King W G, Mattaliano M D, Chan T O, Tsichlis P N, Brugge J S
ARIAD Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, USA.
Mol Cell Biol. 1997 Aug;17(8):4406-18. doi: 10.1128/MCB.17.8.4406.
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.
细胞与纤连蛋白的附着刺激了与整合素相关的磷脂酰肌醇（PI）3激酶与整合素相关的粘着斑激酶（FAK）之间依赖整合素的相互作用，以及Ras/丝裂原活化蛋白（MAP）激酶途径的激活。然而，尚不清楚这种PI 3激酶-FAK相互作用是否会增加3-磷酸化磷脂酰肌醇（3-PPI）的合成，以及活化的PI 3激酶在整合素信号传导中是否发挥作用（如果有）。我们在此证明了PI 3激酶产物PI 3,4-二磷酸[PI(3,4)P2]和PI(3,4,5)P3依赖整合素的积累，以及AKT激酶的激活，AKT激酶是一种丝氨酸/苏氨酸激酶，可通过PI(3,4)P2的结合而被刺激。PI 3激酶抑制剂渥曼青霉素和LY294002显著降低了整合素诱导的3-PPI积累和AKT激酶的激活，而对PI(4,5)P2水平或桩蛋白的酪氨酸磷酸化没有显著影响。这些抑制剂还减少了细胞在纤连蛋白上的粘附/铺展，但对细胞与聚赖氨酸的附着没有影响。有趣的是，渥曼青霉素和LY294002至少抑制了80%的整合素介导的Erk-2、Mek-1和Raf-1激活，但对Ras-GTP负载没有影响。为支持药理学结果，PI 3激酶的显性干扰p85亚基的过表达完全抑制了纤连蛋白对Erk-2和AKT激酶的激活。我们得出结论，整合素介导的与纤连蛋白的粘附导致PI 3激酶产物PI(3,4)P2和PI(3,4,5)P3的积累，以及Raf-1、Mek-1、Erk-2和AKT激酶的PI 3激酶依赖性激活，并且PI 3激酶可能在整合素激活Erk-2 MAP和AKT激酶过程中在Raf-1上游但在Ras下游发挥作用。
