ABSTRACT
pVHL, the product of the VHL tumor suppressor gene, plays an important role in the regulation of cell growth and differentiation of human kidney cells, and inactivation of the VHL gene is the most frequent genetic event in human kidney cancer. The biochemical function of pVHL is unknown. Here we report that pVHL exists in vivo in a complex that displays ubiquitination-promoting activity in conjunction with the universally required components E1, E2, and ubiquitin. pVHL-associated ubiquitination activity requires, at a minimum, pVHL to bind elongin C and Cul-2, relatives of core components of SCF (Skp1-Cdc53/Cul-1-F-box protein) E3 ligase complexes. Notably, certain tumor-derived mutants of pVHL demonstrate loss of associated ubiquitination promoting activity. These results identify pVHL as a component of a potential SCF-like E3 ubiquitin-protein ligase complex and suggest a direct link between pVHL tumor suppressor and the process of ubiquitination.
Subject(s)
Genes, Tumor Suppressor , Ligases/metabolism , Proteins/metabolism , Tumor Suppressor Proteins , Amino Acid Sequence , Base Sequence , Cell Extracts , DNA Primers , Humans , Kidney Neoplasms/metabolism , Ligases/genetics , Molecular Sequence Data , Mutation , Precipitin Tests , Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Tumor Cells, Cultured , Ubiquitin-Protein Ligases , Ubiquitins/metabolism , Von Hippel-Lindau Tumor Suppressor ProteinABSTRACT
In normal and transformed cells, the F-box protein p45(SKP2) is required for S phase and forms stable complexes with p19(SKP1) and cyclin A-cyclin-dependent kinase (CDK)2. Here we identify human CUL-1, a member of the cullin family, and the ubiquitin-conjugating enzyme CDC34 as additional partners of p45(SKP2) in vivo. CUL-1 also associates with cyclin A and p19(SKP1) in vivo and, with p45(SKP2), they assemble into a large multiprotein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F-box protein, a member of the cullin family and a homolog of p19(SKP1)) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45(SKP2)-CUL-1-p19(SKP1) complex may be a human representative of an SCF-type E3 ubiquitin protein ligase. We propose that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Finally, we show that multiprotein complex formation involving p45(SKP2)-CUL-1 and p19(SKP1) is governed, in part, by periodic, S phase-specific accumulation of the p45(SKP2) subunit and by the p45(SKP2)-bound cyclin A-CDK2. The dependency of p45(SKP2)-p19(SKP1) complex formation on cyclin A-CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway.
