Abstract
Treatment with divalent metal ions such as cobalt (Co(2+)) or nickel (Ni(2+)) result in the stabilization of hypoxia-inducible factor-1alpha (HIF1alpha). Recently, HIF1alpha was shown to be ubiquitinated by an E3-ligase complex and be subsequently targeted for proteasomal degradation. In this study, we demonstrated that Co(2+) and Ni(2+) specifically bind to cullin-2. Mutant analysis revealed that cullin-2 possesses at least three sites for the binding. Furthermore, fluorescence spectroscopy revealed that only Co(2+) and Ni(2+) have the binding activity to cullin-2, but other metal ions, including Cu(2+), Ca(2+), Mg(2+), Mn(2+), and Zn(2+), did not. Finally, we found that Co(2+) and Ni(2+) do not bind to any components of the E3-ligase other than cullin-2, suggesting that cullin-2 is a key target of Co(2+) and Ni(2+). Interestingly, Co(2+) did not affect the complex formation of the ligase, suggesting that the metal binding to cullin-2 affects the function, but not the assembly of the E3-ligase.
(c)2002 Elsevier Science.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenoviridae / genetics
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Animals
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Binding Sites
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Blotting, Western
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COS Cells
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Cell Cycle Proteins / chemistry*
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Cell Cycle Proteins / metabolism*
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Cell Line
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Cobalt / chemistry*
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Cobalt / metabolism
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Cullin Proteins*
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DNA, Complementary / metabolism
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Dose-Response Relationship, Drug
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Endothelium, Vascular / cytology
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Genetic Vectors
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Glutathione Transferase / chemistry
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Glutathione Transferase / metabolism
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Humans
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Hypoxia-Inducible Factor 1, alpha Subunit
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Ligases / metabolism
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Magnesium / chemistry
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Manganese / chemistry
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Mutation
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Nickel / chemistry*
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Nickel / metabolism
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Plasmids
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Precipitin Tests
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Protein Binding
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Spectrometry, Fluorescence
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Transcription Factors / chemistry
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Transcription, Genetic
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Transfection
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Ubiquitin-Protein Ligases
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Umbilical Veins / cytology
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Zinc / chemistry
Substances
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CUL2 protein, human
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Cell Cycle Proteins
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Cullin Proteins
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DNA, Complementary
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HIF1A protein, human
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Hypoxia-Inducible Factor 1, alpha Subunit
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Recombinant Fusion Proteins
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Transcription Factors
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Cobalt
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Manganese
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Nickel
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Ubiquitin-Protein Ligases
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Glutathione Transferase
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Ligases
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Magnesium
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Zinc