Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination.
The human papilloma virus E6-associated protein (E6AP) functions as a ubiquitin protein ligase (E3) in the E6-mediated ubiquitination of p53. E6AP is also an E3 in the absence of E6, but its normal cellular substrates have not yet been identified. Here we report the identification of HHR23A, one of the human homologues of the yeast DNA repair protein Rad23, as an E6-independent target of E6AP. HHR23A binds E6AP and is ubiquitinated in vitro in an E6AP-dependent manner. Ubiquitinated forms of endogenous HHR23A are detectable in mammalian cells. Overexpression of wild-type E6AP in vivo enhances the ubiquitination of HHR23A, whereas a dominant negative E6AP mutant inhibits HHR23A ubiquitination. Although HHR23A is a stable protein in non-synchronized cells, its levels are regulated in a cell cycle-dependent manner, with specific degradation occurring during S phase. The S phase degradation of HHR23A could be blocked in vivo by dominant negative E6AP, providing direct evidence for the involvement of E6AP in the regulation of HHR23A. Consistent with a role of the HHR23 proteins in DNA repair, UV-induced DNA damage inhibited HHR23A degradation. Although the precise role of HHR23 proteins in DNA repair and cell cycle progression remains to be elucidated, our data suggest that E6AP-mediated ubiquitination of HHR23A may have important implications in DNA repair and cell cycle progression.
Kumar
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Harvard Medical School, Department of Pathology, Boston, Massachusetts 02115, USA.
Talis
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Howley
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D002453:Cell Cycle; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D014162:Transfection; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D014452:Ubiquitins; C086985:RAD23A protein, human; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D045643:DNA Repair Enzymes