Structural plasticity of D3-D14 ubiquitin ligase in strigolactone signalling.

The strigolactones, a class of plant hormones, regulate many aspects of plant physiology. In the inhibition of shoot branching, the α/ß hydrolase D14-which metabolizes strigolactone-interacts with the F-box protein D3 to ubiquitinate and degrade the transcription repressor D53. Despite the fact that multiple modes of interaction between D14 and strigolactone have recently been determined, how the hydrolase functions with D3 to mediate hormone-dependent D53 ubiquitination remains unknown. Here we show that D3 has a C-terminal α-helix that can switch between two conformational states. The engaged form of this α-helix facilitates the binding of D3 and D14 with a hydrolysed strigolactone intermediate, whereas the dislodged form can recognize unmodified D14 in an open conformation and inhibits its enzymatic activity. The D3 C-terminal α-helix enables D14 to recruit D53 in a strigolactone-dependent manner, which in turn activates the hydrolase. By revealing the structural plasticity of the SCFD3-D14 ubiquitin ligase, our results suggest a mechanism by which the E3 coordinates strigolactone signalling and metabolism.

The pseudophosphatase STYX targets the F-box of FBXW7 and inhibits SCFFBXW7 function.

The F-box protein FBXW7 is the substrate-recruiting subunit of an SCF ubiquitin ligase and a major tumor-suppressor protein that is altered in several human malignancies. Loss of function of FBXW7 results in the stabilization of numerous proteins that orchestrate cell proliferation and survival. Little is known about proteins that directly regulate the function of this protein. In the current work, we have mapped the interactome of the enigmatic pseudophosphatase STYX We reasoned that a catalytically inactive phosphatase might have adopted novel mechanisms of action. The STYX interactome contained several F-box proteins, including FBXW7. We show that STYX binds to the F-box domain of FBXW7 and disables its recruitment into the SCF complex. Therefore, STYX acts as a direct inhibitor of FBXW7, affecting the cellular levels of its substrates. Furthermore, we find that levels of STYX and FBXW7 are anti-correlated in breast cancer patients, which affects disease prognosis. We propose the STYX-FBXW7 interaction as a promising drug target for future investigations.

CRL4 antagonizes SCFFbxo7-mediated turnover of cereblon and BK channel to regulate learning and memory.

Intellectual disability (ID), one of the most common human developmental disorders, can be caused by genetic mutations in Cullin 4B (Cul4B) and cereblon (CRBN). CRBN is a substrate receptor for the Cul4A/B-DDB1 ubiquitin ligase (CRL4) and can target voltage- and calcium-activated BK channel for ER retention. Here we report that ID-associated CRL4CRBN mutations abolish the interaction of the BK channel with CRL4, and redirect the BK channel to the SCFFbxo7 ubiquitin ligase for proteasomal degradation. Glioma cell lines harbouring CRBN mutations record density-dependent decrease of BK currents, which can be restored by blocking Cullin ubiquitin ligase activity. Importantly, mice with neuron-specific deletion of DDB1 or CRBN express reduced BK protein levels in the brain, and exhibit similar impairment in learning and memory, a deficit that can be partially rescued by activating the BK channel. Our results reveal a competitive targeting of the BK channel by two ubiquitin ligases to achieve exquisite control of its stability, and support changes in neuronal excitability as a common pathogenic mechanism underlying CRL4CRBN-associated ID.

Inhibition of Skp1-Cullin-F-box complexes during bovine oocyte maturation and preimplantation development leads to delayed development of embryos†.

The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by the ubiquitin-proteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited them using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cell expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the four-cell to late eight-cell stage, we found a statistically significant delay in their development; some of the treated embryos were, however, able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos; no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a Western blot analysis of selected proteins (SMAD4, ribosomal protein S6, centromeric protein E, P27, NFKB inhibitor alpha, RNA-binding motif protein 19). No statistically significant increase in protein levels was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cell expansion, fertilization, and early preimplantation development of cattle.

The Skp1 Homologs SKR-1/2 Are Required for the Caenorhabditis elegans SKN-1 Antioxidant/Detoxification Response Independently of p38 MAPK.

