Role of p190RhoGAP in β2 Integrin Regulation of RhoA in Human Neutrophils

Abstract

We found that engagement of β2 integrins on human neutrophils induced activation of RhoA, as indicated by the increased ratio of GTP:GTP + GDP recovered on RhoA and translocation of RhoA to a membrane fraction. The clustering of β2 integrins also induced a time-dependent increase in GDP bound to RhoA, which correlated with β2 integrin-induced activation of p190RhoGAP. The activation of p190RhoGAP was completely blocked by [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] (PP1), a selective inhibitor of Src family tyrosine kinases. However, clustering of β2 integrins did not increase the basal tyrosine phosphorylation of p190RhoGAP, nor did it affect the amount of p120RasGAP bound to p190RhoGAP. Instead, the β2 integrin-induced activation of p190RhoGAP was accompanied by increased tyrosine phosphorylation of a p190RhoGAP-associated protein, p120RasGAP, and accumulation of both p120RasGAP and p190RhoGAP in a membrane fraction. PP1 blocked the β2 integrin-induced phosphorylation of p120RasGAP, as well as the translocation of p190RhoGAP and p120RasGAP, but it did not affect the accumulation of RhoA in the membrane fraction. In agreement with the mentioned findings, PP1 also increased the GTP:GTP + GDP ratio recovered on RhoA immunoprecipitated from β2 integrin-stimulated cells. Thus, in neutrophils, β2 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.

Down-regulation of Rac Activity during β2 Integrin-mediated Adhesion of Human Neutrophils

“…Several observations support our conclusion. First, we have previously observed that engagement of ␤ 2 integrins on PMNs increased the activity of p190RhoGAP (a GAP protein for RhoA and Rac) and its translocation to a crude membrane fraction (22). Second, other investigators have detected Rac-GAP activity in a membrane fraction of PMNs and suggested that its modulation could play a role in the regulation of the NADPH oxidase (40).…”

“…We pretreated the PMNs with the following agents: wortmanin or LY294002, both of which inhibit PI 3-kinase by distinct mechanisms and are known to block fMLP-induced generation of phosphatidylinositol 3,4,5-trisphosphate and activation of the respiratory burst in PMNs (37), or PP1, which is a potent and selective inhibitor of Src family tyrosine kinases (38). We have previously shown that PP1 (3 M) blocks basal and ␤ 2 integrin-induced overall tyrosine phosphorylation of proteins in PMNs (22). In our present study, pretreatment of PMNs with wortmanin or LY294002 significantly reduced, but did not totally block, adhesion-induced up-regulation of Cdc42 activity, and it partly reversed adhesion-elicited down-regulation of the activities of Rac1 and Rac2 (Fig.…”

“…We have recently shown that ligation of the ␤ 2 integrins on PMNs resulted in activation of Ras (21) and RhoA (22), but it is not yet known whether other Rho GTPases are also regulated in the course of this event. In the present study, we examined ␤ 2 integrin-dependent regulation of Rac and Cdc42 GTPases in adherent PMNs.…”

The neglected terminators: Rho family GAPs in neutrophils

“…31,32 Although the regulatory function could be demonstrated both with human proteins and with murine cells, contribution of the protein to integrin activation differs in human and murine neutrophils. 12,13 In addition to phosphorylation, GAP activity can be altered in numerous other ways. 33 Neutrophils are characterized by a short lifetime (see Hellebrekers et al, in this issue); hence, expression-level regulation is not what one would typically expect.…”

“…Rac/Rho Human (cell free) NADPH oxidase [6,9,11] Mouse No role in integrin-mediated neutrophil functions and in rheumatoid arthritis [12] Human β2 integrin activation [13] ARHGAP46/GMIP RhoA Mouse/human Azurophilic granule exocytosis. [14] ARHGAP15 Rac Mouse Migration, NADPH oxidase, phagocytosis, abdominal sepsis, retention in bone marrow [15,16] Human/cell culture Migration, actin polymerization [17] ARHGAP13/srGAP1 Cdc42 Mouse/human Neutrophil chemotaxis [18] BCR/ABR Rac/Cdc42 Mouse (double knockout) Acute inflammatory responses [19] BCR Rac/Cdc42 Human (cell free) NADPH oxidase [9] Mouse Septic shock NADPH oxidase [20] ARHGAP12 Rac Human/cell culture Actin polymerization [8] ARHGAP43/SH3BP1 Rac Human/cell culture Actin polymerization [8] ARHGAP21/ ARHGAP10…”