PI3K-dependent phosphorylation of Fbw7 modulates substrate degradation and activity

The Fbw7 tumor suppressor gene encodes the substrate recognition subunit of the SCF ubiquitin ligase, which targets for degradation a range of oncogenic proteins in a phosphorylation-dependent manner. Substrate phosphorylation is thought to be the main mechanism that ensures timely destruction of Fbw7 substrates. We show here that PI3K dependent phosphorylation of Fbw7 stimulates its ability to ubiquitinate and degrade its substrates. Mutation of the phosphorylation site destabilizes Fbw7 and attenuates degradation of cyclin E and Myc leading to the enhanced expression of a subset of Myc target genes. We suggest that PI3K-dependent phosphorylation of Fbw7 controls the balance between turnover of Fbw7 and its substrates to fine-tune their activity.     

Ubiquitination and Degradation of the Hominoid-Specific Oncoprotein TBC1D3 Is Mediated by CUL7 E3 Ligase

Expression of the hominoid-specific TBC1D3 oncoprotein enhances growth factor receptor signaling and subsequently promotes cellular proliferation and survival. Here we report that TBC1D3 is degraded in response to growth factor signaling, suggesting that TBC1D3 expression is regulated by a growth factor-driven negative feedback loop. To gain a better understanding of how TBC1D3 is regulated, we studied the effects of growth factor receptor signaling on TBC1D3 post-translational processing and turnover. Using a yeast two-hybrid screen, we identified CUL7, the scaffolding subunit of the CUL7 E3 ligase complex, as a TBC1D3-interacting protein. We show that CUL7 E3 ligase ubiquitinates TBC1D3 in response to serum stimulation. Moreover, TBC1D3 recruits F-box 8 (Fbw8), the substrate recognition domain of CUL7 E3 ligase, in pull-down experiments and in an in vitro assay. Importantly, alkaline phosphatase treatment of TBC1D3 suppresses its ability to recruit Fbw8, indicating that TBC1D3 phosphorylation is critical for its ubiquitination and degradation. We conclude that serum- and growth factor-stimulated TBC1D3 ubiquitination and degradation are regulated by its interaction with CUL7-Fbw8.     

Inhibition of ubiquitin ligase F-box and WD repeat domain-containing 7α (Fbw7α) causes hepatosteatosis through Krüppel-like factor 5 (KLF5)/peroxisome proliferator-activated receptor γ2 (PPARγ2) pathway but not SREBP-1c protein in mice

F-box and WD repeat domain-containing 7α (Fbw7α) is the substrate recognition component of a ubiquitin ligase that controls the degradation of factors involved in cellular growth, including c-Myc, cyclin E, and c-Jun. In addition, Fbw7α degrades the nuclear form of sterol regulatory element-binding protein (SREBP)-1a, a global regulator of lipid synthesis, particularly during mitosis in cultured cells. This study investigated the in vivo role of Fbw7α in hepatic lipid metabolism. siRNA knockdown of Fbw7α in mice caused marked hepatosteatosis with the accumulation of triglycerides. However, inhibition of Fbw7α did not change the level of nuclear SREBP-1 protein or the expression of genes involved in fatty acid synthesis and oxidation. In vivo experiments on the gain and loss of Fbw7α function indicated that Fbw7α regulated the expression of peroxisome proliferator-activated receptor (PPAR) γ2 and its target genes involved in fatty acid uptake and triglyceride synthesis. These genes included fatty acid transporter Cd36, diacylglycerol acyltransferase 1 (Dgat1), and fat-specific protein 27 (Cidec). The regulation of PPARγ2 by Fbw7α was mediated, at least in part, by the direct degradation of the Krüppel-like factor 5 (KLF5) protein, upstream of PPARγ2 expression. Hepatic Fbw7α contributes to normal fatty acid and triglyceride metabolism, functions that represent novel aspects of this cell growth regulator.     

The Stomatin-Like Protein SLP-1 and Cdk2 Interact with the F-Box Protein Fbw7-γ

Control of cellular proliferation is critical to cell viability. The F-box protein Fbw7 (hAgo/hCdc4/FBXW7) functions as a specificity factor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex and targets several proteins required for cellular proliferation for ubiquitin-mediated destruction. Fbw7 exists as three splice variants but the mechanistic role of each is not entirely clear. We examined the regulation of the Fbw7-γ isoform, which has been implicated in the degradation of c-Myc. We show here that Fbw7-γ is an unstable protein and that its turnover is proteasome-dependent in transformed cells. Using a two-hybrid screen, we identified a novel interaction partner, SLP-1, which binds the N-terminal domain of Fbw7-γ. Overexpression of SLP-1 inhibits the degradation of Fbw7-γ, suggesting that this interaction can happen in vivo. When Fbw7-γ is stabilized by overexpression of SLP-1, c-Myc protein abundance decreases, suggesting that the SCF^Fbw7-γ complex maintains activity. We demonstrate that Cdk2 also binds the N-terminal domain of Fbw7-γ as well as SLP-1. Interestingly, co-expression of Cdk2 and SLP-1 does not inhibit Fbw7-γ degradation, suggesting that Cdk2 and SLP-1 may have opposing functions.     

Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome

We determined composition and relative roles of deubiquitylating proteins associated with the 26S proteasome in mammalian cells. Three deubiquitylating activities were associated with the 26S proteasome: two from constituent subunits, Rpn11/S13 and Uch37, and one from a reversibly associated protein, Usp14. RNA interference (RNAi) of Rpn11/S13 inhibited cell growth, decreased cellular proteasome activity via disrupted 26S proteasome assembly, and inhibited cellular protein degradation. In contrast, RNAi of Uch37 or Usp14 had no detectable effect on cell growth, proteasome structure or proteolytic capacity, but accelerated cellular protein degradation. RNAi of both Uch37 and Usp14 also had no effect on proteasome structure or proteolytic capacity, but inhibited cellular protein degradation. Thus, proper proteasomal processing of ubiquitylated substrates requires Rpn11 plus either Uch37 or Usp14. Although the latter proteins feature redundant deubiquitylation functions, they also appear to exert noncatalyic effects on proteasome activity that are similar to but independent of one another. These results reveal unexpected functional relationships among multiple deubiquitylating proteins and suggest a model for mammalian 26S proteasome function whereby their concerted action governs proteasome function by linking deubiquitylation to substrate hydrolysis.     

Nucleophosmin and its AML-associated mutant regulate c-Myc turnover through Fbw7 gamma

Mutations leading to aberrant cytoplasmic localization of nucleophosmin (NPM) are the most frequent genetic alteration in acute myelogenous leukemia (AML). NPM binds the Arf tumor suppressor and protects it from degradation. The AML-associated NPM mutant (NPMmut) also binds p19Arf but is unable to protect it from degradation, which suggests that inactivation of p19Arf contributes to leukemogenesis in AMLs. We report here that NPM regulates turnover of the c-Myc oncoprotein by acting on the F-box protein Fbw7gamma, a component of the E3 ligase complex involved in the ubiquitination and proteasome degradation of c-Myc. NPM was required for nucleolar localization and stabilization of Fbw7gamma. As a consequence, c-Myc was stabilized in cells lacking NPM. Expression of NPMmut also led to c-Myc stabilization because of its ability to interact with Fbw7gamma and delocalize it to the cytoplasm, where it is degraded. Because Fbw7 induces degradation of other growth-promoting proteins, the NPM-Fbw7 interaction emerges as a central tumor suppressor mechanism in human cancer.     

Mechanisms of Tumor Suppression by the SCFFbw7

SCF ubiquitin ligases regulate the degradation of many proteins involved in thecontrol of cell division and growth. F-box proteins are the SCF components that bind tosubstrates, and this binding is usually signaled by substrate phosphorylation. TheFbw7/hCdc4 F-box protein was first recognized by its binding to cyclin E, and theSCFFbw7 is now known to target c-Myc, c-Jun and Notch for degradation, in addition toits role in cyclin E proteolysis. Fbw7 thus negatively regulates several keyoncoproteins. Accordingly, Fbw7 is a tumor suppressor that is mutated in a widespectrum of human cancers, and Fbw7 functions as a haploinsufficient tumor suppressorin mice. Because there are three Fbw7 isoforms that reside in different subcellularcompartments, as well as multiple Fbw7 substrates that are the products of protooncogenes,the mechanisms of tumor suppression by Fbw7 are complex and incompletelyunderstood. In this review we discuss the activities of the SCFFbw7 in the context of itsrole as a tumor suppressor and highlight recent findings demonstrating that dominantoncogenes disable Fbw7 function.     

Stabilization of Myc through Heterotypic Poly-Ubiquitination by mLANA Is Critical for γ-Herpesvirus Lymphoproliferation

Host colonization by lymphotropic γ-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of γ-herpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic γ-herpesvirus MuHV-4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B-cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological post-translational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly-ubiquitination mediated via the viral E3 ubiquitin-ligase mLANA protein. EC₅S^mLANA modulates cellular control of Myc turnover by antagonizing SCF^Fbw7 mediated proteasomal degradation of Myc, mimicking SCF^β-TrCP. The findings here reported reveal that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease.     

The Drosophila F box protein archipelago regulates dMyc protein levels in vivo

BACKGROUND: The Myc oncoprotein is an important regulator of cellular growth in metazoan organisms. Its levels and activity are tightly controlled in vivo by a variety of mechanisms. In normal cells, Myc protein is rapidly degraded, but the mechanism of its degradation is not well understood. RESULTS: Here we present genetic and biochemical evidence that Archipelago (Ago), the F box component of an SCF-ubiquitin ligase and the Drosophila ortholog of a human tumor suppressor, negatively regulates the levels and activity of Drosophila Myc (dMyc) protein in vivo. Mutations in archipelago (ago) result in strongly elevated dMyc protein levels and increased tissue growth. Genetic interactions indicate that ago antagonizes dMyc function during development. Archipelago binds dMyc and regulates its stability, and the ability of Ago to bind dMyc in vitro correlates with its ability to inhibit dMyc accumulation in vivo. CONCLUSIONS: Our data indicate that archipelago is an important inhibitor of dMyc in developing tissues. Because archipelago can also regulate Cyclin E levels and Notch activity, these results indicate how a single F box protein can be responsible for the degradation of key components of multiple pathways that control growth and cell cycle progression.     

