Phosphatidylinositol 3-Kinase Is Required for Integrin-Stimulated AKT and Raf-1/Mitogen-Activated Protein Kinase Pathway Activation

Abstract

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)P 2 ] and PI(3,4,5)P 3 , as well as activation 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 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)P 2 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)P 2 and PI(3,4,5)P 3 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.Cellular adhesion to extracellular matrix proteins is mediated by a diverse class of ␣␤ heterodimeric receptors known as integrins. In addition to participating in cellular adhesion, these cell surface receptors transduce signals within the cell to regulate intracellular events, including cytoskeletal rearrangements and cell spreading, migration, differentiation, survival, and cell growth. These events are associated with activation of multiple intracellular signal transduction pathways, involving protein phosphorylation, alterations in calcium levels, changes in cytoplasmic pH, cytoskeletal rearrangements and inositol phospholipid turnover (for reviews, see references 17, 40, and 76). Although many signaling enzymes are activated following integrin engagement, little is known about the specific roles of these proteins in mediating integrin-induced changes in cell behavior.One enzyme that has been implicated in integrin-induced signal transduction is phosphatidylinositol 3-kinase (PI 3-kinase). This enzyme catalyzes the phosphorylation of inositol lipids at the D-3 position of the inositol ring, resulting in the formation of the 3-phosphorylated phosphoinositides (3-PPIs): phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol 3,4-bisph...

Integrins in regulation of tissue development and function

“…This is most evident for endothelial and epithelial cells but has also been reported for fibroblasts [39]. Integrin-mediated cell adhesion stimulates PI3K-mediated PKB/AKT activity [40,41] and, in doing so, suppresses caspase levels, promotes a high bcl-2/Bax ratio, and suppresses p53 activity [42,43]. Integrins that are not ligand bound can also trigger apoptosis of fully adherent cells by recruitment and activation of caspase-8 [44][45][46], suggesting that a given integrin expression profile renders a cell dependent on a specific ECM environment for its survival.…”

Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation

“…These studies strongly implicated PKB as a downstream effector of growth-factor-stimulated PI-3K activation in a variety of cell types. Subsequently PKB has been shown to be activated by a wide variety of stimuli including haemopoietic cytokines [IL (interleukin)-2, IL-3, IL-4, IL-5], chemokines [formylmethionylleucylphenylalanine, IL-8, RANTES (regulated on activation, normal T-cell expressed and secreted)], heat shock, hyperosmolarity, hypoxia, integrins, the T-cell antigen receptor and nerve growth factor [32][33][34][35][36][37][38][39][40].…”

Mechanisms of apoptosis by c-Myc

“…A second pathway linked to the executioner machinery in cancer cells is the PI3'K-AKT survival pathway. This pathway can be activated by a variety of stimuli, including integrin-dependent cell adhesion, ligation of the receptors for insulin-like growth factor-1 (IGF1) or IL-3, and activated Ras (Datta et al, 1997;del Peso et al, 1997;Franke et al, 1997;Frisch and Ruoslahti, 1997;Khwaja et al, 1997;King et al, 1997;Liu et al, 1998). Two substrates in the executioner machinery for the AKT kinase which have been identi®ed are Bad and caspase-9 (Datta et al, 1997;del Peso et al, 1997;Cardone et al, 1998).…”