Two diphosphorylated degrons control c-Myc degradation by the Fbw7 tumor suppressor.

c-Myc (hereafter, Myc) is a cancer driver whose abundance is regulated by the SCFFbw7 ubiquitin ligase and proteasomal degradation. Fbw7 binds to a phosphorylated Myc degron centered at threonine 58 (T58), and mutations of Fbw7 or T58 impair Myc degradation in cancers. Here, we identify a second Fbw7 phosphodegron at Myc T244 that is required for Myc ubiquitylation and acts in concert with T58 to engage Fbw7. While Ras-dependent Myc serine 62 phosphorylation (pS62) is thought to stabilize Myc by preventing Fbw7 binding, we find instead that pS62 greatly enhances Fbw7 binding and is an integral part of a high-affinity degron. Crystallographic studies revealed that both degrons bind Fbw7 in their diphosphorylated forms and that the T244 degron is recognized via a unique mode involving Fbw7 arginine 689 (R689), a mutational hotspot in cancers. These insights have important implications for Myc-associated tumorigenesis and therapeutic strategies targeting Myc stability.

Merkel cell polyomavirus Tumor antigens expressed in Merkel cell carcinoma function independently of the ubiquitin ligases Fbw7 and ß-TrCP.

Merkel cell polyomavirus (MCPyV) accounts for 80% of all Merkel cell carcinoma (MCC) cases through expression of two viral oncoproteins: the truncated large T antigen (LT-t) and small T antigen (ST). MCPyV ST is thought to be the main driver of cellular transformation and has also been shown to increase LT protein levels through the activity of its Large-T Stabilization Domain (LSD). The ST LSD was reported to bind and sequester several ubiquitin ligases, including Fbw7 and ß-TrCP, and thereby stabilize LT-t and several other Fbw7 targets including c-Myc and cyclin E. Therefore, the ST LSD is thought to contribute to transformation by promoting the accumulation of these oncoproteins. Targets of Fbw7 and ß-TrCP contain well-defined, conserved, phospho-degrons. However, as neither MCPyV LT, LT-t nor ST contain the canonical Fbw7 phospho-degron, we sought to further investigate the proposed model of ST stabilization of LT-t and transformation. In this study, we provide several lines of evidence that fail to support a specific interaction between MCPyV T antigens and Fbw7 or ß-TrCP by co-immunoprecipitation or functional consequence. Although MCPyV ST does indeed increase LT protein levels through its Large-T Stabilization domain (LSD), this is accomplished independently of Fbw7. Therefore, our study indicates a need for further investigation into the role and mechanism(s) of MCPyV T antigens in viral replication, latency, transformation, and tumorigenesis.

Tumor suppression by the Fbw7 ubiquitin ligase: mechanisms and opportunities.

Tumor suppressors with widespread impact on carcinogenesis control broad spectra of oncogenic pathways. Protein degradation is an emerging mechanism by which tumor suppressors regulate a diversity of pathways and is exemplified by the SCF(Fbw7) ubiquitin ligase. Rapidly accumulating data indicate that SCF(Fbw7) regulates a network of crucial oncoproteins. Importantly, the FBXW7 gene, which encodes Fbw7, is one of the most frequently mutated genes in human cancers. These studies are yielding important new insights into tumorigenesis and may soon enable therapies targeting the Fbw7 pathway. Here, we focus on the mechanisms and consequences of Fbw7 deregulation in cancers and discuss possible therapeutic approaches.

Fbw7 dimerization determines the specificity and robustness of substrate degradation.

The Fbw7 tumor suppressor targets a broad network of proteins for ubiquitylation. Here we show critical functions for Fbw7 dimerization in regulating the specificity and robustness of degradation. Dimerization enables Fbw7 to target substrates through concerted binding to two suboptimal and independent recognition sites. Accordingly, an endogenous dimerization-deficient Fbw7 mutation stabilizes suboptimal substrates. Dimerization increases Fbw7's robustness by preserving its function in the setting of mutations that disable Fbw7 monomers, thereby buffering against pathogenic mutations. Finally, dimerization regulates Fbw7 stability, and this likely involves Fbw7 trans-autoubiquitylation. Our study reveals novel functions of Fbw7 dimerization and an unanticipated complexity in substrate degradation.

Phosphorylation of eukaryotic elongation factor 2 (eEF2) by cyclin A-cyclin-dependent kinase 2 regulates its inhibition by eEF2 kinase.

Protein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A-cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylation in vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2K in vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity.

