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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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PHD3-dependent two-hybrid interaction between pVHL and DGKι. Two-hybrid interactions between BD-pVHL and AD-DGKι in the absence and presence of the indicated PHD isoforms were assayed as in Figure 2a.
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Figure 5: PHD3-dependent two-hybrid interaction between pVHL and DGKι. Two-hybrid interactions between BD-pVHL and AD-DGKι in the absence and presence of the indicated PHD isoforms were assayed as in Figure 2a.

Mentions: We thus further characterized the two-hybrid interaction between DGKι and pVHL. To map the region of pVHL binding more precisely, we generated different truncated versions of DGKι fused to the AD (Figure 4a). While full-length DGKι1-1065 interacted with pVHL in a PHD3- and ELB/ELC-dependent manner (Figure 4b), C-terminally truncated variants lacking the LxxLAP motif at Pro903 failed to interact, demonstrating that the C-terminal LxxLAP motif is required for the interaction with pVHL. Consistently, the C-terminal fragment DGKι879-1065 including the C-terminal LxxLAP motif was sufficient for the pVHL interaction. Next, we directly tested the importance of the LxxLAP motif for pVHL interaction by mutating the critical prolyl residue, either alone (DGKι879-1065PA) or in concert with the N-terminal LxxLAP motif (DGKι1-1065PPAA). Importantly, neither of the two DGKι variants interacted with pVHL (Figure 4b), strongly suggesting that the two-hybrid interaction depends on the PHD3-catalyzed prolyl hydroxylation of residue Pro903. The expression levels of these mutant prey constructs were found to be very similar to those of the respective wild-type constructs (Figure 4c). Furthermore, we analyzed the PHD specificity of the interaction and found that full-length DGKι interacted strongly with pVHL in the presence of PHD3, while only a very weak interaction was detectable in the presence of PHD1 or PHD2 (Figure 5). This result is consistent with the possibility that PHD3 has evolved to recognize a broader range of substrates than the PHD1 and PHD2 isoenzymes (32).Figure 5.


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)

PHD3-dependent two-hybrid interaction between pVHL and DGKι. Two-hybrid interactions between BD-pVHL and AD-DGKι in the absence and presence of the indicated PHD isoforms were assayed as in Figure 2a.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: PHD3-dependent two-hybrid interaction between pVHL and DGKι. Two-hybrid interactions between BD-pVHL and AD-DGKι in the absence and presence of the indicated PHD isoforms were assayed as in Figure 2a.
Mentions: We thus further characterized the two-hybrid interaction between DGKι and pVHL. To map the region of pVHL binding more precisely, we generated different truncated versions of DGKι fused to the AD (Figure 4a). While full-length DGKι1-1065 interacted with pVHL in a PHD3- and ELB/ELC-dependent manner (Figure 4b), C-terminally truncated variants lacking the LxxLAP motif at Pro903 failed to interact, demonstrating that the C-terminal LxxLAP motif is required for the interaction with pVHL. Consistently, the C-terminal fragment DGKι879-1065 including the C-terminal LxxLAP motif was sufficient for the pVHL interaction. Next, we directly tested the importance of the LxxLAP motif for pVHL interaction by mutating the critical prolyl residue, either alone (DGKι879-1065PA) or in concert with the N-terminal LxxLAP motif (DGKι1-1065PPAA). Importantly, neither of the two DGKι variants interacted with pVHL (Figure 4b), strongly suggesting that the two-hybrid interaction depends on the PHD3-catalyzed prolyl hydroxylation of residue Pro903. The expression levels of these mutant prey constructs were found to be very similar to those of the respective wild-type constructs (Figure 4c). Furthermore, we analyzed the PHD specificity of the interaction and found that full-length DGKι interacted strongly with pVHL in the presence of PHD3, while only a very weak interaction was detectable in the presence of PHD1 or PHD2 (Figure 5). This result is consistent with the possibility that PHD3 has evolved to recognize a broader range of substrates than the PHD1 and PHD2 isoenzymes (32).Figure 5.

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