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A c-Cbl yeast two hybrid screen reveals interactions with 14-3-3 isoforms and cytoskeletal components
H Robertson1, W Y Langdon, C B Thien
1Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Victoria, Australia.
Abstract:
The protein product of c-cbl proto-oncogene is known to interact with several proteins, including Grb2, Crk and PI3 kinase, and is thought to regulate signalling by many cell surface receptors. The precise function of c-Cbl in these pathways is not clear, although a genetic analysis in Caenorhabditis elegans suggests that c-Cbl is a negative regulator of the epidermal growth factor receptor. Here we describe a yeast two hybrid screen performed with c-Cbl in an attempt to further elucidate its role in signal transduction. The screen identified interactions involving c-Cbl and two 14-3-3 isoforms, cytokeratin 18, human unconventional myosin IC, and a recently identified SH3 domain containing protein, SH3 P17. We have used the yeast two hybrid assay to localise regions of c-Cbl required for its interaction with each of the proteins. Interaction with 14-3-3 is demonstrated in mammalian cell extracts.
Insights
The c-Cbl proto-oncogene protein regulates cell signaling pathways. Yeast two-hybrid screening identified new binding partners for c-Cbl, including 14-3-3 isoforms, aiding in understanding its role in signal transduction.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenes
Background:
- The c-Cbl proto-oncogene protein is implicated in regulating cell surface receptor signaling.
- Its precise function in signal transduction pathways remains unclear, despite suggestions of a negative regulatory role for the epidermal growth factor receptor.
Purpose of the Study:
- To elucidate the role of c-Cbl in signal transduction.
- To identify novel protein interactors of c-Cbl using a yeast two-hybrid system.
Main Methods:
- Yeast two-hybrid screening was employed to identify c-Cbl interacting proteins.
- Regions of c-Cbl critical for these interactions were mapped.
- Interactions were further validated in mammalian cell extracts.
Main Results:
- The screen identified interactions between c-Cbl and two 14-3-3 isoforms, cytokeratin 18, human unconventional myosin IC, and SH3 P17.
- Specific domains within c-Cbl were localized for interaction with these identified proteins.
- Interaction with 14-3-3 proteins was confirmed in mammalian cell extracts.
Conclusions:
- The study identified several novel interaction partners for c-Cbl, expanding the understanding of its role in signal transduction.
- The findings suggest a complex regulatory network involving c-Cbl and its newly identified binding proteins.
- Further investigation into these interactions can clarify c-Cbl's function in various cellular processes.