Howe A K, Juliano R L
Department of Pharmacology and The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599-7365, USA.
J Biol Chem. 1998 Oct 16;273(42):27268-74. doi: 10.1074/jbc.273.42.27268.
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-1, 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 PKC inhibitors. The combination of PKC 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.
整合素介导的与细胞外基质的黏附激活了经典的丝裂原活化蛋白激酶级联反应，尽管确切机制尚未完全阐明。我们发现，整合素介导的丝裂原活化蛋白激酶上游调节因子Raf-1的激活分为两个阶段。Raf的早期高效激活需要Raf与Ras相互作用，但不受蛋白激酶C（PKC）抑制剂的影响，而较低水平的持续活性则不依赖于Raf与Ras的相互作用，但会被PKC抑制剂降低。PKC抑制和缺乏Ras结合的组合完全阻断了整合素介导的Raf活性。一种缺乏Ras结合能力的膜结合Raf突变体（Raf-R89L-CAAX）的活性也受到黏附的调节。Raf-R89L-CAAX在非黏附细胞中的活性较低，细胞黏附可迅速将其刺激至野生型水平，且其活性维持在接近最大水平的时间比野生型活性更长。细胞松弛素D消除了野生型和突变型Raf蛋白的激活，表明即使Raf定位于细胞膜，细胞骨架组织对于Raf的激活也是必需的。这些数据表明，整合素介导的Raf激活存在不同的初始阶段和持续阶段，分别需要Raf定位于细胞膜以及可能需要PKC活性，并且整合素介导的黏附可能调节负责Raf激活的细胞骨架相关因子。
