PHOSPHATIDYLINOSITOL 3-KINASE IS REQUIRED FOR INTEGLRIN-STIMULATED AKT AND RAF-1 MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY ACTIVATION/

Cell attachment to fibronectin stimulates the integrin-dependent inter action of p85-associated phosphatidylinositol (PI) 3-kinase with integ rin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway, However, it is no t known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstr ate here the integrin dependent accumulation of the PI 3-kinase produc ts, PI 3,4-bisphosphate [PI(3,4)P-2] and PI(3,4,5)P-3, as well as acti vation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P-2. The PI 3-kinase inhibitors wortmannin and L Y294002 significantly decreased the integrin-induced accumulation of t he 3-PPIs and activation of AKT kinase, without having significant eff ects on the levels of PI(4,S)P-2 or tyrosine phosphorylation of paxill in. These inhibitors also reduced cell adhesion/spreading onto fibrone ctin but had no effect on attachment to polylysine, Interestingly, int egrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loa ding, was inhibited at least 80% by wortmannin and LY294002, In suppor t of the pharmacologic results, fibronectin activation of Erk-2 and AK T kinases was completely inhibited by overexpression of a dominant int erfering p85 subunit of PI 3-kinase, We conclude that integrin-mediate d adhesion to fibronectin results in the accumulation of the PI 3-kina se products PI(3,4)P-2 and PI(3,4,5)P-3 as well as the PI 3-kinase-dep endent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in i ntegrin activation of Erk-2 MAP and AKT kinases.