Identification of HHR23A as a Substrate for E6-associated Protein-mediated Ubiquitination

doi:10.1074/jbc.274.26.18785

Identification of HHR23A as a Substrate for E6-associated Protein-mediated Ubiquitination*

From the Harvard Medical School, Department of Pathology, Boston, Massachusetts 02115

Abstract

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.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant RO1 CA64888 (to P. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Supported by Post-doctoral Fellowship PF-4309 from the American Cancer Society.
↵§ To whom correspondence should be addressed: Harvard Medical School, Dept. of Pathology, 200 Longwood Ave., Boston, MA 02115 Tel.: 617-432-2884; Fax: 617-432-2882; E-mail:peter_howley{at}hms.harvard.edu.
↵2 C. Maki, unpublished observations.
↵3 C. Schauber, W. Potts, R. D. Kirkpatrick, R. D. Gietz, L. Chen, and K. Madura, submitted for publication.
↵4 S. Kumar, A. L. Talis, and P. M. Howley, unpublished observations.
Abbreviations:

E1

ubiquitin-activating enzyme

E2

ubiquitin-conjugating enzymes

E3

ubiquitin protein ligase

E6AP

E6-associated protein

GST

glutathioneS-transferase

PAGE

polyacrylamide gel electrophoresis

NER

nucleotide excision repair

WGE

wheat germ extracts

CMV

cytomegalovirus

WT

wild type

HPV

human papilloma virus

HA

hemagglutinin

mAb

monoclonal antibody

XP

Xeroderma pigmentosum

XPC

xeroderma pigmentosum group C
- Received February 10, 1999.
- Revision received March 17, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.