Barberis L, Wary K K, Fiucci G, Liu F, Hirsch E, Brancaccio M, Altruda F, Tarone G, Giancotti F G
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
J Biol Chem. 2000 Nov 24;275(47):36532-40. doi: 10.1074/jbc.M002487200.
It has been proposed that integrins activate ERK through the adaptor protein Shc independently of focal adhesion kinase (FAK) or through FAK acting on multiple target effectors, including Shc. We show that disruption of the actin cytoskeleton by cytochalasin D causes a complete inhibition of FAK but does not inhibit Shc signaling and activation of ERK. We have then generated primary fibroblasts carrying a targeted deletion of the segment of beta(1) subunit cytoplasmic domain required for activation of FAK. Analysis of these cells indicates that FAK is not necessary for efficient tyrosine phosphorylation of Shc, association of Shc with Grb2, and activation of ERK in response to matrix adhesion. In addition, integrin-mediated activation of FAK does not appear to be required for signaling to ERK following growth factor stimulation. To examine if FAK could contribute to the activation of ERK in a cell type-specific manner through the Rap1/B-Raf pathway, we have used Swiss-3T3 cells, which in contrast to primary fibroblasts express B-Raf. Dominant negative studies indicate that Shc mediates the early phase and peak, whereas FAK, p130(CAS), Crk, and Rap1 contribute to the late phase of integrin-dependent activation of ERK in these cells. In addition, introduction of B-Raf enhances and sustains integrin-mediated activation of ERK in wild-type primary fibroblasts but not in those carrying the targeted deletion of the beta(1) cytoplasmic domain. Thus, the Shc and FAK pathways are activated independently and function in a parallel fashion. Although not necessary for signaling to ERK in primary fibroblasts, FAK may enhance and prolong integrin-mediated activation of ERK through p130(CAS), Crk, and Rap1 in cells expressing B-Raf.
有人提出，整合素可通过衔接蛋白Shc独立于粘着斑激酶（FAK）激活细胞外信号调节激酶（ERK），或者通过FAK作用于包括Shc在内的多个靶效应器来激活ERK。我们发现，细胞松弛素D破坏肌动蛋白细胞骨架会导致FAK完全抑制，但不会抑制Shc信号传导和ERK激活。然后，我们生成了携带FAK激活所需的β(1)亚基细胞质结构域片段靶向缺失的原代成纤维细胞。对这些细胞的分析表明，FAK对于Shc的有效酪氨酸磷酸化、Shc与Grb2的结合以及响应基质粘附的ERK激活并非必需。此外，生长因子刺激后向ERK发出信号似乎也不需要整合素介导的FAK激活。为了研究FAK是否可以通过Rap1/B-Raf途径以细胞类型特异性方式促进ERK激活，我们使用了瑞士3T3细胞，与原代成纤维细胞不同，它们表达B-Raf。显性负性研究表明，Shc介导早期阶段和峰值，而FAK、p130(CAS)、Crk和Rap1在这些细胞中促成整合素依赖性ERK激活的后期阶段。此外，引入B-Raf可增强并维持野生型原代成纤维细胞中整合素介导的ERK激活，但在携带β(1)细胞质结构域靶向缺失的细胞中则不然。因此，Shc和FAK途径独立激活并以平行方式发挥作用。虽然FAK对于原代成纤维细胞向ERK发出信号并非必需，但在表达B-Raf的细胞中，FAK可能通过p130(CAS)、Crk和Rap1增强并延长整合素介导的ERK激活。
