Regulation of the cell cycle by SCF-type ubiquitin ligases

Seminars in Cell & Developmental Biology 16 (2005) 323–333

Review

Regulation of the cell cycle by SCF-type ubiquitin ligases Keiichi I. Nakayama a, b , Keiko Nakayama b, c, ∗ a

Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan b CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan c Division of Developmental Genetics, Center for Translational and Advanced Animal Research on Human Diseases, Tohoku University School of Medicine, Sendai, Miyagi 980-8575, Japan Available online 2 March 2005

Abstract Regulation of the cell cycle is dependent on protein degradation by the ubiquitin–proteasome system. Two major ubiquitin ligases, the anaphase-promoting complex or cyclosome (APC/C) and SCF complex, are responsible for the periodic proteolysis of many regulators of the cell cycle. The receptor component of the SCF complex is one of many F-box proteins, three of which—Skp2, Fbw7, and ␤-TrCP—are well characterized and implicated in cell cycle regulation. We have generated mice deficient in Skp2, Fbw7, or ␤-TrCP1 and have identified the roles of these proteins in both cell cycle regulation and mouse development. Clinical evidence also suggests that dysregulation of these F-box proteins contributes to human cancers. © 2005 Elsevier Ltd. All rights reserved. Keywords: Cell cycle; Proteolysis; Ubiquitin; SCF complex; F-box protein

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Skp2 targets CKIs for degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fbw7 contributes to the degradation of growth promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Common targets of Skp2 and Fbw7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Skp2 as an oncoprotein and Fbw7 as a tumor suppressor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . β-TrCP is a versatile F-box protein involved in many signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of β-TrCP in regulation of the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Progression of the eukaryotic cell cycle is controlled by a series of cyclin-dependent kinases (CDKs). The activity of these enzymes is regulated by several mechanisms, including association with regulatory subunits (cyclins), phosphorylation and dephosphorylation and interaction ∗

Corresponding author. Tel.: +81 92 642 6815; fax: +81 92 642 6819. E-mail address: [email protected] (K. Nakayama).

1084-9521/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2005.02.010

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with CDK inhibitors (CKIs) [1,2]. The amounts of cyclins, CKIs and many other cell cycle regulators oscillate during the cell cycle as a result of periodic proteolysis [3]. The ubiquitin–proteasome proteolytic pathway mediates the degradation of such short-lived regulatory proteins and thereby controls their intracellular concentrations [4,5]. The rapidity and substrate specificity of protein degradation by this pathway are consistent with such a role. Despite the structural similarities among cyclins, these proteins are degraded by the ubiquitin–proteasome pathway at different

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Fig. 1. The SCF complexes containing Skp2, Fbw7, or ␤-TrCP target various substrates for ubiquitylation. The SCF complex consists of four components: the invariable subunits Skp1, Cul1 and Rbx1 and a variable F-box protein that serves as a receptor for target proteins and thereby determines target specificity. Among the many F-box proteins, Skp2, Fbw7 and ␤-TrCP have been shown to control the abundance of cell cycle regulators.

stages of the cell cycle, suggesting that the ubiquitylation machinery for each cyclin is distinct and highly specific. The ubiquitin–proteasome pathway of protein degradation comprises two discrete steps: the covalent attachment of multiple ubiquitin molecules to the protein substrate and degradation of the polyubiquitylated protein by the 26S proteasome complex [4,5]. The attachment of ubiquitin to target proteins is mediated by at least three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin ligase (E3). The E3 components are thought to be primarily responsible for the specific recognition of each of the large number of target proteins. These apparently conflicting characteristics, versatility and specificity of E3 enzymes implicate the existence of numerous such ligases, the genes for which likely account for a substantial proportion (more than several percent) of all human genes. Two major types of E3 enzyme are thought to regulate cell cycle progression: the anaphase-promoting complex or cyclosome (APC/C) and the SCF complex [3,6–8]. The APC/C is required for the separation of sister chromatids at anaphase and for the exit of cells from M phase into G1 , events that are mediated by the ubiquitylation of anaphase inhibitors known as securins (Pds1 or Cut2) and of mitotic cyclins, respectively [9–12]. The SCF complex was thought primarily to regulate G1 –S progression, given that it targets G1 cyclins and CKIs for ubiquitylation [13–15]. However, this complex has subsequently been found to play important roles during most phases of the cell cycle. The SCF complex consists of the invariable components Skp1, Cul1 and Rbx1 as well as a variable component, known

