The F-box protein β-TrCP associates with phosphorylated β-catenin and regulates its activity in the cell

Abstract

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.

Blocking Wnt signaling by SFRP-like molecules inhibits in vivo cell proliferation and tumor growth in cells carrying active β-catenin

“…Consistently, in human CRC cells, phosphorylation of b-catenin at S45 does not seem to be required for subsequent phosphorylation at residues S33, S37 or T41, in contrast to the prevailing models in normal cells . However, S33 and S37 are essential for interaction with b-TrCP and subsequent b-catenin ubiquitination and degradation (Hart et al, 1999). Therefore, as expected, in HEK293T cells transiently expressing S37A b-catenin or a dominantnegative form of b-catenin lacking the S33, S37, T41 and S45 residues (D29-48 b-catenin), neither V3Nter nor SFRP-1 could inhibit CRT (not shown).…”

“…Therefore, in parental HCT116 cells, inhibition of Wnt signaling results from the additive effects on both WT and mutant b-catenin alleles. As serines 33 and 37 of b-catenin are essential for b-TrCP-dependant b-catenin degradation (Hart et al, 1999), neither V3Nter nor SFRP-1 inhibited CRT in HEK293T cells expressing mutant S33 or S37 b-catenins, implying a GSK3b-and b-TrCP-dependent pathway. In contrast, in human liver cancer cells expressing either S37A or S33Y b-catenins, SFRP-1 and V3Nter did inhibit CRT, suggesting a GSK3b and b-TrCP-independent pathway.…”

Differential interaction of plakoglobin and β-catenin with the ubiquitin-proteasome system

“…The targeting of b-catenin for degradation involves the phosphorylation of its N-terminus by glycogen synthase kinase 3b (GSK) (Aberle et al, 1997;Yost et al, 1996) which occurs in a multi-protein complex consisting of b-catenin, GSK, the adenoma-tous polyposis coli (APC) tumor suppressor protein and axin/conductin Rubinfeld et al, 1996;Zeng et al, 1997;Ikeda et al, 1998;Behrens et al, 1998;Yamamoto et al, 1998). This complex associates with the ubiquitin ligase, b-TrCP (b-transducin repeat-containing protein) that recognizes the N-terminally phosphorylated forms of bcatenin and regulates its ubiquitination and degradation by the proteasome (Winston et al, 1999;Liu et al, 1999;Latres et al, 1999;Hart et al, 1999;Kitagawa et al, 1999). b-TrCP associates through its F-box with Cul1/Skp1/ROC1 forming the modular ubiquitin ligase SCF complex (Latres et al, 1999;Tan et al, 1999) and via its WD repeat motif with the phosphorylated bcatenin (Winston et al, 1999;Liu et al, 1999;Hart et al, 1999), thus forming a multimolecular complex consisting of b-TrCP/b-catenin/Axin/GSK/APC (Liu et al, 1999;Kitagawa et al, 1999).…”

“…This complex associates with the ubiquitin ligase, b-TrCP (b-transducin repeat-containing protein) that recognizes the N-terminally phosphorylated forms of bcatenin and regulates its ubiquitination and degradation by the proteasome (Winston et al, 1999;Liu et al, 1999;Latres et al, 1999;Hart et al, 1999;Kitagawa et al, 1999). b-TrCP associates through its F-box with Cul1/Skp1/ROC1 forming the modular ubiquitin ligase SCF complex (Latres et al, 1999;Tan et al, 1999) and via its WD repeat motif with the phosphorylated bcatenin (Winston et al, 1999;Liu et al, 1999;Hart et al, 1999), thus forming a multimolecular complex consisting of b-TrCP/b-catenin/Axin/GSK/APC (Liu et al, 1999;Kitagawa et al, 1999). b-TrCP and its Drosophila homolog Slimb were implicated in the regulation of wnt/wg signaling (Jiang and Struhl, 1998) and the DF-b-TrCP mutant, lacking the F-box, was shown to induce axis duplication in Xenopus (Marikawa and Elinson, 1998;Liu et al, 1999), and loss of function mutations in Slimb, induce the accumulation of armadillo in Drosophila (Jiang and Struhl, 1998).…”

Targeted increase in shaggy activity levels blocks wingless signaling

“…This degradation turnover necessitates the phosphorylation of several residues of the Arm N-terminus (Peifer et al, 1994). The kinase Shaggy (Sgg), the Drosophila GSK-3 ortholog, is an antagonist of Wg signal transduction and was shown critical for the phosphorylations and subsequent ubiquination-marking of Arm/␤-catenin and for the integrity of the Arm/␤-catenin degradation-complex (Hart et al, 1999;Liu et al, 2002).…”