S338 phosphorylation of Raf-1 is independent of phosphatidylinositol 3-kinase and Pak3
S338 phosphorylation of Raf-1 is independent of phosphatidylinositol 3-kinase and Pak3
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DOI：
10.1128/mcb.21.7.2423-2434.2001
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发表时间：
2001-04-01
影响因子：
5.3
通讯作者：
Marais, R
中科院分区：
文献类型：
--
作者：
Chiloeches, A;Mason, CS;Marais, R
Raf-I丝氨酸/苏氨酸蛋白激酶需要在位置338（S338）处的丝氨酸的磷酸化来活化。Ras需要将Raf-I募集到质膜，这是S338磷酸化发生的地方。Pak 3可以通过Ras/磷脂酰肌醇3-激酶（P13-K）/-Cdc 42-依赖性途径直接磷酸化S338来刺激Raf-I活性的最近建议引起了很多关注。使用磷酸化特异性抗体S338，我们重新检查了这个模型。使用LY 294002和wortmannin，P13-K的抑制剂，我们发现生长因子介导的S338磷酸化仍然发生，即使当P13-K活性完全黑。虽然高浓度的LY 294002和渥曼青霉素确实抑制S338磷酸化，但它们也抑制Ras活化。此外，我们表明，Pak 3是不激活的条件下，S338磷酸化，但当Pak 3被强烈激活，通过与V12 Cdc 42共表达或突变，使其独立的Cdc 42，它刺激S338磷酸化。然而，这发生在胞质溶胶中并且不刺激Raf-I激酶活性。Pak 3不能激活Raf-1不是由于不能刺激341位酪氨酸的磷酸化，而是可能由于其不能将Raf-1募集到质膜。总之，我们的数据表明，生长因子刺激的Raf-I活性是独立的P13-K活性，并反对Pak 3是生长因子刺激的细胞中S338磷酸化的生理介质。
The Raf-l serine/threonine protein kinase requires phosphorylation of the serine at position 338 (S338) for activation. Ras is required to recruit Raf-l to the plasma membrane, which is where S338 phosphorylation occurs. The recent suggestion that Pak3 could stimulate Raf-l activity by directly phosphorylating S338 through a Ras/phosphatidylinositol 3-kinase (P13-K)/-Cdc42-dependent pathway has attracted much attention. Using a phospho-specific antibody to S338, we have reexamined this model. Using LY294002 and wortmannin, inhibitors of P13-K, we find that growth factor-mediated S338 phosphorylation still occurs, even when P13-K activity is completely blacked. Although high concentrations of LY294002 and wortmannin did suppress S338 phosphorylation, they also suppressed Ras activation. Additionally, we show that Pak3 is not activated under conditions where S338 is phosphorylated, but when Pak3 is strongly activated, by coexpression with V12Cdc42 or by mutations that make it independent of Cdc42, it did stimulate S338 phosphorylation. However, this occurred in the cytosol and did not stimulate Raf-l kinase activity. The inability of Pak3 to activate Raf-1 was not due to an inability to stimulate phosphorylation of the tyrosine at position 341 but may be due to its inability to recruit Raf-l to the plasma membrane. Taken together, our data show that growth factor-stimulated Raf-l activity is independent of P13-K activity and argue against Pak3 being a physiological mediator of S338 phosphorylation in growth factor-stimulated cells.