as an F-box protein, that binds to Skp1 through its F-box motif and is responsible for substrate recognition (Fig. 1) [7]. More than 70 F-box proteins have been identified in humans. This large number of F-box proteins, in combination with the core complex of Skp1, Cul1 and Rbx1 as well as associated E2 proteins, thus provides the basis for multiple substrate-specific ubiquitylation pathways. Only a few F-box proteins have been well characterized, however, the substrates and physiological functions of the others being largely unknown. In this article, the properties of three mammalian F-box proteins—Skp2, Fbw7 and ␤-TrCP1—that have been implicated in cell cycle regulation by the results of both biochemical and genetic studies are reviewed. Furthermore, the relation between dysregulation of these F-box proteins and human cancer will also be addressed.

2. Skp2 targets CKIs for degradation The discovery of Skp2 preceded the identification of Fbox proteins as components of the SCF ubiquitin ligase. A substantial fraction of cyclin A–Cdk2 complexes was found to be associated with three proteins (p9, p19 and p45) in transformed cells [16]. Although p9, p19 and p45 are now known as Cks1 (or Cks2), Skp1 and Skp2, respectively, the functions of p19 (Skp1) and p45 (Skp2) were not known at the time of their discovery. Several important characteristics of Skp2 were, however, described in this early study, including the facts that the abundance of Skp2 is greatly increased in many transformed cells and that Skp2 expression is maximal in S and G2 phases of the cell cycle, not in G1 .

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Three laboratories subsequently showed independently that Skp2 binds to and mediates the ubiquitylation of the CKI p27 [17–19], which was known to be degraded via the ubiquitin–proteasome pathway in G1 phase [20,21]. Furthermore, Skp2 was found to target only p27 molecules in which threonine-187 are phosphorylated. These observations were consistent with the previous demonstration that the stability of p27 is regulated by phosphorylation on threonine-187, probably by the cyclin E–Cdk2 complex [22–24]. This biochemical evidence that Skp2 might function as a specificity factor in p27 ubiquitylation was reinforced by genetic evidence showing that p27 accumulates at high levels in mice that lack Skp2 [8,25]. Skp2 was also found to require a cofactor, Cks1, for the recognition of phosphorylated p27 [26,27]. It thus became widely accepted that Skp2 mediates p27 degradation at the G1 –S transition of the cell cycle to activate cyclin E–Cdk2 kinase complex. The story turned out not to be quite so simple, however. In normal cells, the amount of p27 is high during G0 phase of the cell cycle but decreases rapidly on re-entry of cells into G1 phase [28]. For example, mitogenic activation of resting lymphocytes or re-exposure of serum-deprived embryonic fibroblasts to serum induces rapid degradation of p27 between 3 and 9 h after stimulation. However, Skp2 is not expressed until early S phase (18–24 h after stimulation), unequivocally later than the degradation of p27 apparent at G0 –G1 [28,29] (Fig. 2A). Moreover, p27 is exported from the nucleus to the cytoplasm at G0 –G1 [29–32], whereas Skp2 is restricted to the nucleus [33,34]. These discrepancies between the temporal and spatial patterns of p27 expression and those of Skp2 expression suggested the existence of Skp2-independent pathway for the degradation of p27. Indeed, the down-regulation of p27 at the G0 –G1 transition occurs normally in Skp2−/− cells and is sensitive to proteasome inhibitors, indicating that p27 is degraded at G0 –G1 by a proteasome-dependent, but Skp2-independent, mechanism [28] (Fig. 2B). Biochemical analysis of crude extracts of Skp2−/− cells revealed the presence in the cytoplasmic fraction of a Skp2-independent E3 activity that mediates the ubiquitylation of p27. This ubiquitylation of p27 is not dependent on the phosphorylation of threonine-187, which is a prerequisite for Skp2-mediated ubiquitylation. We have recently purified an E3 enzyme, designated Kip1 ubiquitylation-promoting complex (KPC), that interacts with and ubiquitylates p27 in G1 phase and is localized to the cytoplasm of mammalian cells [35]. In contrast to the normal degradation of p27 at G1 phase apparent in Skp2−/− cells, these cells exhibit an abnormal accumulation of p27 during S and G2 phases [28]. This abnormality is associated with prominent cellular phenotypes—including nuclear enlargement, polyploidy and an increased number of centrosomes—that are likely due to overreplication of chromosomes and centrosomes [25] (Table 1). We have recently shown that Skp2−/− ;p27−/− mice do not exhibit such overreplication phenotypes, suggesting that p27 accumulation in S and G2 phases is required for

