. Recently we identified the focal adhesion protein, GIT ArfGAP2 (GIT2) as one of the Rac1 interactors in human podocytes by proximity-dependent biotin identification (BioID) and proteomics. Methods: Systemic and podocyte-specific GIT2 knockout mice were generated and assessed for kidney phenotypes. Human podocytes with GIT2 knockdown and overexpression were established using lentiviral transduction and characterized. Results: GIT2 was enriched in glomeruli including podocytes in the mouse kidney. Gene deletion of Git2 in podocytes caused exacerbated proteinuria and foot process effacement when subjected to the minimal change disease model and the salt-sensitive hypertension model, which were improved by pharmacological inhibition of Rac1. In cultured podocytes, GIT2 knockdown resulted in Rac1-dependent cell spreading with marked lamellipodial protrusions, accelerated focal adhesion disassembly, and a shorter focal adhesion lifetime. In GIT2 knockdown podocytes, tyrosine phosphorylation of the focal adhesion protein, p130 Crk-associated substrate (Cas), was significantly increased, accompanied by impaired localization of the tyrosine phosphatase, PTP1B, to focal adhesions. These phenotypes observed in GIT2 knockdown podocytes were reversed by GIT2 overexpression. Conclusions: The results indicate that GIT2 facilitates translocation of PTP1B to focal adhesions where it dephosphorylates p130Cas, thereby suppressing local Rac1 activity and protecting against podocyte injury and proteinuria.