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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Figure 1

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Disruption of elongin B and elongin C interaction by an elongin C–derive...
Disruption of elongin B and elongin C interaction by an elongin C–derived peptide. (a) 35S-labeled elongin B in vitro translate was incubated with glutathione Sepharose preloaded with GST-elongin C in the absence of peptide (lane 2), in the presence of increasing amounts of a synthetic peptide corresponding to elongin C residues 17–50 (lanes 3–5; 0.5, 2.5, 10 μg, respectively), or in the presence of 10 μg of a sequence-scrambled elongin (17–50) peptide. (b) 35S-labeled elongin C in vitro translate was incubated with glutathione Sepharose preloaded with GST-elongin B in the absence (lane 2) or presence (lanes 3–6) of elongin C peptides as in a. Bound proteins were resolved by SDS-PAGE and detected by fluorography. Comparable recovery of the GST fusion proteins in each lane was confirmed by Coomassie blue staining. Twenty-five percent of the in vitro translate used per binding reaction was loaded directly in lane 1.