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Fig. 2. Ubiquitylation of the CKI p27 mediated via Skp2-dependent and Skp2-independent pathways. (A) Expression of p27 and Skp2 during the cell cycle. Freshly isolated mouse lymphocytes in G0 phase were stimulated with phorbol ester and calcium ionophore for the indicated times, after which cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins. Whereas p27 is rapidly destroyed between 3 and 9 h after the onset of mitogenic stimulation and its abundance then remains low up to 48 h, Skp2 is not expressed in G0 and G1 phases but is abundant during S and G2 phases. (B) Degradation of p27 at G1 phase occurs normally in Skp2−/− lymphocytes. Cell lysates were then subjected to immunoblot analysis with antibodies to p27 (upper panels). The degradation of p27 in G1 phase occurs similarly in both Skp2+/+ and Skp2−/− cells, whereas p27 accumulates in S and G2 phases only in the Skp2−/− cells. These data suggest that the degradation of p27 in G1 phase is independent of Skp2, whereas that in S and G2 phases is dependent on Skp2 (lower panel). (C) Regulation of p27 degradation both by KPC to control Cdk2 activity at the G1 –S transition and by Skp2 to control Cdc2 activity during G2 –M progression.

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Table 1 Phenotypes of mice that lack the F-box proteins Skp2, Fbw7, or ␤-TrCP1 F-box protein

General defects

Tissue or cell abnormalities

Protein accumulation

Ref.

Skp2

Reduced body size; reduced fertility Embryonic death (E10.5); retarded embryogenesis

Slow growth rate; nuclear enlargement; polyploidy; centrosome overduplication Defect in vascular remodelling; abnormal arteriovenous differentiation; cardiac developmental defect; impaired hematopoiesis Slow growth rate; polyploidy; centrosome overduplication; defective spermatogenesis

p27, p21, p57, p130, cyclin E, Cdk9, c-Myc

[25]

Notch4, c-Myc, cyclin Ea

[63,64]

I␬B␣ and I␬B␤ (transient), ␤-catenin (nuclear), Emi1

[147,148]

Fbw7

␤-TrCP1

a

Reduced male fertility

Cyclin E accumulation was apparent only in the placenta, not in the embryo.

their development [36]. A substantial amount of p27 associates with Cdc2 in the cells of Skp2−/− mice, resulting in a reduction in Cdc2-associated kinase activity. The lack of p27 degradation in G2 phase in Skp2−/− cells may thus result in suppression of Cdc2 activity and consequent inhibition of entry into M phase. These data suggest that p27 proteolysis is necessary for the activation not only of Cdk2 but also of Cdc2, and that Skp2 contributes to regulation of G2 –M progression by mediating the degradation of p27 (Fig. 2C). In addition to p27, other cell cycle regulators have been implicated as potential substrates of Skp2. These proteins include the p27-related CKIs p21 [37,38] and p57 [39], p130 [40,41], cyclin A [25], cyclin D1 [37], free cyclin E [25], E2F-1 [42], Orc1 [43], Cdt1 [44–47], Cdk9 [48], c-Myc [49,50] and B-Myb [51] (Fig. 3). Of these proteins, cyclin A, cyclin D1, E2F-1 and Cdt1 do not accumulate in Skp2−/− cells [25] (unpublished observations), suggesting either that they are not bonafide substrates of Skp2 or that there is redundancy that allows for their ubiquitylation in the absence of Skp2. The fact that most of the cellular and histopathologic defects apparent in Skp2-deficient mice are not observed in Skp2−/− ; p27−/− mice suggests that p27 is the major substrate of Skp2 [36]. However, the observation that the Skp2−/− ; p27−/− double mutant appears similar but

not identical to the p27−/− single mutant constitutes genetic evidence for the notion that, although p27 might be the main target of Skp2, Skp2 likely also mediates the ubiquitylation of other substrates in the physiological context.

