Back to Search
Start Over
GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 1993 Sep 15; Vol. 90 (18), pp. 8683-6. - Publication Year :
- 1993
-
Abstract
- Ras is involved in signal transduction of various factors for growth, differentiation, and oncogenesis. Recent studies have revealed several proteins that function upstream and downstream of the Ras signaling pathway. However, its immediate downstream target molecular has not yet been identified. In an effort to identify the Ras-associated downstream proteins, we added recombinant Ha-Ras in a GTP-bound form to cell-free lysates and used several antibodies against Ras to immunoprecipitate Ras complexes. We found that a serine/threonine kinase, Raf-1, was coimmunoprecipitated with Ha-Ras by two anti-Ras antibodies (LA069 and Y13-238), whereas a neutralizing antibody against Ras (Y13-259) could not precipitate Raf-1. The coimmunoprecipitation was observed with a complex of Ras and guanosine 5'-[gamma- thio]triphosphate but not with a complex of Ras and guanosine 5'-[beta-thio]diphosphate. The GTP-dependent association of Ha-Ras with Raf-1 was observed with lysates of various types of cultured cells, including NIH 3T3, pheochromocytoma (PC) 12, Ba/F3, and Jurkat T cells, and also with crude extracts from rat brain. Furthermore, Raf-1 was precipitated with a transforming Ha-Ras mutant ([Val12]Ras) and wild-type Ha-Ras but not with an effector-region mutant ([Leu35,ARg37]Ras) that lacks transforming activity. These results indicate that Ras.GTP physically associates with Raf either directly or through other component(s) and strongly suggest that Raf functions in close downstream proximity to Ras in mammalian cells.
- Subjects :
- 3T3 Cells
Animals
Brain metabolism
Cell Line
GTP-Binding Proteins isolation & purification
GTP-Binding Proteins metabolism
Guanosine 5'-O-(3-Thiotriphosphate) metabolism
Guanosine Diphosphate analogs & derivatives
Guanosine Diphosphate metabolism
Humans
Immunoblotting
Lymphocytes
Mice
PC12 Cells
Protein Binding
Protein Serine-Threonine Kinases metabolism
Proto-Oncogene Proteins isolation & purification
Proto-Oncogene Proteins c-raf
Proto-Oncogene Proteins p21(ras) isolation & purification
Rats
Rats, Wistar
Signal Transduction
Thionucleotides metabolism
Tumor Cells, Cultured
Guanosine Triphosphate metabolism
Proto-Oncogene Proteins metabolism
Proto-Oncogene Proteins p21(ras) metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0027-8424
- Volume :
- 90
- Issue :
- 18
- Database :
- MEDLINE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 8378348
- Full Text :
- https://doi.org/10.1073/pnas.90.18.8683