SKN-1/Nrf are the primary antioxidant/detoxification response transcription factors in animals and they promote health and longevity in many contexts. SKN-1/Nrf are activated by a remarkably broad-range of natural and synthetic compounds and physiological conditions. Defining the signaling mechanisms that regulate SKN-1/Nrf activation provides insights into how cells coordinate responses to stress. Nrf2 in mammals is regulated in part by the redox sensor repressor protein named Keap1. In C. elegans, the p38 MAPK cascade in the intestine activates SKN-1 during oxidative stress by promoting its nuclear accumulation. Interestingly, we find variation in the kinetics of p38 MAPK activation and tissues with SKN-1 nuclear accumulation among different pro-oxidants that all trigger strong induction of SKN-1 target genes. Using genome-wide RNAi screening, we identify new genes that are required for activation of the core SKN-1 target gene gst-4 during exposure to the natural pro-oxidant juglone. Among 10 putative activators identified in this screen was skr-1/2, highly conserved homologs of yeast and mammalian Skp1, which function to assemble protein complexes. Silencing of skr-1/2 inhibits induction of SKN-1 dependent detoxification genes and reduces resistance to pro-oxidants without decreasing p38 MAPK activation. Global transcriptomics revealed strong correlation between genes that are regulated by SKR-1/2 and SKN-1 indicating a high degree of specificity. We also show that SKR-1/2 functions upstream of the WD40 repeat protein WDR-23, which binds to and inhibits SKN-1. Together, these results identify a novel p38 MAPK independent signaling mechanism that activates SKN-1 via SKR-1/2 and involves WDR-23.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.

Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

Forkhead box O3 plays a role in skeletal muscle atrophy through expression of E3 ubiquitin ligases MuRF-1 and atrogin-1 in Cushing's syndrome.

Cushing's syndrome is caused by overproduction of the adrenocorticotropic hormone (ACTH), which stimulates the adrenal grand to make cortisol. Skeletal muscle wasting occurs in pathophysiological response to Cushing's syndrome. The forkhead box (FOX) protein family has been implicated as a key regulator of muscle loss under conditions such as diabetes and sepsis. However, the mechanistic role of the FOXO family in ACTH-induced muscle atrophy is not understood. We hypothesized that FOXO3a plays a role in muscle atrophy through expression of the E3 ubiquitin ligases, muscle RING finger protein-1 (MuRF-1), and atrogin-1 in Cushing's syndrome. For establishment of a Cushing's syndrome animal model, Sprague-Dawley rats were implanted with osmotic minipumps containing ACTH (40 ng·kg-1·day-1). ACTH infusion significantly reduced muscle weight. In ACTH-infused rats, MuRF-1, atrogin-1, and FOXO3a were upregulated and the FOXO3a promoter was targeted by the glucocorticoid receptor (GR). Transcriptional activity and expression of FOXO3a were significantly decreased by the GR antagonist RU486. Treatment with RU486 reduced MuRF-1 and atrogin-1 expression in accordance with reduced enrichment of FOXO3a and Pol II on the promoters. Knockdown of FOXO3a prevented dexamethasone-induced MuRF-1 and atrogin-1 expression. These results indicate that FOXO3a plays a role in muscle atrophy through expression of MuRF-1 and atrogin-1 in Cushing's syndrome.

Ubiquitin-mediated response to microsporidia and virus infection in C. elegans.

Microsporidia comprise a phylum of over 1400 species of obligate intracellular pathogens that can infect almost all animals, but little is known about the host response to these parasites. Here we use the whole-animal host C. elegans to show an in vivo role for ubiquitin-mediated response to the microsporidian species Nematocida parisii, as well to the Orsay virus, another natural intracellular pathogen of C. elegans. We analyze gene expression of C. elegans in response to N. parisii, and find that it is similar to response to viral infection. Notably, we find an upregulation of SCF ubiquitin ligase components, such as the cullin ortholog cul-6, which we show is important for ubiquitin targeting of N. parisii cells in the intestine. We show that ubiquitylation components, the proteasome, and the autophagy pathway are all important for defense against N. parisii infection. We also find that SCF ligase components like cul-6 promote defense against viral infection, where they have a more robust role than against N. parisii infection. This difference may be due to suppression of the host ubiquitylation system by N. parisii: when N. parisii is crippled by anti-microsporidia drugs, the host can more effectively target pathogen cells for ubiquitylation. Intriguingly, inhibition of the ubiquitin-proteasome system (UPS) increases expression of infection-upregulated SCF ligase components, indicating that a trigger for transcriptional response to intracellular infection by N. parisii and virus may be perturbation of the UPS. Altogether, our results demonstrate an in vivo role for ubiquitin-mediated defense against microsporidian and viral infections in C. elegans.