3. Fbw7 contributes to the degradation of growth promoters Fbw7 (also known as SEL-10, hCdc4, or hAgo) was first identified in Caenorhabditis elegans as a negative regulator of Notch (LIN-12) [52]. Biochemical evidence has suggested that both Notch1 [53–55] and Notch4 [52,55] as well as presenilin [56] and cyclin E [57–59] are targets for ubiquitylation mediated by mammalian Fbw7. Furthermore, the products of two proto-oncogenes, c-Jun and c-Myc, have recently been added to the list of proteins that are ubiquitylated via the Fbw7-dependent pathway [60–62]. We and others have generated mice deficient in Fbw7 and found that the embryos die in utero at embryonic day 10.5 manifesting marked abnormalities in vascular development [63,64] (Table 1). Vascular remodeling was shown to be impaired in the brain and yolk sac, and the major trunk veins were not formed. Notch4, an endothelial cell-specific mammalian isoform of Notch,

Fig. 3. Various targets of Skp2 and Fbw7. The size of the circles represents the proposed relative physiological importance of the substrates.

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accumulates in Fbw7−/− embryos, resulting in an increased expression of Hey1, a transcriptional repressor that acts downstream of Notch and is implicated in vascular development. Expression of Notch1, -2, or -3 or of cyclin E was unaffected in Fbw7−/− embryos [63], whereas the abundance of cyclin E was increased only in the placenta [64]. Mammalian Fbw7 thus appears to play an indispensable role in negative regulation of the Notch4–Hey1 signaling pathway and is required for vascular development during embryogenesis.

4. Common targets of Skp2 and Fbw7 At least two cell cycle regulators, cyclin E and c-Myc, are targeted by both Skp2 and Fbw7 (Fig. 3). The mechanisms for the degradation of cyclin E appear complex. The Skp2–Cul1 complex and Cul3 interact with the free, nonphosphorylated form of cyclin E [25,65–67], thereby mediating its ubiquitylation-dependent proteolysis. In parallel, SCFFbw7 is thought to target phosphorylated cyclin E complexed with Cdk2 [57–59,68,69]. Fbw7 recognizes cyclin E phosphorylated on threonine-380, which is a critical determinant of cyclin E stability. However, mouse embryos that lack Fbw7 (which die in utero) show neither accumulation of cyclin E nor an increase in Cdk2 activity [63], whereas Skp2−/− mice manifest marked accumulation of cyclin E in cell nuclei. Thus, Fbw7 appears to be dispensable for cyclin E degradation, at least until mid-embryogenesis. In contrast, depletion of Fbw7 by RNA interference resulted in the accumulation of cyclin E in cultured cells [58,61], suggesting that Fbw7 might be required for cyclin E turnover in adult tissues. Given that p27 accumulates to a high level in Skp2−/− cells, it is possible that phosphorylation of cyclin E by associated Cdk2 is inhibited by this CKI, resulting in stabilization of cyclin E. The accumulation of cyclin E in these cells has thus been thought to be secondary to the increase in the abundance of p27. Our recent study with Skp2−/− ; p27−/− mice, however, showed that cyclin E degradation is also impaired in the double-mutant mice, providing genetic evidence that the altered expression of cyclin E in Skp2−/− cells is independent of p27 accumulation [36]. Our previous biochemical observations that Skp2 interacts with free cyclin E and promotes its ubiquitylation both in vitro and in vivo, and that cyclin E degradation is impaired, resulting in loss of periodicity of cyclin E expression, in Skp2−/− cells [25], are therefore consistent with the genetic evidence that Skp2 directly targets cyclin E. It remains possible that the accumulation of CKIs such as p21, p57 and p130 in Skp2−/− mice contributes to the stabilization of cyclin E, although our observation that the kinase activity of Cdk2 is unchanged in Skp2−/− cells suggests against this possibility [36]. c-Myc has been shown to undergo ubiquitylation and degradation by the proteasome [70,71]. The region of c-Myc that signals its ubiquitylation (the degron) overlaps with the transactivation domain [72]. Two highly conserved sequence

