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A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1
1Department of Biological Chemistry, UCLA School of Medicine 90024.
Abstract:
The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.
Insights
Researchers identified Rin1, a human protein that directly interacts with Ras proteins, particularly when Ras is bound to GTP. This interaction suggests Rin1 may function as a Ras effector or regulator in mammalian cells.
Area of Science:
- Molecular Biology
- Cell Signaling
- Yeast Genetics
Background:
- Human protein overexpression can disrupt the Ras signal transduction pathway in yeast.
- Functional interference occurs at the effector level, downstream of activating mutations.
Purpose of the Study:
- To identify and characterize human proteins that interact with Ras.
- To elucidate the mechanism of Ras-protein interactions in yeast and mammalian systems.
Main Methods:
- In vivo and in vitro experiments using yeast Saccharomyces cerevisiae and human H-Ras.
- Analysis of protein-protein interactions using Ras mutants (effector and dominant negative).
- Competition assays with known Ras effectors like Raf1.
Main Results:
- A human cDNA clone (99) encodes a protein, named Rin1, that directly binds yeast Ras2p and human H-Ras.
- Rin1-Ras interaction is enhanced by GTP-bound Ras and is specific to functional Ras proteins.
- Rin1 shares an interaction profile with known Ras effectors (Raf1, adenylyl cyclase) and competes with Raf1 for H-Ras binding.
- Rin1 localizes to the plasma membrane, similar to H-Ras.
Conclusions:
- Rin1 is a novel Ras-interacting protein identified through functional studies in yeast.
- Rin1 exhibits characteristics of a Ras effector or regulator in mammalian cells.
- Rin1's plasma membrane localization suggests a role in Ras-mediated signaling at the cell surface.