Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.
Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not 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 demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent 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 integrin activation of Erk-2 MAP and AKT kinases.
King
W G
WG
ARIAD Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, USA.
Mattaliano
M D
MD
Chan
T O
TO
Tsichlis
P N
PN
Brugge
J S
JS
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006160:Guanosine Triphosphate / Q000378:metabolism; D016023:Integrins / Q000502:physiology*; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D009025:Morpholines / Q000494:pharmacology; D051419:Paxillin; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011107:Polylysine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction / Q000502:physiology*; D000077191:Wortmannin; D018631:ras Proteins / Q000378:metabolism
D000730:Androstadienes; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015815:Cell Adhesion Molecules; D002867:Chromones; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006160:Guanosine Triphosphate; D016023:Integrins; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D009025:Morpholines; D051419:Paxillin; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D011107:Polylysine; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; D000077191:Wortmannin; D018631:ras Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one