Chaudhary A, King W G, Mattaliano M D, Frost J A, Diaz B, Morrison D K, Cobb M H, Marshall M S, Brugge J S
Department of Cell Biology, Harvard Medical School, Boston, 02115, USA.
Curr Biol. 2000 May 4;10(9):551-4. doi: 10.1016/s0960-9822(00)00475-9.
We have previously shown that inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and that Raf is the critical target of PI 3-kinase regulation [1]. To investigate how PI 3-kinase regulates Raf, we examined sites on Raf1 required for regulation by PI 3-kinase and explored the mechanisms involved in this regulation. Serine 338 (Ser338), which was critical for fibronectin stimulation of Raf1, was phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) was independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, was also dependent on PI 3-kinase activity and expression of a kinase-inactive Pak-1 mutant blocked phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins.
我们之前已经表明，抑制磷脂酰肌醇（PI）3激酶会严重减弱整合素/纤连蛋白受体结合后细胞外信号调节激酶（Erk）的激活，并且Raf是PI 3激酶调节的关键靶点[1]。为了研究PI 3激酶如何调节Raf，我们检查了PI 3激酶调节Raf1所需的位点，并探索了这种调节所涉及的机制。丝氨酸338（Ser338）对纤连蛋白刺激Raf1至关重要，在纤连蛋白受体结合后以PI 3激酶依赖的方式被磷酸化。此外，含有磷酸模拟突变（S338D）的Raf1突变体的纤连蛋白激活不依赖于PI 3激酶。此外，整合素诱导的丝氨酸/苏氨酸激酶Pak-1的激活也依赖于PI 3激酶活性，并且激酶失活的Pak-1突变体的表达阻断了Raf1 Ser338的磷酸化，已证明Pak-1可使Raf1 Ser338磷酸化。这些结果表明，PI 3激酶通过丝氨酸/苏氨酸激酶Pak调节Raf1 Ser338的磷酸化。因此，通过PI 3激酶和Pak对Raf1 Ser338的磷酸化提供了一个共刺激信号，该信号与Ras一起导致整合素对Raf1激酶活性的强烈激活。
