Cloning of human ubiquitin-conjugating enzymes UbcH6 and UbcH7 (E2-F1) and characterization of their interaction with E6-AP and RSP5.
E6-AP, a 100-kDa cellular protein, was originally identified through its interaction with the E6 protein of the oncogenic human papillomavirus types 16 and 18. The complex of E6-AP and E6 specifically interacts with p53 and mediates ubiquitination of p53 in concert with the E1 ubiquitin-activating enzyme and the E2 ubiquitin-conjugating enzyme UbcH5. Recent results suggest that E6-AP is representative of a family of putative ubiquitin-protein ligases. Members of this family are characterized by a conserved C-terminal region, termed hect domain. In this paper, we describe the isolation of two human E2s, designated as UbcH6 and UbcH7, that in addition to UbcH5 can interact with E6-AP. UbcH6 is a novel member of an evolutionally conserved subfamily of E2s that includes UbcH5 and Saccharomyces cerevisiae UBC4. Although UbcH7 does not appear to be a member of this subfamily, UbcH7 efficiently substitutes for UbcH5 in E6-AP-dependent ubiquitination. Surprisingly, UbcH6 was only weakly active in this particular assay. In addition, UbcH5 but not UbcH6 or UbcH7 efficiently interacts with the heet protein RSP5. These results indicate that E6-AP can interact with at least two species of E2 and that different hect proteins may interact with different E2s.
Nuber
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U
Deutsches Krabsforschungszentrum, Angewandte Tumorvirologie, Heidelberg, Federal Republic of Germany.
Schwarz
S
S
Kaiser
P
P
Schneider
R
R
Scheffner
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014764:Viral Proteins / Q000378:metabolism*
D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; C489963:UBE2E1 protein, human; C489967:UBE2J1 protein, human; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; C469287:RSP5 protein, S cerevisiae