Role of p190RhoGAP in beta(2) integrin regulation of RhoA in human neutrophils

We found that engagement of beta (2) integrins on human neutrophils induced activation of RhoA, as indicated by the increased ratio of GTP:GTP + GDP r ecovered on RhoA and translocation of RhoA to a membrane fraction. The clus tering of beta (2) integrins also induced a time-dependent increase in GDP bound to RhoA, which correlated with beta (2) integrin-induced activation o f p190RhoGAP. The activation of p190RhoGAP was completely blocked by [4-ami no-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] (PP1), a select ive inhibitor of Src family tyrosine kinases. However, clustering of beta ( 2) integrins did not increase the basal tyrosine phosphorylation of p190Rho GAP, nor did it affect the amount of p120RasGAP bound to p190RhoGAP. Instea d, the beta (2) integrin-induced activation of p190RhoGAP was accompanied b y increased tyrosine phosphorylation of a p190RhoGAP-associated protein, p1 20RasGAP, and accumulation of both p120RasGAP and p190RhoGAP in a membrane fraction. PP1 blocked the beta (2) integrin-induced phosphorylation of p120 RasGAP, as well as the translocation of p190RhoGAP and p120RasGAP, but it d id not affect the accumulation of RhoA in the membrane fraction. In agreeme nt with the mentioned findings, PP1 also increased the GTP:GTP + GDP ratio recovered on RhoA immunoprecipitated from beta (2) integrin-stimulated cell s. Thus, in neutrophils, beta (2) integrin-induced activation of p190RhoGAP requires a signal from a Src family tyrosine kinase, but it does not occur via the signaling pathway responsible for activation of RhoA.