Identification of a novel binding partners for tumor suppressor PTEN by a yeast two-hybrid approach.
OBJECTIVE
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).


CONCLUSIONS
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.
Gorbenko
Olena
O
Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine.
Kuznetsov
Vitaliy
V
Kukharenko
Olexandr
O
Zhyvoloup
Alexandr
A
Panasyuk
Ganna
G
Nemazanyy
Ivan
I
Filonenko
Valeriy
V
Gout
Ivan
I
D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D016147:Genes, Tumor Suppressor; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D018076:DNA, Complementary; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase