Distinct mechanisms mediate the initial and sustained phases of integrin-mediated activation of the Raf/MEK/mitogen-activated protein kinase cascade
Distinct mechanisms mediate the initial and sustained phases of integrin-mediated activation of the Raf/MEK/mitogen-activated protein kinase cascade
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DOI：
10.1074/jbc.273.42.27268
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发表时间：
1998-10-16
影响因子：
4.8
通讯作者：
Juliano, RL
中科院分区：
文献类型：
--
作者：
Howe, AK;Juliano, RL
整合素介导的细胞外基质粘附激活经典的促分裂原活化蛋白激酶级联反应，尽管确切的机制还没有完全解决。我们表明，整联蛋白介导的有丝分裂原活化蛋白激酶上游调节因子Raf-l的激活分两个阶段发生。Raf的有效早期激活需要Raf-Ras相互作用，但不受蛋白激酶C（PKC）抑制剂的影响，而较低的，持续的活性水平是独立的Raf-Ras相互作用，但减少PEC抑制剂。PEC抑制和Ras结合缺乏的组合完全阻断了整合素介导的Raf活性。Ras结合缺陷的膜结合Raf突变体（Raf-R89 L-CAAX）的活性也受粘附调节。Raf-R89 L-CAAX活性在非贴壁细胞中较低，被细胞迅速刺激至野生型水平。粘附，并保持在接近最大水平的时间比野生型活动。野生型和突变体Raf蛋白的活化被细胞松弛素D消融，表明细胞骨架组织是Raf活化所必需的，即使是靶向膜时。这些数据表明，不同的初始和持续阶段的整合素介导的Raf激活，需要Raf膜定位和可能的PKC活性，分别，和整合素介导的粘附可能调节细胞因子相关的因子（S）负责Raf激活。
Integrin-mediated adhesion to the extracellular matrix activates the canonical mitogen-activated protein kinase cascade, although the exact mechanism is not fully resolved. We show that integrin-mediated activation of Raf-l, an upstream regulator of mitogen-activated protein kinase, occurs in two phases. Efficient early activation of Raf required Raf-Ras interaction but was not affected by protein kinase C (PKC) inhibitors, while a lower, sustained level of activity was independent of Raf-Ras interaction but was reduced by PEC inhibitors. The combination of PEC inhibition and lack of Ras binding completely blocked integrin-mediated Raf activity. The activity of a membrane-bound Raf mutant that is deficient in Ras binding (Raf-R89L-CAAX) was also regulated by adhesion. Raf-R89L-CAAX activity was low in nonadherent cells, was rapidly stimulated to wild-type levels by cell. adhesion, and remained at nearly maximal levels longer than wild-type activity. The activation of wild-type and mutant Raf proteins was ablated by cytochalasin D, demonstrating that cytoskeletal organization is required for activation of Raf, even when targeted to the membrane. These data suggest distinct initial and sustained phases of integrin-mediated Raf activation that require Raf membrane localization and possibly PKC activity, respectively, and that integrin-mediated adhesion may regulate a cytoskeleton-associated factor(s) responsible for Raf activation.