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elements, Myc box 1 (MB1) and MB2, in the transactivation domain have been implicated both in the proteolysis of cMyc as well as in its transactivation and oncogenic activities [73,74]. In particular, phosphorylation of threonine-58 and serine-62 in MB1 is an important determinant of c-Myc stability [75–77]. Consistent with their effect on c-Myc stability, these two residues are frequently mutated in various human tumors [78]. We and others recently showed that Skp2 binds to c-Myc via its MB2 and helix–loop–helix–leucine zipper (HLH-Zip) domains and thereby mediates its ubiquitylation and degradation. Unexpectedly, Skp2 was also found to increase the transactivation activity of c-Myc, suggesting that Skp2 is a transcriptional cofactor [49,50]. In addition, Fbw7 interacts with and promotes the degradation of c-Myc in a manner dependent on phosphorylation of MB1 [61,62]. The peptide sequence surrounding threonine-58 of c-Myc conforms to a motif known as the Cdc4 phospho-degron (CPD) [79], as does the cyclin E degron surrounding threonine-380. Accumulation of c-Myc is also apparent in mouse Skp2−/− or Fbw7−/− cells [61]. These observations suggest that two Fbox proteins, Fbw7 and Skp2, differentially regulate c-Myc stability by targeting MB1 and MB2 domains, respectively.

5. Skp2 as an oncoprotein and Fbw7 as a tumor suppressor It has been widely accepted that p27 functions as a tumor suppressor on the basis not only of its activity as a CKI but also of evidence both from mouse models [80–83] and from clinical studies of human cancer patients showing a marked correlation between reduced p27 levels and poor prognosis [84–87]. Indeed, a reduction in the abundance of p27 is common in many types of human malignancies [88]. Unlike other tumor suppressors, such as p53 or Rb, however, deletion of the p27 gene is an uncommon event in the development of human cancers [89–93], suggesting that dysregulation of p27 expression in human tumors is often due to post-transcriptional mechanisms. It has become evident that expression of the F-box protein SKP2 is inversely correlated with p27 in many cancers [94–103] and also with the grade of malignancy in certain human tumors [94,95,101–103]. In addition, frequent amplification and overexpression of the SKP2 gene has been observed in primary small cell lung cancers [100], and in cell lines expressing high-risk human papilloma virus [104]. The oncogenic potential of Skp2 has also been demonstrated in mice [94,105]. Double-transgenic mice that coexpress Skp2 and activated N-Ras in the T cell lineage develop T cell lymphoma at a rate of 75%, which is three times that observed in mice harboring the N-Ras transgene alone [94]. Such evidence supports the notion that Skp2 is a growth promoter and an oncoprotein. Given that Skp2 targets not only p27 but also p21, p57, p130, cyclin E, c-Myc and other substrates, it has been

