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
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1Graduate Program in Biochemistry and Molecular Biology, Rutgers University, Piscataway, New Jersey 08854.
Abstract:
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.
Insights
Ras-15A protein inhibits normal Ras protein function in mammalian cells by sequestering GDP-GTP exchange factors. This mechanism differs from Ras-17N, which uses reversible sequestration.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- Ras proteins are key regulators of cell growth and proliferation.
- Oncogenic Ras mutations are common in human cancers.
- Understanding Ras regulation is crucial for developing cancer therapies.
Purpose of the Study:
- To investigate the mechanism by which Ras-15A inhibits endogenous Ras protein function in mammalian cells.
- To compare the inhibitory mechanism of Ras-15A with that of Ras-17N.
- To determine if Ras-15A interferes with upstream activation of Ras proteins.
Main Methods:
- Expression of Ras-15A in mammalian cells.
- Assessing cell growth and transcriptional activation of a Ras-responsive promoter.
- Investigating physical interactions between Ras proteins and CDC25 exchange protein.
- Analyzing the effect of guanine nucleotides (GDP/GTP) on protein complex stability.
Main Results:
- Ras-15A potently inhibits endogenous Ras function in normal mammalian cells but not in cells with oncogenic Ras.
- Ras-15A blocks Ras-mediated growth and transcriptional activation by interfering with upstream signaling.
- Ras-15A forms stable, non-productive complexes with CDC25, sequestering GDP-GTP exchange factors irreversibly.
- Ras-17N also binds CDC25 but is released by GDP/GTP, suggesting a reversible sequestration mechanism.
Conclusions:
- Ras-15A inhibits normal Ras protein function by irreversibly sequestering GDP-GTP exchange factors, blocking Ras activation.
- The mechanism of Ras-15A differs from Ras-17N, which uses reversible sequestration.
- These findings provide insights into Ras signaling regulation and potential therapeutic strategies targeting Ras-driven cancers.