Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein
Michael Ohh, … , Joan Weliky Conaway, William G. Kaelin Jr.
Michael Ohh, … , Joan Weliky Conaway, William G. Kaelin Jr.
Published December 1, 1999
Citation Information: J Clin Invest. 1999;104(11):1583-1591. https://doi.org/10.1172/JCI8161.
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Article

Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein

Abstract

The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17–50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157–171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.

Authors

Michael Ohh, Yuichiro Takagi, Teijiro Aso, Charles E. Stebbins, Nikola P. Pavletich, Bert Zbar, Ronald C. Conaway, Joan Weliky Conaway, William G. Kaelin Jr.

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Inhibition of elongin/SIII activity by pVHL-derived peptides. (a) Runoff...
Inhibition of elongin/SIII activity by pVHL-derived peptides. (a) Runoff transcription reactions containing elongin A and elongin BC were performed as described in Methods. All reactions included 50 μM ATP, 50 μM GTP, 10 μM CTP, 10 μCi [α-32P] CTP, and 2 μM UTP. The reaction in the first lane did not include VHL peptide. Reactions in the remaining lanes included 12 nM, 40 nM, 120 nM, or 400 nM of the indicated peptide. AdML indicates the position of the approximately 250 nucleotides runoff transcript initiated at the AdML promoter. (b) Runoff transcription reactions were performed in the presence of elongin A or elongin A and elongin BC as indicated, with 0, 100 nM, 200 nM, and 400 nM of the indicated peptide, with 50 μM ATP, 50 μM GTP, 10 μM CTP, 10 μCi [α-32P] CTP, and 2 μM UTP. Reactions labeled High NTPs were performed in the absence of elongin A and elongin BC and in the presence of 0 (–) or 600 nM (+) of the wild-type peptide and 50 μM ATP, 50 μM GTP, 10 μM CTP, 10 μCi [α-32P] CTP, and 50 μM UTP. Elo, elongin.