Formation of the Ras dimer is essential for Raf-1 activation

被引:124
作者
Inouye, K [1 ]
Mizutani, S [1 ]
Koide, H [1 ]
Kaziro, Y [1 ]
机构
[1] Tokyo Inst Technol, Fac Biosci & Biotechnol, Midori Ku, Yokohama, Kanagawa 2268501, Japan
关键词
D O I
10.1074/jbc.275.6.3737
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:3737 / 3740
页数:4
相关论文
共 27 条