Skp1: Implications in cancer and SCF-oriented anti-cancer drug discovery.

In the last decade, the ubiquitin proteasome system (UPS), in general, and E3 ubiquitin ligases, in particular, have emerged as valid drug targets for the development of novel anti-cancer therapeutics. Cullin RING Ligases (CRLs), which can be classified into eight groups (CRL1-8) and comprise approximately 200 members, represent the largest family of E3 ubiquitin ligases which facilitate the ubiquitination-derived proteasomal degradation of a myriad of functionally and structurally diverse substrates. S phase kinase-associated protein 1 (Skp1)-Cullin1-F-Box protein (SCF) complexes are the best characterized among CRLs, which play crucial roles in numerous cellular processes and physiological dysfunctions, such as in cancer biology. Currently, there is growing interest in developing SCF-targeting anti-cancer therapies for clinical application. Indeed, the research in this field has seen some progress in the form of cullin neddylation- and Skp2-inhibitors. However, it still remains an underdeveloped area and needs to design new strategies for developing improved form of therapy. In this review, we venture a novel strategy that rational pharmacological targeting of Skp1, a central regulator of SCF complexes, may provide a novel avenue for SCF-oriented anti-cancer therapy, expected: (i) to simultaneously address the critical roles that multiple SCF oncogenic complexes play in cancer biology, (ii) to selectively target cancer cells with minimal normal cell toxicity, and (iii) to offer multiple chemical series, via therapeutic interventions at the Skp1 binding interfaces in SCF complex, thereby maximizing chances of success for drug discovery. In addition, we also discuss the challenges that might be posed regarding rational pharmacological interventions against Skp1.

Clenbuterol changes phosphorylated FOXO1 localization and decreases protein degradation in the sartorius muscle of neonatal chicks.

To investigate the intracellular signaling mechanisms by which clenbuterol reduces muscle protein degradation, we examined the phosphorylation level and intracellular localization of FOXO1 in the sartorius muscle of neonatal chicks. One-day-old chicks were given a single intraperitoneal injection of clenbuterol (0.1 mg/kg body weight). Three hours after injection, AKT protein was phosphorylated in the sartorius muscle by clenbuterol injection. Coincidentally, clenbuterol increased cytosolic level of phosphorylated FOXO1 protein, while it decreased nuclear level of FOXO1 protein in the sartorius muscle. Furthermore, clenbuterol decreased the expression of mRNAs for muscle-specific ubiquitin ligases (atrogin-1/MAFbx and MuRF1) in the sartorius muscle accompanied by decreased plasma 3-methylhistidine concentration, an index of muscle protein degradation, at 3 h after injection. These results suggested that, in the sartorius muscle of the chicks, clenbuterol changed the intracellular localization of phosphorylated FOXO1, and consequently decreased protein degradation via suppressing the expression of genes encoding muscle-specific ubiquitin ligases.

Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signaling pathway.

Control of cardiac muscle mass is thought to be determined by a dynamic balance of protein synthesis and degradation. Recent studies have demonstrated that atrophy-related forkhead box O 3a (FOXO3a)/muscle atrophy F-box (MAFbx) signaling pathway plays a central role in the modulation of proteolysis and exert inhibitory effect on cardiomyocyte hypertrophy. In this study, we tested the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation attenuates cardiomyocyte hypertrophy by regulating FOXO3a/MAFbx signaling pathway and its downstream protein degradation. The results showed that activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated cardiomyocyte hypertrophy induced by angiotensin II (Ang II). The antihypertrophic effects of AICAR were blunted by AMPK inhibitor Compound C. In addition, AMPK dramatically increased the activity of transcription factor FOXO3a, up-regulated the expression of its downstream ubiquitin ligase MAFbx, and enhanced cardiomyocyte proteolysis. Meanwhile, the effects of AMPK on protein degradation and cardiomyocyte hypertrophy were blocked after MAFbx was silenced by transfection of cardiomyocytes with MAFbx-siRNA. These results indicate that AMPK plays an important role in the inhibition of cardiomyocyte hypertrophy by activating protein degradation via FOXO3a/MAFbx signaling pathway.

Resveratrol prevents dexamethasone-induced expression of the muscle atrophy-related ubiquitin ligases atrogin-1 and MuRF1 in cultured myotubes through a SIRT1-dependent mechanism.

