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

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