Formation of the Ras dimer is essential for Raf-1 activation
Formation of the Ras dimer is essential for Raf-1 activation
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DOI：
10.1074/jbc.275.6.3737
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发表时间：
2000-02-11
影响因子：
4.8
通讯作者：
Kaziro, Y
中科院分区：
文献类型：
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作者：
Inouye, K;Mizutani, S;Kaziro, Y
尽管已充分确定Ras需要膜定位来激活其靶分子Raf - 1，但对这一要求的原因尚未完全理解。在本研究中，我们发现从Sf9细胞中纯化的修饰Ras在掺入脂质体时，能够在无细胞体系中激活Raf - 1。利用一种双功能交联剂和一种蛋白质片段互补分析，我们分别在脂质体和完整细胞中检测到Ras的二聚体形成。这些结果表明，Ras在脂质膜中的二聚化对于Raf - 1的激活至关重要。为支持这一点，我们发现当与谷胱甘肽S - 转移酶（GST）融合时，在大肠杆菌中表达的未加工Ras能够绕过对脂质体的需求。我们之前报道的一种Ras依赖性Raf - 1激活剂（Mizutani, S., Koide, H., and Kaziro, Y. (1998) Oncogene 16, 2781 - 2786）对于GST - Ras激活Raf - 1仍然是必需的。此外，在人胚肾（HEK）293细胞中，利用一部分回旋酶B或雌激素受体强制未修饰的致癌Ras突变体二聚化，也导致了Raf - 1的激活。从这些结果中，我们得出结论：膜定位使Ras能够形成二聚体，这对于Raf - 1的激活是必要的，尽管不是充分的。
Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood. in this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cell-free system, when incorporated into liposome. Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively. These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1. To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed has expressed in Escherichia coli could bypass the requirement for liposome. A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y. (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras. Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1. From these results, we conclude that membrane localization allows has to form a dimer, which is essential, although not sufficient, for Raf-1 activation.