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Raf-1 interferes with Ras and Rap1A effector functions in yeast
R Ruggieri1, S G Macdonald, M Callow
1Onyx Pharmaceuticals, Richmond, California.
Abstract:
Raf-1 is a serine/threonine kinase that acts downstream of Ras in mitogenic signal transduction pathways, but the mechanism by which Ras transmits signals to Raf-1 is not known. We have examined the interaction between Raf-1 and human H-ras in three different systems that utilize H-ras-induced phenotypes in Saccharomyces cerevisiae. In each system, the effects of H-ras depend on guanosine triphosphate and appear to be mediated through the H-ras effector binding region. H-ras effector function was blocked in each case by expression of the N-terminal regulatory domain of Raf-1. These inhibitory effects did not require the Raf-1 kinase domain. Raf-1 also blocked Rap1A effector function in S. cerevisiae. Raf-1, therefore, appears to interact with H-Ras and Rap1A in these in vivo systems with properties that suggest it is an immediate downstream effector.
Insights
This study reveals that Raf-1 kinase interacts with Ras proteins, acting as a direct downstream effector in cell signaling pathways. These findings clarify how Ras transmits signals, impacting our understanding of mitogenic signal transduction.
Area of Science:
- Cellular biology
- Molecular genetics
- Signal transduction pathways
Background:
- Raf-1 is a serine/threonine kinase functioning downstream of Ras in mitogenic signal transduction.
- The precise mechanism of Ras signal transmission to Raf-1 remains unclear.
- Ras proteins are key regulators of cellular signaling, influencing growth and differentiation.
Purpose of the Study:
- To investigate the interaction between Raf-1 and human H-Ras.
- To elucidate the mechanism by which Ras transmits signals to Raf-1.
- To determine if Raf-1 acts as an immediate downstream effector of Ras.
Main Methods:
- Utilized three Saccharomyces cerevisiae systems exhibiting H-Ras-induced phenotypes.
- Examined the effects of H-Ras in a guanosine triphosphate-dependent manner.
- Assessed the impact of the Raf-1 N-terminal regulatory domain on H-Ras and Rap1A effector function.
Main Results:
- H-Ras effector function was mediated through its effector binding region and dependent on guanosine triphosphate.
- Expression of the Raf-1 N-terminal regulatory domain inhibited H-Ras effector function.
- The inhibitory effects of Raf-1 did not necessitate its kinase domain and also blocked Rap1A effector function.
Conclusions:
- Raf-1 interacts with H-Ras and Rap1A in vivo.
- Raf-1 functions as an immediate downstream effector of Ras and Rap1A.
- These findings provide mechanistic insights into Ras-mediated signaling pathways.