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

Abstract

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

Figure 1

FWD1 is an F-box/WD40-repeat protein related to βTrCP and Slimb. Alignment of the amino acid sequences of FWD1 (Mus musculus), βTrCP (Homo sapiens and Xenopus laevis), Slimb (Drosophila melanogaster), and FWD2 (M. musculus) is shown. Similar amino acids among more than three members are boxed. The seven copies of the WD40 repeats in all four proteins are underlined; the single F-box in each protein is double-underlined. The overall identity to FWD1 is 99% (for human βTrCP), 86% (for Xenopus βTrCP), 70% (for Drosophila Slimb), and 13% (for Mus FWD2).

Figure 2

FWD1 associates with Skp1, Cul1, and IκBα. (A) FWD1 binds to Skp1. Cells (293T) were transfected with expression plasmids encoding Myc-Skp1 and Flag-p57 (lane 1), Flag-FWD1 (lane 2), or Flag-FWD1 (ΔF) [lane 3; FWD1 (ΔF) is mutant FWD1 that lacks an F-box domain]. Cell lysates were immunoprecipitated via a Myc tag on Skp1 or via a Flag tag on p57, FWD1, or FWD1 (ΔF), then immunoblotted, and probed with anti-Flag (Upper) or anti-Myc (Lower) antibodies; 10% of the input lysates was also immunoblotted and probed with antibodies to show the expression level of those proteins. The position of each protein is indicated. (B) Coimmunoprecipitation of Skp1 and Cul1 with FWD1. Cells (293T) were transfected as indicated at the top of each lane. As controls, Flag-p27 (lane 1) and HA-E2-25k (lane 4) were used as indicated. Cell lysates were immunoprecipitated via a Flag tag on FWD1, then immunoblotted, and probed with anti-HA (Top), anti-Myc (Middle), and anti-Flag (Bottom); 10% of the input lysates was also shown (Right, lanes 5–8) in identical order. (C) FWD1 associates with phosphorylated IκBα. Cells (293T) were transfected with expression plasmids as indicated at the top of each lane. SA indicates the mutant IκBα in which both Ser-32 and Ser-36 are replaced with Ala, and KN indicates the kinase-negative mutant IKK-2 (K44M). Myc-FWD2 (lanes 1 and 2) or Myc-FWD1 (lanes 3–8) also were introduced. Cell lysates were immunoprecipitated via Myc tag on FWD2 or FWD1, then immunoblotted, and probed with anti-Flag to detect total IκBα or anti-phosphorylated IκBα (Upper). IKK-2 seems to interact weakly with FWD1, probably through IκBα, but it is invisible in this figure (data not shown); 10% of the input lysates also was immunoblotted and probed with anti-Flag to show the expression levels of IκBα and IKK-2 or anti-Myc for FWD1/2 (Lower). The positions of native and phosphorylated IκBα are indicated.

Figure 3

FWD1 facilitates IκBα ubiquitination. (A) FWD1 promotes IκBα ubiquitination in vivo. Cells (293T) were transfected with expression plasmids alone (mock; lanes 1 and 4) or plasmids encoding Flag-FWD2 (lanes 2 and 5) or Flag-FWD1 (lanes 3 and 6) in combination with wild-type IκBα (lanes 1 to 3) or 32/36 SA mutant IκBα (lanes 4 to 6). An expression plasmid encoding IKK-2 also was transfected in all lanes. Cell lysates were immunoprecipitated via a Myc tag on wild-type or mutant IκBα, then immunoblotted, and probed with anti-ubiquitin, anti-Myc to detect total IκBα, and anti-Flag to detect FWD1 or FWD2, which is associated with IκBα; 10% of the input lysates was immunoblotted and probed with anti-Flag to show the expression level of FWD1 and FWD2. (B) The F-box of FWD1 is required for ubiquitination but not for association with IκBα. Cells (293T) were transfected with expression plasmids encoding Flag-FWD2 (lane 1), Flag-FWD1 (lane 2), or Flag-FWD1 (ΔF) (lane 3). Expression plasmids encoding Myc-IκBα and IKK-2 were transfected in all lanes. Cell lysates were immunoprecipitated via a Myc tag on IκBα, then immunoblotted, and probed with anti-ubiquitin, anti-Myc to detect whole amount of IκBα, and anti-Flag to detect FWD2, FWD1, or FWD1 (ΔF). (C) In vitro ubiquitination of IκBα is facilitated dramatically by recombinant FWD1. Purified recombinant proteins were generated as described and mixed in combination as indicated with S100 lysate from NIH 3T3 cells. Reaction mixtures were immunoprecipitated with anti-rabbit IgG (lane 1) or anti-IκBα (lanes 2 to 7), then subjected to immunoblotting, and probed with anti-ubiquitin (Upper) or anti-Myc to indicate IκBα and FWD1 in the precipitates (Lower).

Figure 4

FWD1 induces rapid degradation of IκBα. (A) Pulse–chase analysis of the turnover rate of IκBα radiolabeled with [³⁵S]methionine/cysteine in 293T cells that were transfected with expression plasmids alone or plasmids encoding FWD1, IKK-2, or FWD1/IKK-2, in combination with the Myc-tagged IκBα. Cell lysates were immunoprecipitated via a Myc tag on IκBα, then subjected to SDS/PAGE, and autoradiographed. (B) A dominant-negative form of FWD1 inhibits the degradation induced by IKK-2. Wild-type FWD1 displayed promoted degradation induced by IKK-2. In contrast, FWD1 (ΔF) had a significant inhibitory effect on IKK-2-induced degradation. (C) FWD1 facilitates nuclear translocation of NF-κB. Cos7 cells were transfected with expression plasmids encoding Flag-tagged FWD1 (Top), IKK-2 (Middle), or both (Bottom). After 48 h, the cells were fixed and stained with anti-p65/RelA to examine the subcellular distribution of p65/RelA (Left), with anti-Flag to identify the transfected cells (Center), and with Hoechst 33258 dye to show nuclei (Right). Filled arrowheads indicate the transfected cells, and open arrowheads indicate nontransfected cells. Introduction of either FWD1 or IKK-2 alone is not sufficient for nuclear translocation of p65/RelA, whereas coexpression of FWD1 and IKK-2 leads to translocation of p65/RelA to the nucleus (Bottom Left).

Figure 5

A model for the function of SCF^FWD1 in ubiquitination. (A) Alignment of amino acid sequences necessary for association with FWD1 in IκBα (human), β-catenin (mouse), and Vpu (HIV). Serines subject to phosphorylation are indicated by arrows. (B) External signals activate the corresponding kinase, which phosphorylates the substrates at the DSGXXS motif. The phosphorylated substrate is attracted by FWD1. FWD1 links the target protein to the Skp1/Cullin/E2 ubiquitination apparatus, leading to the formation of a multiple ubiquitin chain. The multiubiquitinated protein is subject to the proteolysis by the 26S proteasome.