The SCFβ-TRCP–ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro

The transcription factor NF-κB has a central role in cellular stress and inflammatory responses by controlling cytokine-inducible gene expression and lymphocyte stimulation by antigens (Baeuerle and Baltimore 1996; Gilmore et al. 1996). In addition, NF-κB is required to block cell death in response to tumor necrosis factor α (TNFα) and ionizing radiation, suggesting that it acts to regulate the transcription of survival genes (Beg and Baltimore 1996; Liu et al. 1996; Van Antwerp et al. 1996; Wang et al. 1996). NF-κB is a ubiquitous heterodimeric complex composed of a p65/RelA subunit and a p50 subunit. This complex is normally sequestered in an inactive form in the cytoplasm through interaction with members of a family of inhibitory proteins, the IκBs (Beg et al. 1992; for review, see Baeuerle and Baltimore 1996). These proteins, when associated with NF-κB, obscure the nuclear localization signal in NF-κB and also block the ability of NF-κB to bind DNA. In response to TNFα and other signals, IκBα is rapidly phosphorylated on two serine residues near the amino terminus (Ser-32 and Ser-36 in IκBα) (Beg et al. 1993; Finco et al. 1994; Alkalay et al. 1995; Brown et al. 1995; Chen et al. 1995, 1996; DiDonato et al. 1995; Lin et al. 1995). This modification triggers the rapid destruction of IκBα by ubiquitin-mediated proteolysis, thereby allowing NF-κB nuclear translocation and target gene expression (Chen et al. 1995; Scherer et al. 1995; for review, see Hochstrasser 1996). Recent work has uncovered two IκBα kinases, IκKα and IκKβ, that are responsible for signal-dependent phosphorylation of IκBα (DiDonato et al. 1997; Mercurio et al. 1997; Regnier et al. 1997; Woronicz et al. 1997; Zandi et al. 1997, 1998). These proteins are part of a 700-kD protein complex that is assembled through two structural components IκKγ/NEMO and IKAP (Cohen et al. 1998; Rothwarf et al. 1998; Yamaoka et al. 1998) and are activated by cytokines. In vitro, both IκKα and IκKβ can phosphorylate IκBα specifically on serines 32 and 36, but both kinases are required for efficient IκBα phosphorylation in vivo (Zandi et al. 1997). Although the pathways leading to IκBα phosphorylation have been described in detail, little is known about the molecules responsible for ubiquitination. Ubiquitin-mediated proteolysis involves a cascade of ubiquitin transfer reactions in which the ubiquitin-activating enzyme E1 uses ATP to form a high-energy thiolester bond with ubiquitin, which is then transferred to members of the E2 ubiquitin-conjugating enzyme family (Hershko et al. 1983; Hochstrasser 1995). Ubiquitin is then transferred from the E2 to lysine residues in the target through an E3–ubiquitin ligase. E3s serve as adaptors that interact with both the target protein and the appropriate E2, thereby providing specificity to the ubiquitin transfer reaction. In some cases, the E3 is also involved in ubiquitin transfer (Scheffner et al. 1995; Rolfe et al. 1995). Multiple rounds of ubiquitination of the initial conjugates lead to polyubiquitination, which targets the protein for proteolysis by the 26S proteasome. Recent studies have elaborated a modular ubiquitin ligase complex, the SCF–ubiquitin ligase, which mediates phosphorylation-dependent ubiquitination of a large number of proteins (for review, see Elledge and Harper 1998; Patton et al. 1998b). The SCF is composed of Skp1, Cdc53/Cul1, and a specificity-conferring F-box protein (Bai et al. 1996; Feldman et al. 1997; Skowyra et al. 1997; Patton et al. 1998a). F-box proteins contain two domains, an F-box motif that binds Skp1 and allows assembly into Skp1/Cdc53 complexes, and a second protein–protein interaction domain that interacts specifically with one or more target proteins (Bai et al. 1996). Cdc53/Cul1, in turn, interacts with both the E2 and the Skp1/F-box protein complex (Skowyra et al. 1997; Patton et al. 1998a). SCF complexes mediate phosphorylation-dependent destruction of a wide array of regulatory proteins in yeast, including the Cdk inhibitors Sic1, Far1, and Rum1, G1 cyclins, the transcription factor Gcn4, and the DNA replication initiator proteins Cdc6 and Cdc18 (for review, see Elledge and Harper 1998; Patton et al. 1998b). In contrast with yeast, targets of vertebrate SCF complexes remain largely unknown. Previously, we identified four vertebrate proteins that contain the F-box motif, linking them to the ubiquitin pathway: mammalian cyclin F, Skp2, MD6, and Xenopus β-TRCP (β-transducin repeat-containing protein; Bai et al. 1996). β-TRCP was originally identified as a suppressor of a temperature-sensitive mutation in the budding yeast CDC15 gene (Spevak et al. 1993), but its mechanism of suppression has not been determined. Recent genetic evidence has implicated Xenopus β-TRCP and its Drosphila homolog, slimb, in control of proteolysis in the Hedgehog and Wingless/Wnt signaling pathways (Jiang and Struhl 1998; Marikawa and Elinson 1998). We have used biochemical approaches to examine whether IκBα ubiquitination might involve an SCF–ubiquitin ligase. Here we report that mammalian β-TRCP binds to the IκBα destruction motif in a phosphorylation-dependent manner, thereby recruiting IκBα into an SCF–ubiquitin ligase complex. Moreover, SCFβ-TRCP components cofractionate with IκBα–ubiquitin ligase activity from tissue culture cells and SCFβ-TRCP can stimulate ubiquitination of phosphorylated but not unphosphorylated IκBα in an in vitro reconstitution assay. We also demonstrate that the same SCFβ-TRCP complex recognizes a similar destruction motif in β-catenin, a component of the TCF/Lef transcription factor complex that functions downstream of Wingless/Wnt (for review, see Peifer 1997) and whose levels are also controlled by phosphorylation-dependent ubiquitin-mediated proteolysis (Aberle et al. 1997). Our results, together with the effects of loss-of-function mutations in the Drosophila β-TRCP homolog slimb (Jiang and Struhl 1998), suggest that a single SCFβ-TRCP complex functions in diverse signaling pathways that impinge on transcription control mediated by cytokines (NF-κB), Wnt/Wingless (β-catenin), and Hedgehog [Cubitus interruptus (Ci)].