Receptor for activated C kinase1 (RACK1) binds with G beta1 and restricts focal adhesion kinase activation and restoration of endothelial barrier function

Abstract

We showed that sustained activation of focal adhesion kinase (FAK) is required for reversing the increase in endothelial permeability induced by inflammatory mediator thrombin. However, mechanism that sustain FAK activation is not known and therefore will help in identifying signaling cues responsible for restoring endothelial barrier function. Gβ1 and receptor for activated C kinase (RACK1) belong to large family of WD40 repeat proteins and act as scaffold proteins to orchestrate downstream signaling events. We therefore investigated the contribution of RACK1 and Gβ1 in regulating FAK activation and recovery of endothelial barrier function. We show that RACK1 is associated with Gβ1 in untreated cells. Thrombin induced a time‐dependent reversible dissociation of RACK1 from Gβ1, enabling it to interact with upstream FAK regulator Fyn. Inhibiting endogenous expression of Gβ1 had no effect on thrombin‐induced increase in endothelial permeability but markedly impaired endothelial barrier repair. Knockdown of Fyn in endothelial cells also prevented barrier recovery post‐thrombin challenge, linking Gβ1 to the regulation of Fyn function. In contrast, knock down of RACK1 increased basal phosphorylation of FAK and potentiated endothelial barrier recovery. Thus, these data demonstrate that RACK1 prevents the access of Gβ1 from forming a complex with Fyn, which is required for FAK activation and restoration of endothelial barrier function.Supported by NIH grants: HL71794 and HL84153