Formation of the Ras Dimer Is Essential for Raf-1 Activation
| Autor: | Kaoru Inouye, Shin Mizutani, Hiroshi Koide, Yoshito Kaziro |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Liposome
Oncogene Activator (genetics) Chemistry Recombinant Fusion Proteins Cell Membrane HEK 293 cells Mutant Sf9 Cell Biology Biochemistry Molecular biology Cell Line Enzyme Activation Proto-Oncogene Proteins c-raf Enzyme activator Protein-fragment complementation assay Liposomes Mutation Escherichia coli ras Proteins Humans Dimerization Molecular Biology Glutathione Transferase |
| Zdroj: | Journal of Biological Chemistry. 275:3737-3740 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.275.6.3737 |
| Popis: | 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 Ras 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 Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation. |
| Databáze: | OpenAIRE |
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