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Published on: August 4, 2019
Identification of a novel binding partners for tumor suppressor PTEN by a yeast two-hybrid approach
Olena Gorbenko1, Vitaliy Kuznetsov, Olexandr Kukharenko
1Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine.
Aim:
To identify novel PTEN-binding partners.
Methods:
The technique of yeast two-hybrid screening was used in this study. A panel of bait constructs was created, containing the C-terminal domain of PTEN, full length PTEN, activated and phosphatase-dead mutants. The expression of LexA-fused baits, their nuclear localization and autoactivation potential were tested according to the standard protocol of Duplex A system. CDNA libraries from Colon Cancer, HeLa and Mouse Embryo were screened with two selected bait constructs. Isolated positive clones were further analysed by mating assay and identified by automated DNA sequencing and database searching.
Results:
Extensive screening of cDNA libraries with the full length and the C-terminal domain of PTEN led to the identification of 43 positive clones, which were confirmed in mating assay. Sequence analysis indicated that two clones encode AEBP1 (Adipocyte Enhancer Binding Protein 1).
Conclusion:
Our data indicate that the interaction between PTEN and AEBP1 is mediated by their C-terminal and N-terminal domains, respectively. The functional importance of PTEN-AEBP1 interaction is currently under investigation.
Insights
Researchers identified Adipocyte Enhancer Binding Protein 1 (AEBP1) as a novel PTEN-binding partner using yeast two-hybrid screening. This interaction is mediated by the C-terminal domain of PTEN and the N-terminal domain of AEBP1.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- PTEN is a critical tumor suppressor gene.
- Identifying PTEN-binding partners is crucial for understanding its cellular functions.
Purpose of the Study:
- To discover novel proteins that interact with PTEN.
- To characterize the interaction domains between PTEN and its binding partners.
Main Methods:
- Yeast two-hybrid screening was employed.
- Bait constructs included full-length PTEN, its C-terminal domain, and mutants.
- CDNA libraries from Colon Cancer, HeLa, and Mouse Embryo were screened.
Main Results:
- Forty-three positive clones were identified and confirmed.
- Sequence analysis revealed two clones encoding Adipocyte Enhancer Binding Protein 1 (AEBP1).
Conclusions:
- PTEN interacts with AEBP1.
- The interaction is mediated by PTEN's C-terminal and AEBP1's N-terminal domains.
- The functional significance of this interaction requires further investigation.
