Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.
It has recently been shown that Ras proteins interact directly with Raf serine/threonine kinases in vitro and in the yeast two-hybrid system, leading to speculation that Raf proteins function as effectors for Ras. Here it is demonstrated that the endogenous Raf-1 protein co-immunoprecipitates with Ras from mammalian cells when the non-neutralizing anti-Ras monoclonal antibody Y13-238 is used. The formation of a Ras-Raf complex is absolutely dependent on prior treatment of the cells with a stimulus that activates Ras: phorbol ester or anti-T cell receptor antibody in the case of human peripheral blood T lymphoblasts, or epidermal growth factor in the case of Rat-1 fibroblasts. Up to 3% of cellular Raf-1 can be found in association with Ras. The association is not competed by addition of exogenous GST-Raf to the cell lysates and is therefore unlikely to be due to Ras-Raf binding after cell lysis. Specific interaction of Ras and Raf therefore occurs in intact mammalian cells in response to stimuli that cause Ras to become GTP-bound.
Hallberg
B
B
Imperial Cancer Research Fund, London, United Kingdom.
Rayter
S I
SI
Downward
J
J
D000818:Animals; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000502:physiology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D051381:Rats; D013601:T-Lymphocytes / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D066246:ErbB Receptors; D019908:Proto-Oncogene Proteins c-raf; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)