Resveratrol (3,5,4'-trihydroxystilbene) has been ascribed multiple beneficial biological effects but the influence of resveratrol on glucocorticoid-induced muscle atrophy is not known. We examined the effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression, FOXO1 acetylation, protein degradation and atrophy in cultured L6 myotubes. In addition, the role of the deacetylase SIRT1 in the effects of resveratrol was determined by transfecting myotubes with SIRT1 siRNA. The catabolic effects of dexamethasone were prevented by resveratrol and the protective effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression were abolished in myotubes transfected with SIRT1 siRNA. Results suggest that resveratrol can prevent glucocorticoid-induced muscle wasting and that this effect is at least in part SIRT1-dependent.

miR-19a-3p Functions as an Oncogene by Regulating FBXO32 Expression in Multiple Myeloma.

BACKGROUND: Multiple myeloma remains a virtually incurable hematologic malignancy, which is featured with the aberrant growth of malignant plasma cells. AIMS: To elucidate the functions of miR-19a-3p in multiple myeloma. STUDY DESIGN: Cell study. METHODS: Cell counting kit-8 assay was performed to detect cell viability, and flow cytometry was conducted to detect cell apoptosis. Bioinformatics analysis predicted miR-19a-3p-associated biological function, pathway, core regulatory network, and target genes. Luciferase reporter assay verified the target sequence of miR-19a-3p regulating FBXO32. RESULTS: miR-19a-3p is upregulated in multiple myeloma cells (p<0.01) and patients with multiple myeloma (p<0.001). Overexpressed miR-19a-3p significantly increased cell viability (p<0.05) and inhibited cell apoptosis (p<0.01). FBXO32 is a target gene of miR-19a-3p (p<0.01). Moreover, FBXO32 is downregulated in MM, and it significantly decreased cell viability (p<0.05) and promoted cell apoptosis (p<0.01). FBXO32 significantly rescued the influence of miR-19a-3p-inhibiting cell apoptosis (p<0.05). CONCLUSION: miR-19a-3p promoted cell proliferation and inhibited cell apoptosis by degrading the target FBXO32 mRNA in multiple myeloma.

E3 ubiquitin ligase Atrogin-1 mediates adaptive resistance to KIT-targeted inhibition in gastrointestinal stromal tumor.

KIT/PDGFRA oncogenic tyrosine kinase signaling is the central oncogenic event in most gastrointestinal stromal tumors (GIST), which are human malignant mesenchymal neoplasms that often feature myogenic differentiation. Although targeted inhibition of KIT/PDGFRA provides substantial clinical benefit, GIST cells adapt to KIT/PDGFRA driver suppression and eventually develop resistance. The specific molecular events leading to adaptive resistance in GIST remain unclear. By using clinically representative in vitro and in vivo GIST models and GIST patients' samples, we found that the E3 ubiquitin ligase Atrogin-1 (FBXO32)-the main effector of muscular atrophy in cachexia-resulted in the most critical gene derepressed in response to KIT inhibition, regardless the type of KIT primary or secondary mutation. Atrogin-1 in GISTs is transcriptionally controlled by the KIT-FOXO3a axis, thus indicating overlap with Atrogin-1 regulation mechanisms in nonneoplastic muscle cells. Further, Atrogin-1 overexpression was a GIST-cell-specific pro-survival mechanism that enabled the adaptation to KIT-targeted inhibition by apoptosis evasion through cell quiescence. Buttressed on these findings, we established in vitro and in vivo the preclinical proof-of-concept for co-targeting KIT and the ubiquitin pathway to maximize the therapeutic response to first-line imatinib treatment.

The bacterial effector Cif interferes with SCF ubiquitin ligase function by inhibiting deneddylation of Cullin1.

Cycle inhibiting factor (Cif) is one of the effectors delivered into epithelial cells by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) via the type III secretion system (TTSS). Cif family proteins, which inhibit host cell-cycle progression via mechanisms not yet precisely understood, are highly conserved among EPEC, EHEC, Yersinia pseudotuberculosis, Photorhabdus luminescens and Burkholderia pseudomallei. Levels of several proteins relevant to cell-cycle progression are modulated by Cullin-RING ligases (CRLs), which in turn are activated by conjugation and deconjugation of NEDD8 to Cullins. Here we show that Cif interacts with NEDD8 and interferes with SCF (Skp1-Cullin1-F-box protein) complex ubiquitin ligase function. We found that neddylated Cullin family proteins accumulated and ubiquitination of p27 decreased in cells infected with EPEC. Consequently, Cif stabilized SCF substrates such as CyclinD1, Cdt1, and p27, and caused G1 cell-cycle arrest. Using time-lapse-imaging of fluorescent ubiquitination-based cell-cycle indicator (Fucci)-expressing cells, we were able to monitor cell-cycle progression during EPEC infection and confirmed the arrest of infected cells at G1. Our in vitro and in vivo data show that Cif-NEDD8 interaction inhibits deneddylation of Cullins, suppresses CRL activity and induces G1 arrest. We thus conclude that the bacterial effector Cif interferes with neddylation-mediated cell-cycle control.

