Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. We reasoned that the human homologs of Slimb - beta-TrCP and its isoform beta-TrCP2 (KIAA0696) - might interact with beta-catenin. We found that the binding of beta-TrCP to beta-catenin was direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promoted the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated beta-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 did not associate with beta-catenin. We conclude that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin.

The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein ␤TrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IB␣ and ␤-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation and on ATF4 serine residue 219 present in the context of DSGXXXS, which is similar but not identical to the motif found in other substrates of ␤TrCP. ATF4 ubiquitination in HeLa cells is enhanced in the presence of ␤TrCP. The F-box-deleted ␤TrCP protein behaves as a negative transdominant mutant that inhibits ATF4 ubiquitination and degradation and, subsequently, enhances its activity in cyclic AMP-mediated transcription. ATF4 represents a novel substrate for the SCF ␤TrCP complex, which is the first mammalian E3 ubiquitin ligase identified so far for the control of the degradation of a bZIP transcription factor.Proteasome-mediated protein degradation requires the covalent attachment of polyubiquitin to the substrate proteins (11,25,38). The cascade of ubiquitin transfer reactions involves the ubiquitin-activating enzyme E1, an E2 ubiquitinconjugating enzyme that operates with specificity factor E3. The selectivity of the reaction is due to the E3 ubiquitin ligase, which interacts with both E2 and the substrate.SCF (Skp1/Cullin/F-box protein) complexes were initially shown to function as E3 ubiquitin ligases for a variety of phosphorylated proteins involved in the yeast cell cycle (1,15,35,52,60). The core components of these complexes include Skp1, Cul-1 (Cdc53), and the newly identified protein Rbx1 (Roc1 or Hrt1), which is thought to stabilize the interaction between Cul-1 and the E2 enzyme Cdc34 (12,14,30,61). The SCF complexes also contain a variable receptor subunit, an F-boxcontaining protein, that provides substrate specificity. The Fbox motif serves to anchor the receptor subunit to the SCF complex by its interaction with Skp1 (12,14,52,60). For ubiquitination, substrate proteins are recruited to the SCF E3 ubiquitin ligase complexes through interaction with the substrate binding domain (WD-40 or leucine-rich repeats) of the F-box receptor subunits. SCF complexes are the largest and most versatile class of E3 ubiquitin ligases. To date, only two human SCF complexes, SCF Skp2 and SCF ␤TrCP , have been analyzed in detail and have had some of their substrates characterized (6, 7, 23, 36, 39, 41-44, 49, 62, 63, 67, 68). We made the first identification of human ␤TrCP (beta-transducin repeat-containing protein) as the F-box receptor component of the E3 ubiquitin ligase SCF ␤TrCP responsible for the degradation of CD4 induced by the human immunodeficiency virus type 1 (HIV-1) protein Vpu (43). Subsequently, we and others showed that SCF ␤TrCP is also responsible for phosphorylationdependent ubiquitination and then for the degradation of 36,39,42,62,67,68). Vpu, IB␣, and ␤-catenin shar...