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Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases
S B Cantor1, T Urano, L A Feig
1Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Abstract:
Ral proteins constitute a distinct family of Ras-related GTPases. Although similar to Ras in amino acid sequence, Ral proteins are activated by a unique nucleotide exchange factor and inactivated by a distinct GTPase-activating protein. Unlike Ras, they fail to promote transformed foci when activated versions are expressed in cells. To identify downstream targets that might mediate a Ral-specific function, we used a Saccharomyces cerevisiae-based interaction assay to clone a novel cDNA that encodes a Ral-binding protein (RalBP1). RalBP1 binds specifically to the active GTP-bound form of RalA and not to a mutant Ral with a point mutation in its putative effector domain. In addition to a Ral-binding domain, RalBP1 also contains a Rho-GTPase-activating protein domain that interacts preferentially with Rho family member CDC42. Since CDC42 has been implicated in bud site selection in S. cerevisiae and filopodium formation in mammalian cells, Ral may function to modulate the actin cytoskeleton through its interactions with RalBP1.
Insights
Researchers identified RalBP1, a novel protein that binds to active Ral proteins. This discovery suggests Ral proteins may influence the actin cytoskeleton via RalBP1 and CDC42 interactions.
Area of Science:
- Molecular and Cellular Biology
- Ras GTPase signaling pathways
Background:
- Ral proteins are Ras-related GTPases with distinct activation and inactivation mechanisms.
- Unlike Ras, activated Ral proteins do not induce transformed foci in cells, suggesting unique downstream functions.
Purpose of the Study:
- To identify downstream targets mediating Ral protein-specific functions.
- To clone and characterize novel Ral-binding proteins.
Main Methods:
- Utilized a Saccharomyces cerevisiae-based interaction assay to screen for Ral-binding proteins.
- Cloned a novel cDNA encoding Ral-binding protein 1 (RalBP1).
- Performed binding assays to confirm specificity of RalBP1 for active RalA and its interaction with CDC42.
Main Results:
- Identified and cloned RalBP1, a novel cDNA encoding a Ral-binding protein.
- RalBP1 specifically binds to the active GTP-bound form of RalA.
- RalBP1 possesses a Rho-GTPase-activating protein domain that preferentially interacts with CDC42.
Conclusions:
- RalBP1 is a downstream target of Ral proteins.
- Ral proteins may modulate the actin cytoskeleton through RalBP1 and its interaction with CDC42.
- This interaction provides insight into Ral-specific cellular functions distinct from Ras.