Nucleolar targeting of the fbw7 ubiquitin ligase by a pseudosubstrate and glycogen synthase kinase 3.

E3 ubiquitin ligases catalyze protein degradation by the ubiquitin-proteasome system, and their activity is tightly controlled. One level of regulation involves subcellular localization, and the Fbw7 tumor suppressor exemplifies this type of control. Fbw7 is the substrate-binding component of an SCF ubiquitin ligase that degrades critical oncoproteins. Alternative splicing produces three Fbw7 protein isoforms that occupy distinct compartments: Fbw7α is nucleoplasmic, Fbw7ß is cytoplasmic, and Fbw7γ is nucleolar. We found that cancer-associated Fbw7 mutations that disrupt substrate binding prevent Fbw7γ nucleolar localization, implicating a substrate-like interaction in nucleolar targeting. We identified EBNA1-binding protein 2 (Ebp2) as the critical nucleolar factor that directly mediates Fbw7 nucleolar targeting. Ebp2 binds to Fbw7 like a substrate, and this is mediated by an Ebp2 degron that is phosphorylated by glycogen synthase kinase 3. However, despite these canonical substrate-like interactions, Fbw7 binding is largely uncoupled from Ebp2 turnover in vivo. Ebp2 thus acts like a pseudosubstrate that directly recruits Fbw7 to nucleoli.

Identification of CDK2 substrates in human cell lysates.

BACKGROUND: Protein phosphorylation regulates a multitude of biological processes. However, the large number of protein kinases and their substrates generates an enormously complex phosphoproteome. The cyclin-dependent kinases--the CDKs--comprise a class of enzymes that regulate cell cycle progression and play important roles in tumorigenesis. However, despite intense study, only a limited number of mammalian CDK substrates are known. A comprehensive understanding of CDK function requires the identification of their substrate network. RESULTS: We describe a simple and efficient approach to identify potential cyclin A-CDK2 targets in complex cell lysates. Using a kinase engineering strategy combined with chemical enrichment and mass spectrometry, we identified 180 potential cyclin A-CDK2 substrates and more than 200 phosphorylation sites. About 10% of these candidates function within pathways related to cell division, and the vast majority are involved in other fundamental cellular processes. We have validated several candidates as direct cyclin A-CDK2 substrates that are phosphorylated on the same sites that we identified by mass spectrometry, and we also found that one novel substrate, the ribosomal protein RL12, exhibits site-specific CDK2-dependent phosphorylation in vivo. CONCLUSIONS: We used methods entailing engineered kinases and thiophosphate enrichment to identify a large number of candidate CDK2 substrates in cell lysates. These results are consistent with other recent proteomic studies, and suggest that CDKs regulate cell division via large networks of cellular substrates. These methods are general and can be easily adapted to identify direct substrates of many other protein kinases.

Transvection mediated by the translocated cyclin D1 locus in mantle cell lymphoma.

In mantle cell lymphoma (MCL) and some cases of multiple myeloma (MM), cyclin D1 expression is deregulated by chromosome translocations involving the immunoglobulin heavy chain (IgH) locus. To evaluate the mechanisms responsible, gene targeting was used to study long-distance gene regulation. Remarkably, these targeted cell lines lost the translocated chromosome (t(11;14)). In these MCL and MM cells, the nonrearranged cyclin D1 (CCND1) locus reverts from CpG hypomethylated to hypermethylated. Reintroduction of the translocated chromosome induced a loss of methylation at the unrearranged CCND1 locus, providing evidence of a transallelic regulatory effect. In these cell lines and primary MCL patient samples, the CCND1 loci are packaged in chromatin-containing CCCTC binding factor (CTCF) and nucleophosmin (NPM) at the nucleolus. We show that CTCF and NPM are bound at the IgH 3' regulatory elements only in the t(11;14) MCL cell lines. Furthermore, NPM short hairpin RNA produces a specific growth arrest in these cells. Our data demonstrate transvection in human cancer and suggest a functional role for CTCF and NPM.

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.

Cyclin E phosphorylation regulates cell proliferation in hematopoietic and epithelial lineages in vivo.

