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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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DGKι possesses two LxxLAP motifs. Sequence alignments of the N-terminal (top) and C-terminal (bottom) PHD recognition motifs found in HIF1α, HIF2α and DGKι. The LxxLAP consensus motif is underlined, with the target prolyl residue in bold.
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Figure 3: DGKι possesses two LxxLAP motifs. Sequence alignments of the N-terminal (top) and C-terminal (bottom) PHD recognition motifs found in HIF1α, HIF2α and DGKι. The LxxLAP consensus motif is underlined, with the target prolyl residue in bold.

Mentions: A particularly interesting new interactor is diacylglycerolkinase iota (DGKι), a member of an enzyme family involved in the regulation of intracellular diacylglycerol and phosphatidic acid levels (20,34). DGKι is predominantly expressed in brain and retina, two tissues that are most frequently affected in VHL disease. Intriguingly, DGKι contains two LxxLAP motifs at residues Pro147 and Pro903 that are potential targets for PHD-catalyzed prolyl hydroxylation (Figure 3), one of which was present in the original isolate from the two-hybrid screen (DGKι668–1065; Figure 4a and Table 2).Figure 3.


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)

DGKι possesses two LxxLAP motifs. Sequence alignments of the N-terminal (top) and C-terminal (bottom) PHD recognition motifs found in HIF1α, HIF2α and DGKι. The LxxLAP consensus motif is underlined, with the target prolyl residue in bold.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: DGKι possesses two LxxLAP motifs. Sequence alignments of the N-terminal (top) and C-terminal (bottom) PHD recognition motifs found in HIF1α, HIF2α and DGKι. The LxxLAP consensus motif is underlined, with the target prolyl residue in bold.
Mentions: A particularly interesting new interactor is diacylglycerolkinase iota (DGKι), a member of an enzyme family involved in the regulation of intracellular diacylglycerol and phosphatidic acid levels (20,34). DGKι is predominantly expressed in brain and retina, two tissues that are most frequently affected in VHL disease. Intriguingly, DGKι contains two LxxLAP motifs at residues Pro147 and Pro903 that are potential targets for PHD-catalyzed prolyl hydroxylation (Figure 3), one of which was present in the original isolate from the two-hybrid screen (DGKι668–1065; Figure 4a and Table 2).Figure 3.

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