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Identification of Ce-AF-6, a novel Caenorhabditis elegans protein, as a putative Ras effector
Y Watari1, K Kariya, M Shibatohge
1Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
Abstract:
Mammalian Ras proteins associate with multiple effectors, including Raf, Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase and AF-6. In the nematode Caenorhabditis elegans, LIN-45/Raf has been identified genetically as an effector of LET-60/Ras. To search for other effectors in C. elegans, we carried out a yeast two-hybrid screening for LET-60-associating proteins. The screening identified a novel protein, designated Ce-AF-6, which exhibited a strong structural homology with human AF-6, rat Afadin and Drosophila melanogaster Canoe and possessed both the Ras-associating (RA) domain and the PSD-95/DlgA/ZO-1 (PDZ) domain. Ce-AF-6 associated with human Ha-Ras in a GTP-dependent manner, with an efficiency comparable to that of human Raf-1 Ras-binding domain. When the effects of mutations of the Ras effector region residues were examined for associations with various effectors, Ce-AF-6 was found to possess a distinct and the most rigorous requirement for the effector region residues. These results strongly suggest that Ce-AF-6 is a putative effector of Ras that possesses a distinct recognition mechanism for association with Ras.
Insights
Researchers identified Ce-AF-6, a novel Ras effector in C. elegans. This protein shows distinct binding requirements, suggesting a unique mechanism for Ras signal transduction.
Area of Science:
- Molecular Biology
- Cell Signaling
- Genetics
Background:
- Ras proteins are key regulators of cell signaling, interacting with multiple downstream effectors.
- In C. elegans, LIN-45/Raf is a known effector of LET-60/Ras.
- The search for additional Ras effectors in C. elegans is crucial for understanding Ras pathway complexity.
Purpose of the Study:
- To identify novel effectors of LET-60/Ras in Caenorhabditis elegans.
- To characterize the interaction between LET-60/Ras and its newly identified binding partners.
Main Methods:
- Yeast two-hybrid screening was employed to identify proteins that associate with LET-60/Ras.
- Structural homology analysis was performed for the identified protein.
- GTP-dependent binding assays and mutation analysis of Ras effector region residues were conducted.
Main Results:
- A novel protein, Ce-AF-6, was identified as a LET-60/Ras-associating protein.
- Ce-AF-6 shares structural homology with mammalian AF-6 and contains Ras-associating (RA) and PDZ domains.
- Ce-AF-6 demonstrated GTP-dependent association with human Ha-Ras and exhibited unique requirements for Ras effector region residues.
Conclusions:
- Ce-AF-6 is a putative novel effector of Ras in C. elegans.
- Ce-AF-6 possesses a distinct recognition mechanism for association with Ras.
- This finding expands the known repertoire of Ras effectors and their interaction mechanisms.