IDENTIFICATION OF A NOVEL BINDING PARTNERS FOR TUMOR SUPPRESSOR PTEN BY A YEAST TWO-HYBRID APPROACH
Olena Gorbenko1, Vitaliy Kuznetsov1, Olexandr Kukharenko1, Alexandr Zhyvoloup1, Ganna Panasyuk1, Ivan Nemazanyy1, Valeriy Filonenko1, Ivan Gout1,2*
1Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine 2University College London, Department of Biochemistry and Molecular Biology, London WC1E 6BT, United Kingdom
Abstract. 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.