USP4 inhibits p53 through deubiquitinating and stabilizing ARF-BP1

Tumour suppressor p53 levels in the cell are tightly regulated by controlled degradation through ubiquitin ligases including Mdm2, COP1, Pirh2, and ARF-BP1. The ubiquitination process is reversible via deubiquitinating enzymes, such as ubiquitin-specific peptidases (USPs). In this study, we identified ubiquitin-specific peptidase 4 (USP4) as an important regulator of p53. USP4 interacts directly with and deubiquitinates ARF-BP1, leading to the stabilization of ARF-BP1 and subsequent reduction of p53 levels. Usp4 knockout mice are viable and developmentally normal, but showed enhanced apoptosis in thymus and spleen in response to ionizing radiation. Compared with wild-type mouse embryonic fibroblasts (MEFs), Usp4-/- MEFs exhibited retarded growth, premature cellular senescence, resistance to oncogenic transformation, and hyperactive DNA damage checkpoints, consistent with upregulated levels and activity of p53 in the absence of USP4. Finally, we showed that USP4 is overexpressed in several types of human cancer, suggesting that USP4 is a potential oncogene.     

A nucleolar isoform of the Fbw7 ubiquitin ligase regulates c-Myc and cell size

The human tumor suppressor Fbw7/hCdc4 functions as a phosphoepitope-specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination of cyclin E , Notch , c-Jun and c-Myc . Fbw7 loss in cancer may thus have profound effects on the pathways that govern cell division, differentiation, apoptosis, and cell growth. Fbw7-inactivating mutations occur in human tumor cell lines and primary cancers , and Fbw7 loss in cultured cells causes genetic instability . In mice, deletion of Fbw7 leads to embryonic lethality associated with defective Notch and cyclin E regulation . The human Fbw7 locus encodes three protein isoforms (Fbw7alpha, Fbw7beta, and Fbw7gamma) . We find that these isoforms occupy discrete subcellular compartments and have identified cis-acting localization signals within each isoform. Surprisingly, the Fbw7gamma isoform is nucleolar, colocalizes with c-Myc when the proteasome is inhibited, and regulates nucleolar c-Myc accumulation. Moreover, we find that knockdown of Fbw7 increases cell size consistent with its ability to control c-Myc levels in the nucleolus. We suggest that interactions between c-Myc and Fbw7gamma within the nucleolus regulate c-Myc's growth-promoting function and that c-Myc activation is likely to be an important oncogenic consequence of Fbw7 loss in cancers.     

Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiquitin ligase

The SCF(FBW7) ubiquitin ligase degrades proteins involved in cell division, growth, and differentiation and is commonly mutated in cancers. The Fbw7 locus encodes three protein isoforms that occupy distinct subcellular localizations, suggesting that each has unique functions. We used gene targeting to create isoform-specific Fbw7-null mutations in human cells and found that the nucleoplasmic Fbw7alpha isoform accounts for almost all Fbw7 activity toward cyclin E, c-Myc, and sterol regulatory element binding protein 1. Cyclin E sensitivity to Fbw7 varies during the cell cycle, and this correlates with changes in cyclin E-cyclin-dependent kinase 2 (CDK2)-specific activity, cyclin E autophosphorylation, and CDK2 inhibitory phosphorylation. These data suggest that oscillations in cyclin E-CDK2-specific activity during the cell cycle regulate the timing of cyclin E degradation. Moreover, they highlight the utility of adeno-associated virus-mediated gene targeting in functional analyses of complex loci.     

Induction of apoptosis by Drosophila Myc

Myc proteins are essential regulators of cellular growth and proliferation during normal development. Activating mutations in myc genes result in excessive growth and are frequently associated with human cancers. At the same time, forced expression of Myc sensitizes vertebrate cells towards different pro-apoptotic stimuli. Recently, the ability of overexpressed Myc to induce cell-autonomous apoptosis has been shown to be evolutionarily conserved in Drosophila Myc (dMyc). Here, we show that dMyc induced apoptosis is accompanied by the induction of Drosophila p53 mRNA, but that dp53 activity is not essential for dMyc's ability to induce apoptosis. Conversely, larvae carrying a hypomorphic dmyc mutation are more resistant to the apoptosis-promoting effects of X-irradiation. These data suggest that the control of apoptosis is a physiological function of Myc and that dMyc might play a role in the response to DNA damage.