Abstract
Little attention has been paid to the specificity between E2 and the target protein during ubiquitination, although RING-E3 induces a potential intra-molecular reaction by mediating the direct transfer of ubiquitin from E2 to the target protein. We have constructed artificial E2 fusion proteins in which a target protein (p27) is tethered to one of six E2s via a flexible linker. Interestingly, only three E2s (UbcH5b, hHR6b, and Cdc34) are able to ubiquitinate p27 via an intra-molecular reaction in this system. Although the first ubiquitination of p27 (p27-Ub) by Cdc34 is less efficient than that of UbcH5b and hHR6b, the additional ubiquitin attachment to p27-Ub by Cdc34 is highly efficient. The E2 core of Cdc34 provides specificity to p27, and the residues 184-196 are required for possessive ubiquitination by Cdc34. We demonstrate direct E2 specificity for p27 and also show that differential ubiquitin linkages can be dependent on E2 alone.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Anaphase-Promoting Complex-Cyclosome
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Cyclin-Dependent Kinase Inhibitor p27
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Humans
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In Vitro Techniques
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Intracellular Signaling Peptides and Proteins / chemistry
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism*
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Lysine / chemistry
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Models, Biological
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Proliferating Cell Nuclear Antigen / chemistry
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Proliferating Cell Nuclear Antigen / genetics
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Proliferating Cell Nuclear Antigen / metabolism
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Substrate Specificity
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Ubiquitin-Conjugating Enzymes / chemistry
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Ubiquitin-Conjugating Enzymes / genetics
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Ubiquitin-Conjugating Enzymes / metabolism*
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Ubiquitin-Protein Ligase Complexes / chemistry
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Ubiquitin-Protein Ligase Complexes / genetics
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Ubiquitin-Protein Ligase Complexes / metabolism
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Ubiquitin-Protein Ligases / chemistry
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism
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Ubiquitination / physiology*
Substances
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CDKN1B protein, human
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Intracellular Signaling Peptides and Proteins
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Proliferating Cell Nuclear Antigen
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Recombinant Fusion Proteins
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Cyclin-Dependent Kinase Inhibitor p27
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CDC34 protein, human
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UBE2B protein, human
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UBE2D2 protein, human
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Ubiquitin-Conjugating Enzymes
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Ubiquitin-Protein Ligase Complexes
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Anaphase-Promoting Complex-Cyclosome
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Ubiquitin-Protein Ligases
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Lysine