Huntingtin Is Ubiquitinated and Interacts with a Specific Ubiquitin-conjugating Enzyme

doi:10.1074/jbc.271.32.19385

Huntingtin Is Ubiquitinated and Interacts with a Specific Ubiquitin-conjugating Enzyme*

From the ^‡ Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4,
the ^§ Department of Biochemistry, School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205-2179, and
the ^¶ Department of Human Genetics, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3

^” Established investigator of the British Columbia Children's Hospital. To whom correspondence should be addressed:
Dept. of Medical Genetics, University of British Columbia, 416-2125 East Mall, Vancouver, B.C. V6T 1Z4, Canada.
Tel.: 604-822-9240; Fax: 604-822-9238.

Abstract

Using the yeast two-hybrid system, we have identified a human ubiquitin-conjugating enzyme (hE2-25K) as a protein that interacts with the gene product for Huntington disease (HD) (Huntingtin). This protein has complete amino acid identity with the bovine E2-25K protein and has striking similarity to the UBC-1, −4 and −5 enzymes of Saccharomyces cerevisiae. This protein is highly expressed in brain and a slightly larger protein recognized by an anti-E2-25K polyclonal antibody is selectively expressed in brain regions affected in HD. The huntingtin-E2-25K interaction is not obviously modulated by CAG length. We also demonstrate that huntingtin is ubiquitinated. These findings have implications for the regulated catabolism of the gene product for HD.

Footnotes

↵^∥ Recipient of a Research Career Development Award from the National Institutes of Health.
↵* This study was supported in part by grants from the National Centre of Excellence (Genetics), the Medical Research Council (MRC) (Canada) (to M. R. H. and D. G.), and the National Institutes of Health (to C. M. P.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U58522[GenBank].
↵¹ The abbreviations used are:

HD

Huntington disease

HIP

huntingtin interacting protein

HIP-2

HIP consisting of amino acids 33-200 of hE2-25K

E2

ubiquitin-conjugating enzyme (where relevant, initial letter indicates species of origin: b, bovine, m, murine, h, human)

GSH

glutathione

GST

glutathione-S-transferase

RT-PCR

reverse transcriptase-polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

FISH

fluorescent in situ hybridization

FITC

fluorescein isothiocyanate

PVDF

polyvinylidine difluoride

CAPS

3-(cyclohexylamino)propanesulfonic acid

DRPLA

dentatorubropallidoluysian atrophy

BD

binding domain

AD

activating domain
↵² D. Gietz, unpublished data.
↵³ G. Xia and C. Pickart, unpublished experiments.
↵⁵ J. Chen and C. Pickart, unpublished experiments.
↵⁴ L. Mastrandrea and C. Pickart, unpublished experiments.
- Received March 22, 1996.