Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

^*Department of Molecular and Cellular Biology and ^§Laboratory of Embryonic and Genetic Engineering, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; ^†CREST, Japan Science and Technology Corporation, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan; ^¶Department of Viral Oncology, Cancer Institute, Japanese Foundation for Cancer Research, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan; ^‖Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan; ^**Section of Pathology, Institute of Immunological Science, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan; and ^‡‡Department of Pediatrics, All Children’s Hospital, University of South Florida, 801 Sixth Street South, St. Petersburg, FL 33701

Contributed by Robert A. Good

Abstract

Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-κB through control of IκB protein stability.

Footnotes

↵ ‡ S.H. and M.K. contributed equally to this work.
↵ †† To whom reprint requests should be addressed. e-mail: nakayak1{at}bioreg.kyushu-u.ac.jp.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF081887, for mouse FWD1; AF083214, for mouse Skp1; AF083216, for mouse Cul 1; and AF083215, for mouse Skp2).
ABBREVIATIONS:

NF-κB,

nuclear factor kappa B;

IκB,

inhibitory subunit of NF-κB;

IKK,

IκB kinase;

SCF,

Skp1/Cull 1/F-box;

FWD1,

F-box/WD40-repeat protein 1;

SCFFWD1,

SCF protein FWD1;

FWD1 (ΔF),

mutant FWD1 lacking an F-box domain