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A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein.

Bex C, Knauth K, Dambacher S, Buchberger A - Nucleic Acids Res. (2007)

Bottom Line: The usefulness of yeast two-hybrid (Y2H) approaches, on the other hand, has been limited by the failure of pVHL to adopt its native structure and by the absence of prolylhydroxylase activity critical for pVHL substrate recognition.Therefore, we modified the Y2H system to faithfully reconstitute the physical interaction between pVHL and its substrates.Our approach relies on the coexpression of pVHL with the cofactors Elongin B and Elongin C and with HIF1/2alpha prolylhydroxylases.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Biochemistry, Department of Molecular Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.

ABSTRACT
The von Hippel-Lindau tumor suppressor protein (pVHL) is inactivated in the hereditary cancer syndrome von Hippel-Lindau disease and in the majority of sporadic renal carcinomas. pVHL is the substrate-binding subunit of the CBC(VHL) ubiquitin ligase complex that negatively regulates cell growth by promoting the degradation of hypoxia-inducible transcription factor subunits (HIF1/2alpha). Proteomics-based identification of novel pVHL substrates is hampered by their short half-life and low abundancy in mammalian cells. The usefulness of yeast two-hybrid (Y2H) approaches, on the other hand, has been limited by the failure of pVHL to adopt its native structure and by the absence of prolylhydroxylase activity critical for pVHL substrate recognition. Therefore, we modified the Y2H system to faithfully reconstitute the physical interaction between pVHL and its substrates. Our approach relies on the coexpression of pVHL with the cofactors Elongin B and Elongin C and with HIF1/2alpha prolylhydroxylases. In a proof-of-principle Y2H screen, we identified the known substrates HIF1/2alpha and new candidate substrates including diacylglycerol kinase iota, demonstrating that our strategy allows detection of stable interactions between pVHL and otherwise elusive cellular targets. Additional future applications may include structure/function analyses of pVHL-HIF1/2alpha binding and screens for therapeutically relevant compounds that either stabilize or disrupt this interaction.

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pVHL and DGKι interact in human cells. Lysates (Input) of human 293T cells overexpressing HA-tagged DGKι in the absence (–) or presence of the indicated FLAG-tagged pVHL isoform were subjected to immunoprecipitation using anti-FLAG antibody (IP: FLAG). Immunoprecipitated pVHL and DGKι were detected by western blot (WB) using anti-FLAG and anti-DGKι antibodies as indicated. The asterisk marks the light chain of the anti-FLAG antibody used for immunoprecipitation.
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Figure 6: pVHL and DGKι interact in human cells. Lysates (Input) of human 293T cells overexpressing HA-tagged DGKι in the absence (–) or presence of the indicated FLAG-tagged pVHL isoform were subjected to immunoprecipitation using anti-FLAG antibody (IP: FLAG). Immunoprecipitated pVHL and DGKι were detected by western blot (WB) using anti-FLAG and anti-DGKι antibodies as indicated. The asterisk marks the light chain of the anti-FLAG antibody used for immunoprecipitation.

Mentions: Finally, we wished to test whether pVHL and DGKι also interact in human cells. To this end, 293T cells were transiently transfected with expression plasmids for full-length DGKι and the long and short isoforms of pVHL, respectively. DGKι was efficiently co-immunoprecipitated with both pVHL isoforms, but not in the negative control lacking exogenous pVHL (Figure 6). Thus, the novel two-hybrid interactor DGKι physically associates with pVHL in human cells, further strengthening the validity of our two-hybrid approach.Figure 6.


A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein.

Bex C, Knauth K, Dambacher S, Buchberger A - Nucleic Acids Res. (2007)

pVHL and DGKι interact in human cells. Lysates (Input) of human 293T cells overexpressing HA-tagged DGKι in the absence (–) or presence of the indicated FLAG-tagged pVHL isoform were subjected to immunoprecipitation using anti-FLAG antibody (IP: FLAG). Immunoprecipitated pVHL and DGKι were detected by western blot (WB) using anti-FLAG and anti-DGKι antibodies as indicated. The asterisk marks the light chain of the anti-FLAG antibody used for immunoprecipitation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175351&req=5

Figure 6: pVHL and DGKι interact in human cells. Lysates (Input) of human 293T cells overexpressing HA-tagged DGKι in the absence (–) or presence of the indicated FLAG-tagged pVHL isoform were subjected to immunoprecipitation using anti-FLAG antibody (IP: FLAG). Immunoprecipitated pVHL and DGKι were detected by western blot (WB) using anti-FLAG and anti-DGKι antibodies as indicated. The asterisk marks the light chain of the anti-FLAG antibody used for immunoprecipitation.
Mentions: Finally, we wished to test whether pVHL and DGKι also interact in human cells. To this end, 293T cells were transiently transfected with expression plasmids for full-length DGKι and the long and short isoforms of pVHL, respectively. DGKι was efficiently co-immunoprecipitated with both pVHL isoforms, but not in the negative control lacking exogenous pVHL (Figure 6). Thus, the novel two-hybrid interactor DGKι physically associates with pVHL in human cells, further strengthening the validity of our two-hybrid approach.Figure 6.

Bottom Line: The usefulness of yeast two-hybrid (Y2H) approaches, on the other hand, has been limited by the failure of pVHL to adopt its native structure and by the absence of prolylhydroxylase activity critical for pVHL substrate recognition.Therefore, we modified the Y2H system to faithfully reconstitute the physical interaction between pVHL and its substrates.Our approach relies on the coexpression of pVHL with the cofactors Elongin B and Elongin C and with HIF1/2alpha prolylhydroxylases.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Biochemistry, Department of Molecular Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.

ABSTRACT
The von Hippel-Lindau tumor suppressor protein (pVHL) is inactivated in the hereditary cancer syndrome von Hippel-Lindau disease and in the majority of sporadic renal carcinomas. pVHL is the substrate-binding subunit of the CBC(VHL) ubiquitin ligase complex that negatively regulates cell growth by promoting the degradation of hypoxia-inducible transcription factor subunits (HIF1/2alpha). Proteomics-based identification of novel pVHL substrates is hampered by their short half-life and low abundancy in mammalian cells. The usefulness of yeast two-hybrid (Y2H) approaches, on the other hand, has been limited by the failure of pVHL to adopt its native structure and by the absence of prolylhydroxylase activity critical for pVHL substrate recognition. Therefore, we modified the Y2H system to faithfully reconstitute the physical interaction between pVHL and its substrates. Our approach relies on the coexpression of pVHL with the cofactors Elongin B and Elongin C and with HIF1/2alpha prolylhydroxylases. In a proof-of-principle Y2H screen, we identified the known substrates HIF1/2alpha and new candidate substrates including diacylglycerol kinase iota, demonstrating that our strategy allows detection of stable interactions between pVHL and otherwise elusive cellular targets. Additional future applications may include structure/function analyses of pVHL-HIF1/2alpha binding and screens for therapeutically relevant compounds that either stabilize or disrupt this interaction.

Show MeSH
Related in: MedlinePlus