Phosphorylations within N- and C-terminal degrons independently control the binding of cyclin E to the SCF(Fbw7) and thus its ubiquitination and proteasomal degradation. We have now determined the physiologic significance of cyclin E degradation by this pathway. We describe the construction of a knockin mouse in which both degrons were mutated by threonine to alanine substitutions (cyclin E(T74A T393A)) and report that ablation of both degrons abolished regulation of cyclin E by Fbw7. The cyclin E(T74A T393A) mutation disrupted cyclin E periodicity and caused cyclin E to continuously accumulate as cells reentered the cell cycle from quiescence. In vivo, the cyclin E(T74A T393A) mutation greatly increased cyclin E activity and caused proliferative anomalies. Cyclin E(T74A T393A) mice exhibited abnormal erythropoiesis characterized by a large expansion of abnormally proliferating progenitors, impaired differentiation, dysplasia, and anemia. This syndrome recapitulates many features of early stage human refractory anemia/myelodysplastic syndrome, including ineffective erythropoiesis. Epithelial cells also proliferated abnormally in cyclin E knockin mice, and the cyclin E(T74A T393A) mutation delayed mammary gland involution, implicating cyclin E degradation in this anti-mitogenic response. Hyperproliferative mammary epithelia contained increased apoptotic cells, suggesting that apoptosis contributes to tissue homeostasis in the setting of cyclin E deregulation. Overall these data show the critical role of both degrons in regulating cyclin E activity and reveal that complete loss of Fbw7-mediated cyclin E degradation causes spontaneous and cell type-specific proliferative anomalies.

FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation.

FBW7 (F-box and WD repeat domain-containing 7) is the substrate recognition component of an evolutionary conserved SCF (complex of SKP1, CUL1 and F-box protein)-type ubiquitin ligase. SCF(FBW7) degrades several proto-oncogenes that function in cellular growth and division pathways, including MYC, cyclin E, Notch and JUN. FBW7 is also a tumour suppressor, the regulatory network of which is perturbed in many human malignancies. Numerous cancer-associated mutations in FBW7 and its substrates have been identified, and loss of FBW7 function causes chromosomal instability and tumorigenesis. This Review focuses on structural and functional aspects of FBW7 and its role in the development of cancer.

FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to gamma-secretase inhibitors.

gamma-secretase inhibitors (GSIs) can block NOTCH receptor signaling in vitro and therefore offer an attractive targeted therapy for tumors dependent on deregulated NOTCH activity. To clarify the basis for GSI resistance in T cell acute lymphoblastic leukemia (T-ALL), we studied T-ALL cell lines with constitutive expression of the NOTCH intracellular domain (NICD), but that lacked C-terminal truncating mutations in NOTCH1. Each of the seven cell lines examined and 7 of 81 (8.6%) primary T-ALL samples harbored either a mutation or homozygous deletion of the gene FBW7, a ubiquitin ligase implicated in NICD turnover. Indeed, we show that FBW7 mutants cannot bind to the NICD and define the phosphodegron region of the NICD required for FBW7 binding. Although the mutant forms of FBW7 were still able to bind to MYC, they do not target it for degradation, suggesting that stabilization of both NICD and its principle downstream target, MYC, may contribute to transformation in leukemias with FBW7 mutations. In addition, we show that all seven leukemic cell lines with FBW7 mutations were resistant to the MRK-003 GSI. Most of these resistant lines also failed to down-regulate the mRNA levels of the NOTCH targets MYC and DELTEX1 after treatment with MRK-003, implying that residual NOTCH signaling in T-ALLs with FBW7 mutations contributes to GSI resistance.

Fbw7/hCDC4 dimerization regulates its substrate interactions.

BACKGROUND: The Fbw7 ubiquitin ligase promotes the rapid degradation of several important oncogenes, such as cyclin E, c-Myc, c-Jun, and Notch. The two fission yeast homologs of Fbw7, pop1 and pop2, have previously been shown to dimerize. In this study, we asked whether Fbw7 can also dimerize and how dimerization affects Fbw7 function. RESULTS: We found that Fbw7 binds efficiently to itself through a domain just upstream of its F-box. We further show that dimerization is essential for the stable interaction of Fbw7 with the cyclin E T380 phospho-degron. Surprisingly, the requirement for dimerization can be suppressed by an additional phosphorylation of this phospho-degron at the +4 position (S384), which creates a binding site with higher affinity for monomeric Fbw7. CONCLUSION: Degradation of cyclin E by the Fbw7 pathway can, thus, be conditionally regulated either by Fbw7 dimerization or by hyperphosphorylation of the T380 phospho-degron. Other substrates, which cannot accommodate an extra phosphate in their phospho-degrons, or which don't provide a negatively charged amino acid in the +4 position, may be absolutely dependent on Fbw7 dimerization for their turnover. Our results point to an additional level of regulation for substrate interaction and turnover by Fbw7.

Kinase-independent function of cyclin E.

