Ras-15A protein shares highly similar dominant-negative biological properties with Ras-17N and forms a stable, guanine-nucleotide resistant complex with CDC25 exchange factor.
We show that expression of Ras-15A, previously shown to be a dominant-negative mutant in yeast, is a potent inhibitor of endogenous Ras protein function in mammalian cells. Expression of Ras-15A did not inhibit the growth of cells containing an oncogenic ras gene nor did it interfere with the ability of transiently expressed oncogenic ras or raf genes to activate transcription from a Ras-responsive ets1/AP-1 promoter. In contrast, expression of Ras-15A completely blocked growth of normal cells and activation of the ets1/AP-1 promoter by transiently overexpressed SOS1 and normal Ras proteins. These results suggest that Ras-15A, like Ras-17N, blocks endogenous Ras function by interfering with upstream activation of Ras proteins rather than downstream effects. To test whether Ras-15A and Ras-17N interfere with Ras function by blocking GDP-GTP exchange proteins, we examined their physical interaction with the CDC25 exchange protein. All three proteins formed stable complexes with CDC25 in the absence of guanine-nucleotides, but only Ras-15A was not released from CDC25 by physiological concentrations of GDP or GTP. These results establish that Ras-15A blocks the activation of normal Ras proteins by sequestering GDP-GTP exchange factors into non-productive complexes. In contrast, it would appear that the similar biological properties of Ras-17N are mediated by a reversible, competitive sequestration of exchange factors.
Chen
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Graduate Program in Biochemistry and Molecular Biology, Rutgers University, Piscataway, New Jersey 08854.
Huff
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SY
Lai
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CC
Der
C J
CJ
Powers
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D016475:3T3 Cells; D000818:Animals; D002461:Cell Line, Transformed; D011905:Genes, ras; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1
D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D010749:Phosphoprotein Phosphatases; D016283:Proto-Oncogene Proteins p21(ras)