RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination

RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination

Laboratory of Immune Cell Biology, Division of Basic Sciences, National Cancer Institute, Building 10, Room 1B34, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1152

Communicated by Richard D. Klausner, National Institutes of Health, Bethesda, MD (received for review July 6, 1999)

Abstract

A RING finger-containing protein (AO7) that binds ubiquitin-conjugating enzymes (E2s) and is a substrate for E2-dependent ubiquitination was identified. Mutations of cation-coordinating residues within AO7’s RING finger abolished ubiquitination, as did chelation of zinc. Several otherwise-unrelated RING finger proteins, including BRCA1, Siah-1, TRC8, NF-X1, kf-1, and Praja1, were assessed for their ability to facilitate E2-dependent ubiquitination. In all cases, ubiquitination was observed. The RING fingers were implicated directly in this activity through mutations of metal-coordinating residues or chelation of zinc. These findings suggest that a large number of RING finger-containing proteins, with otherwise diverse structures and functions, may play previously unappreciated roles in modulating protein levels via ubiquitination.

Footnotes

↵ * K.L.L. and J.P.J. contributed equally to this work.
↵ † Present address: Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.
↵ ‡ To whom correspondence should be addressed. E-mail: amw{at}nih.gov.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF171060).
ABBREVIATIONS:

Ub,

ubiquitin;

HECT,

homologous to E6-AP C terminus;

APC,

anaphase-promoting complex;

GS,

glutathione Sepharose;

GST,

glutathione S-transferase;

HA,

hemagglutinin;

DTPA,

disodiumtriaminopentaacetic acid;

TPEN,

tetrakis(2-pyridylmethyl)ethylenediamine