E-type cyclins are thought to drive cell-cycle progression by activating cyclin-dependent kinases, primarily CDK2. We previously found that cyclin E-null cells failed to incorporate MCM helicase into DNA prereplication complex during G(0) --> S phase progression. We now report that a kinase-deficient cyclin E mutant can partially restore MCM loading and S phase entry in cyclin E-null cells. We found that cyclin E is loaded onto chromatin during G(0) --> S progression. In the absence of cyclin E, CDT1 is normally loaded onto chromatin, whereas MCM is not, indicating that cyclin E acts between CDT1 and MCM loading. We observed a physical association of cyclin E with CDT1 and with MCMs. We propose that cyclin E facilitates MCM loading in a kinase-independent fashion, through physical interaction with CDT1 and MCM. Our work indicates that-in addition to their function as CDK activators-E cyclins play kinase-independent functions in cell-cycle progression.

Zcchc8 is a glycogen synthase kinase-3 substrate that interacts with RNA-binding proteins.

Phosphorylation of c-Myc on threonine 58 (T58) stimulates its degradation by the Fbw7-SCF ubiquitin ligase. We used a phosphorylation-specific antibody raised against the c-Myc T58 region to attempt to identify other proteins regulated by the Fbw7 pathway. We identified two predominant proteins recognized by this antibody. The first is Ebna1 binding protein 2, a nucleolar protein that, in contrast with a previous report, is likely responsible for the nucleolar staining exhibited by this antibody. The second is Zcchc8, a nuclear protein that is highly phosphorylated in cells treated with nocodazole. We show that Zcchc8 is directly phosphorylated by GSK-3 in vitro and that GSK-3 inhibition prevents Zcchc8 phosphorylation in vivo. Moreover, we found that Zcchc8 interacts with proteins involved in RNA processing/degradation. We suggest that Zcchc8 is a GSK-3 substrate with a role in RNA metabolism.

Cell cycle: how cyclin E got its groove back.

CDK1 has long been known to orchestrate the passage of mammalian cells into and through mitosis. Recent work revisits the idea that CDK1, in conjunction with cyclin E, participates in S-phase entry as well. The new results shed light on a recent cell-cycle mystery, and provide another dramatic example of apparent functional redundancy among cyclins and cyclin-dependent kinases.

Ras activity regulates cyclin E degradation by the Fbw7 pathway.

The Skp1-Cullin1 F-box protein-Fbw7 ubiquitin ligase regulates phosphorylation-dependent cyclin E degradation, and disruption of this pathway is associated with genetic instability and tumorigenesis. Fbw7 is a human tumor suppressor that is targeted for mutation in primary cancers. However, mechanisms other than mutation of Fbw7 may also disrupt cyclin E proteolysis in cancers. We show that oncogenic Ha-Ras activity regulates cyclin E degradation by the Fbw7 pathway. Activated Ras impairs Fbw7-driven cyclin E degradation, and, conversely, inhibition of normal Ras activity decreases cyclin E abundance. Moreover, activation of the mitogen-activated protein kinase pathway is the essential Ras function that inhibits cyclin E turnover, and activated Ha-Ras expression inhibits both the binding of cyclin E to Fbw7 and cyclin E ubiquitination. Last, we found that oncogenic Ras activity potentiates cyclin E-induced genetic instability but only when cyclin E is susceptible to degradation by Fbw7. Thus, we conclude that Ras activity regulates Fbw7-mediated cyclin E proteolysis and suggest that impaired cyclin E proteolysis is a mechanism through which Ras mutations promote tumorigenesis.

The SV40 large T antigen contains a decoy phosphodegron that mediates its interactions with Fbw7/hCdc4.

Cell transformation by simian virus 40 (SV40) results mostly from the highly oncogenic activities of the large T antigen (LT), which corrupts the cellular checkpoint mechanisms that guard cell division and the transcription, replication, and repair of DNA. The most prominent LT targets are the retinoblastoma protein (pRb) and p53. Here we report that LT binds directly to Fbw7, the substrate recognition component of the SCF(Fbw7) ubiquitin ligase and a human tumor suppressor. LT binding mislocalizes the nucleolar Fbw7gamma isoform to the nucleoplasm. Interestingly, the binding of LT to Fbw7 occurs via a decoy phospho-epitope within the C terminus of LT that closely mimics the consensus Cdc4 phospho-degron found within Fbw7 substrates. We demonstrate that, using this mode of interaction, LT can interfere with Fbw7-driven cyclin E turnover in vivo and causes increased cyclin E-associated kinase activity. Our data suggest that LT competes with cellular proteins for Fbw7 binding in a substrate-like fashion.

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.