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unclear to what extent the degradation of regulators other than p27 by the Skp2-mediated pathway contributes to cancer development. Enhanced degradation of p21, p57 and p130 by Skp2-dependent proteolysis might be expected to show a cooperative effect with the reduction in p27 levels in this regard. Conversely, down-regulation of cyclin E and c-Myc by Skp2 might antagonize the oncogenic effect attributable to Skp2-mediated depletion of these CKIs. As described above, however, Skp2 does not appear to regulate the activity of cyclin E directly but rather affects it indirectly through control of the pool of free cyclin E [25,36]. Furthermore, although Skp2 mediates ubiquitylation and degradation of c-Myc, it enhances c-Myc-induced S phase transition and increases the transactivation activity of c-Myc [49,50]. The effects of Skp2 on cyclin E and c-Myc thus do not appear to inhibit progression of the cell cycle. These results suggest that the main function of Skp2 is to mediate degradation of tumor suppressor proteins (p27, p21, p57 and p130) in order to promote cell cycle progression, and activation of the oncoprotein c-Myc may further enhance this effect. In contrast to the complexity of the role of Skp2, which targets both positive and negative regulators of the cell cycle, the concept of Fbw7 as an oncosuppressor protein is relatively straightforward. All of the identified substrates of Fbw7—including cyclin E, Notch1, Notch4, presenilin, cMyc and c-Jun—appear to function as cell cycle activators and oncogenic proteins (Fig. 3). The genetic inactivation of Fbw7 in cancer cells was recently found to confer a striking phenotype characterized by the presence of micronuclei and chromosomal instability [106]. Overexpression of cyclin E was able to mimic the induction of micronucleus formation observed in the Fbw7-deficient cells [107], whereas depletion of cyclin E in the Fbw7-ablated cancer cells reduced the extent of micronucleus formation [106]. These data suggest that dysregulation of cyclin E abundance due to the loss of Fbw7 may lead to tumorigenesis. Indeed, the prevalence of FBW7 mutations in human cancers has recently turned out to be higher than expected a few years ago. Whereas such mutations were originally identified in a few breast and ovarian cancer cell lines [57,59], they have subsequently been found in endometrial and colorectal cancers [106,108]. We have recently identified the mouse Fbw7 gene as a p53-dependent tumor suppressor gene [109]. Fbw7+/− mice have greater susceptibility to radiation-induced tumorigenesis, but most tumors retain and express the wild-type allele, indicating that Fbxw7 is a haploinsufficient tumor suppressor gene. Dysregulation not only of cyclin E but also of other substrates of Fbw7 may be integral to cancer development. Truncated Notch proteins exhibit transforming activity both in vitro [110–112] and in animal models [113–115]. Furthermore, dysregulated expression of wild-type Notch or of Notch ligands or downstream targets has been detected in many human malignancies [116–121]; Notch1 was recently shown to be a downstream effector of oncogenic Ras; and depletion of Notch1 in Ras-transformed human cells was sufficient to abolish key elements of the neoplastic phenotype

in vitro and in vivo [122]. Notch4 was originally identified as Int3, a proto-oncogene that is a frequent target for integration of mouse mammary tumor virus in mammary carcinomas [123–125]. The expression level of c-Myc is increased in many malignant tumors. Given that many c-MYC mutations affect the stability of the encoded protein [74,78], its turnover is thought to be a critical determinant of carcinogenesis. The c-Jun oncoprotein is a major component of the transcription factor AP-1, the constitutive activation of which is apparent in various types of human tumor cells, suggesting that AP-1 plays an important role in human oncogenesis [126]. Mutation of human FBW7 may thus result in impairment of the degradation of these various substrates and their consequent accumulation, which, together with the accumulation of cyclin E, might then contribute to carcinogenesis. 6. ␤-TrCP is a versatile F-box protein involved in many signaling pathways ␤-TrCP proteins are highly conserved through evolution and include Drosophila Slimb [127], Xenopus ␤-TrCP [128], as well as mammalian ␤-TrCP1 (also termed Fbw1a or FWD1) [129–137] and ␤-TrCP2 (also known as Fbw1b or HOS) [138,139]. The consensus sequence recognized by ␤-TrCP is the DSG(X)2+n S destruction motif, the serines of which are phosphorylated by specific kinases [140]. In Drosophila, deletion of the Slimb gene results in accumulation of the ␤-catenin ortholog Armadillo [127], suggesting that mammalian ␤-TrCP proteins might mediate the ubiquitylation of ␤-catenin, an important mediator of Wnt signaling. Both ␤-catenin and I␬B, an inhibitor of the transcription factor NF-␬B, were known to be ubiquitylated, and the degrons of both proteins are similar to each other and evolutionarily conserved [141]. These observations led many laboratories to determine whether ␤-TrCP targets ␤-catenin and I␬B␣ for ubiquitylation and degradation, and this was indeed found to be the case [130–133,135–139]. Human ␤-TrCP1 was also shown to interact with the Vpu protein of human immunodeficiency virus-type 1, which contains a DSGXXS motif [129]. Additional substrates that are degraded as a result of ␤-TrCP-dependent ubiquitylation have been found to include I␬B␤ and I␬B␧ [134], the transcription factor ATF4 (CREB2) [142], the p105 [143] and p100 [144] subunits of NF-␬B and the discs large (hDlg) tumor suppressor [145] (Fig. 1). 7. Role of ␤-TrCP in regulation of the cell cycle The observation that cells of Drosophila Slimb mutants exhibit additional centrosomes and mitotic defects indicated that ␤-TrCP might participate in regulation of cell division [146]. Cells of mice that lack ␤-TrCP1 (β-TrCP1–/– mice) also manifest a partial defect in the ability to eliminate ␤catenin and I␬B in certain situations [147] (Table 1). Although both Wnt–␤-catenin and NF-␬B signaling pathways

