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RalGDS-like factor (Rlf) is a novel Ras and Rap 1A-associating protein
R M Wolthuis1, B Bauer, L J van 't Veer
1Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
Abstract:
The small GTPase Rap 1A is a close relative of Ras that, when overexpressed, is able to revert oncogenic transformation induced by active Ras. We screened a mouse embryonic cDNA library using the yeast two-hybrid system and isolated the cDNA of a novel Rap 1A-interacting protein. The open reading frame encodes for an 84 kDa protein with a Cdc25-homology domain which shares approximately 30% identity with Ral guanine nucleotide dissociation stimulator (RalGDS) and RalGDS-like (Rg1). The C-terminal region reveals a striking conservation of sequences with the Ras-binding domain of RalGDS. We designated this protein Rlf, for RalGDS-like factor. In the yeast system, Rlf interacts with Rap 1A, H-Ras and R-Ras, but not with Rac and Rho. In addition, we found that Rlf interacts with Rap 1Aval12 but not with Rap 1AAsn17. In vitro binding studies revealed that a C-terminally located 91 amino acid region of Rlf is sufficient for direct association with the GTP-bound form of Ras and Rap 1A. The observed dissociation constants are 0.6 microM and 0.4 microM, respectively. No significant association with Ras-GDP or Rap 1A-GDP could be detected. These binding characteristics indicate that Rlf is a putative effector for Ras and Rap 1A.
Insights
Researchers identified a novel Rap 1A-interacting protein, Rlf, which acts as a Ral guanine nucleotide dissociation stimulator-like factor. Rlf directly binds to the GTP-bound forms of Ras and Rap 1A, suggesting it functions as an effector for these small GTPases.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- The small GTPase Rap 1A, related to Ras, can reverse Ras-induced oncogenic transformation.
- Understanding Rap 1A and Ras signaling pathways is crucial for cancer research.
Purpose of the Study:
- To identify novel proteins that interact with Rap 1A.
- To characterize the function and binding properties of a newly discovered Rap 1A-interacting protein.
Main Methods:
- Yeast two-hybrid screening of a mouse embryonic cDNA library.
- In vitro binding assays to determine protein-protein interactions and binding affinities.
Main Results:
- A novel Rap 1A-interacting protein, designated Rlf (RalGDS-like factor), was identified.
- Rlf shares homology with Ral guanine nucleotide dissociation stimulator (RalGDS) and contains a Cdc25-homology domain.
- Rlf specifically binds to the GTP-bound forms of Ras and Rap 1A with high affinity, but not their GDP-bound forms.
Conclusions:
- Rlf is a putative effector for Ras and Rap 1A.
- The discovery of Rlf provides new insights into the regulatory mechanisms of Ras and Rap 1A signaling pathways.