Clustering of β2-integrins on human neutrophils activates dual signaling pathways to Ptdlns 3-kinase

The β₂-integrins on leukocytes can serve as a signaling unit during cell adhesion and locomotion, and to further clarify this important property we investigated the possible mechanisms of β₂-integrin-induced activation of PtdIns 3-kinase. It has previously been demonstrated that clustering of β₂-integrins activates p21(ras) by a tyrosine kinase-dependent pathway, and here we show that active p21(ras) interacts with its downstream target, PtdIns 3-kinase. Engagement of β₂-integrins also activates the tyrosine kinases p58(c-fgr) and p59/61(hck) and causes them to associate with the p85 subunit of PtdIns 3-kinase. These findings suggest a mechanism whereby p58(c- fgr) and p59/61(hck) are directly involved in the activation of PtdIns 3- kinase. No coupling between p58(c-fgr) and p59/61(hck) could be detected; hence these kinases probably trigger independent but parallel signals to PtdIns 3-kinase. The effect of β₂-integrin clustering on PtdIns 3-kinase activity was monitored as the activation of protein kinase B (PKB). Stimulation of PKB by β₂-integrins was abolished by genistein and wortmannin but not by using methyl transferase inhibitors to abrogate the influence of p21(ras)-related proteins. Thus, even if PtdIns 3-kinase is not activated by p21(ras), it can maintain full enzyme activity due to the mentioned interaction with p58(c-fgr) or p59/61(hck). These tyrosine kinases apparently activate similar pathways that operate in parallel and therefore have the potential to substitute for each other in mediating adhesion and regulating cell locomotion. (C) 2000 Academic Press.