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converge to regulate the activity of the promoter of the cyclin D1 gene and cell cycle progression, recent evidence suggests that ␤-TrCP also exerts direct control of cell cycle regulators. Defects in male fertility accompanied by testicular accumulation of spermatocytes in metaphase I were observed in β-TrCP1−/− mice [148]. Furthermore, β-TrCP1−/− fibroblasts manifest polyploidy, centrosome overduplication, impaired progression through mitosis and a reduced growth rate [147,148]. Evidence suggests that these abnormalities are attributable to stabilization of Emi1, an inhibitor of the APC/C. Emi1 inhibits the APC/C in S and G2 phases of the cell cycle to ensure that the cyclin B–Cdc2 complex does not prematurely activate the APC/C in early mitosis [149]. ␤TrCP recognizes Emi1 after phosphorylation of a DSGXXS consensus site of the latter by Cdc2 in M phase [148,150]. In cells of β-TrCP1−/− mice, abnormal accumulation of Emi1 stabilizes APC/C substrates such as cyclin B and results in mitotic catastrophe, including centrosome overduplication. Down-regulation of Wee1, a kinase that phosphorylates and thereby inhibits the activity of Cdc2, at the onset of mitosis is required for the rapid activation of Cdc2. Human somatic Wee1 (Wee1A) is down-regulated by ubiquitindependent degradation that is associated with its phosphorylation. Ubiquitylation of Wee1A was originally reported to be mediated by the F-box protein Tome-1, which is targeted by the APC/C for destruction in G1 phase [151]. Recently, however, ␤-TrCP was also shown to function as an E3 in Wee1A ubiquitylation [152]. Although Wee1A lacks a canonical DSGXXS motif, two serine residues at positions 53 and 123 were found to be the most important phosphorylation sites for the recognition of Wee1A by ␤-TrCP. Polo-like kinase 1 (Plk1) and Cdc2 are responsible for the phosphorylation of serine-53 and serine-123 of Wee1A, respectively [152]. The stability of Wee1A is increased by mutation of these residues or by depletion of ␤-TrCP by RNA interference. Indeed, ␤TrCP-dependent degradation of Wee1A appears to be critical for the normal onset of M phase. These findings suggest the existence of a feedback loop between Cdc2 and Wee1A that ensures the rapid activation of Cdc2 when cells are ready to divide. ␤-TrCP, thus, appears to play an important role in determination of the timing of the onset of mitosis by regulating the abundance of Emi1 and Wee1A. ␤-TrCP also regulates the abundance of Cdc25A, a protein phosphatase that dephosphorylates and thereby activates CDKs, both during S phase and in response to DNA damage [153]. Cdc25A contains a consensus motif (DSGXXXXS) for ubiquitylation by ␤-TrCP. In response to DNA damage or stalled DNA replication, the protein kinases ATM and ATR activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. Phosphorylation of Cdc25A on serine-82, the serine within the DSGXXXXS motif, allows the protein to be recognized by ␤-TrCP and ubiquitylated. Depletion of ␤-TrCP1 and ␤-TrCP2 by RNA interference resulted in accumulation of Cdc25A in cells progressing through S phase and prevented the degradation of Cdc25A induced by ionizing radiation.

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