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Quantitative analysis of mutually competitive binding of human Raf-1 and yeast adenylyl cyclase to Ras proteins
1Department of Physiology II, Kobe University School of Medicine, Japan.
Abstract:
Ras proteins appear to have two distinct downstream effectors, adenylyl cyclase in Saccharomyces cerevisiae and a product of raf-1 protooncogene in higher organisms. We found that in vitro activation of adenylyl cyclase by yeast Ras2 and human H-Ras proteins is subject to competitive inhibition by its leucine-rich repeats domain and by the N-terminal regulatory domain of human Raf-1 protein. Kinetic analyses of the inhibition patterns enabled us to determine exact dissociation constants (Kd) of the two polypeptides for Ras2 and H-Ras. The leucine-rich repeats domain bound to the posttranslationally modified Ras2 with the Kd of approximately 13 nM, which was close to the value (7 nM) of the whole adenylyl cyclase. The Kd of Raf-1 for the modified H-Ras, 3.5 nM, was significantly lower than that for Ras2, 24 nM, whereas adenylyl cyclase bound preferentially to Ras2. Similar inhibition was also observed in vivo by suppression of RAS2Val-19-dependent heat shock sensitivity and of Ras-dependent cAMP response to glucose upon overexpression of Raf-1 in yeast. These results indicate that the leucine-rich repeats domain contains the Ras protein-binding site and that Raf-1 and adenylyl cyclase, sharing no structural homology with each other, bind to a similar, if not identical, region of Ras with comparable affinities.
Insights
Ras proteins interact with similar binding sites on their downstream effectors, adenylyl cyclase and Raf-1 protein. This interaction is crucial for cellular signaling pathways in yeast and higher organisms.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Ras proteins are key regulators of cellular processes.
- Ras proteins have distinct downstream effectors, including adenylyl cyclase and Raf-1.
- Understanding Ras effector interactions is vital for deciphering cellular signaling.
Purpose of the Study:
- To investigate the binding interaction between Ras proteins and their effectors, adenylyl cyclase and Raf-1.
- To determine the specific Ras-binding site on these effectors.
- To compare the binding affinities of Ras proteins to adenylyl cyclase and Raf-1.
Main Methods:
- In vitro kinetic analyses of enzyme activation and inhibition.
- Determination of dissociation constants (Kd) for Ras-effector interactions.
- In vivo studies in Saccharomyces cerevisiae to assess functional consequences of Ras-Raf-1 interactions.
Main Results:
- The leucine-rich repeats domain of adenylyl cyclase and the N-terminal regulatory domain of Raf-1 protein competitively inhibit Ras activation of adenylyl cyclase.
- Dissociation constants (Kd) revealed specific binding affinities: Raf-1 binds H-Ras (3.5 nM) more strongly than Ras2 (24 nM), while adenylyl cyclase preferentially binds Ras2 (Kd ~13 nM).
- Overexpression of Raf-1 in yeast suppressed Ras2-dependent heat shock sensitivity and glucose-induced cAMP response, confirming in vivo interaction.
Conclusions:
- The leucine-rich repeats domain of adenylyl cyclase contains the Ras protein-binding site.
- Raf-1 and adenylyl cyclase bind to a similar, potentially identical, region on Ras proteins.
- Despite lacking structural homology, these distinct effectors interact with Ras via comparable affinities, highlighting a conserved binding mechanism.