ACTIVATION OF PHOSPHOINOSITIDE 3-KINASE IS REQUIRED FOR PDGF-STIMULATED MEMBRANE RUFFLING

Background: There is substantial evidence that phosphoinositide 3-kina se (PI 3-kinase) is a critical component of signalling pathways used b y the cell-surface receptors for a variety of mammalian growth factors and other hormones. The physiological product of this enzyme is a hig hly polar membrane lipid called phosphatidylinositol (3,4,5)-trisphosp hate This lipid has been postulated to act as a second-messenger in ce lls but its putative targets are still unknown. Results: A particular rearrangement of actin filaments, which results in membrane ruffling, is elicited by the activation of PDGF beta-receptors expressed in cult ured porcine aortic endothelial cells. We have found that this consequ ence of PDGF beta-receptor activation is inhibited by three independen t manipulations of PI 3-kinase activity: firstly, by the deletion of t yrosine residues in the PDGF beta-receptor to which PI 3-kinase binds; secondly, by the overexpression of a mutant 85 kD PI 3-kinase regulat ory subunit to which the catalytic kinase subunit cannot bind; and thi rdly, by the addition of the fungal metabolite wortmannin, which is a potent inhibitor of the catalytic activity of PI 3-kinase. Conclusions : These results argue strongly that phosphatidylinositol (3,4,5)-trisp hosphate synthesis is required for growth-factor-stimulated membrane r uffling in porcine aortic endothelial cells, and suggest that synthesi s of this lipid may be part of a signalling pathway leading to direct or indirect activation of the small GTP-binding protein Rac.