Hsp27 inhibits IKKbeta-induced NF-kappaB activity and skeletal muscle atrophy.

Heat shock protein 25/27 (Hsp25/27) is a cytoprotective protein that is ubiquitously expressed in most cells, and is up-regulated in response to cellular stress. Previous work, in nonmuscle cells, has shown that Hsp27 inhibits TNF-alpha-induced NF-kappaB activation. During skeletal muscle disuse, Hsp25/27 levels are decreased and NF-kappaB activity increased, and this increase in NF-kappaB activity is required for disuse muscle atrophy. Therefore, the purpose of the current study was to determine whether electrotransfer of Hsp27 into the soleus muscle of rats, prior to skeletal muscle disuse, is sufficient to inhibit skeletal muscle disuse atrophy and NF-kappaB activation. The 35% disuse muscle-fiber atrophy observed in nontransfected fibers was attenuated by 50% in fibers transfected with Hsp27. Hsp27 also inhibited the disuse-induced increase in MuRF1 and atrogin-1 transcription by 82 and 40%, respectively. Furthermore, disuse- and IKKbeta-induced NF-kappaB transactivation were abolished by Hsp27. In contrast, Hsp27 had no effect on Foxo transactivation. In conclusion, Hsp27 is a negative regulator of NF-kappaB in skeletal muscle, in vivo, and is sufficient to inhibit MuRF1 and atrogin-1 and attenuate skeletal muscle disuse atrophy.

Overexpression of the transcriptional coregulator Cited2 protects against glucocorticoid-induced atrophy of C2C12 myotubes.

In patients with various catabolic conditions, glucocorticoid excess induces skeletal muscle wasting by accelerating protein degradation via the ubiquitin-proteasome pathway. Although the transcriptional coactivator p300 has been implicated in this pathological process, regulatory mechanisms and molecular targets of its action remain unclear. Here we show that CREB-binding protein (CBP)/p300-interacting transactivator with ED-rich tail 2 (Cited2), which binds to the cysteine-histidine-rich region 1 of p300 and CBP, regulates muscle mass in vitro. Adenovirus-mediated overexpression of wild-type Cited2 significantly blocked morphological alterations of C2C12 myotubes with a concomitant decrease in myosin heavy chain protein in response to synthetic glucocorticoid dexamethasone, which were attributable to the reduced induction of atrophy-related ubiquitin ligases MuRF1 and MAFbx. These myotube-sparing effects were less pronounced, however, with a carboxyl-terminally truncated mutant of Cited2 that lacked the ability to bind p300. These results suggest that the gain of Cited2 function counteracts glucocorticoid-induced muscle atrophy through inhibition of proteolysis mediated by p300-dependent gene transcription.

Indoxyl sulfate induces myotube atrophy by ROS-ERK and JNK-MAFbx cascades.

Accumulations of uremic toxins has been widely recognized as the major trigger of skeletal muscle loss in chronic kidney disease (CKD), which is defined as uremic sarcopenia. Current study was aimed to examine the effects of representative uremic toxin, indoxyl sulfate (IS), on C2C12 myotubes. The incubation of IS (from 0.1 mM to 1.2 mM) exerted the reduction in myotube diameter without cell survival impairment. Elevated oxidative stress and mitogen-activated protein kinase (MAPKs) phosphorylation were observed after IS stimulation for 1 and 24 h. After N-acetylcysteine (NAC) treatment as antioxidants, the recovery in IS-induced decrease myotube diameter and ERK phosphorylation was observed. This findings were implicit the transduction of p-ERK in IS-induced ROS toxicity. Moreover, the increase of LC3ß was found closely with IS treatment in C2C12 myotubes. The reverse effect of NAC on LC3ß expression revealed the ROS-responsibility in autophagy regulation of CKD myopathy. The evaluation of IS-treated proteasome system showed increased phospho-myosin light chain, along with the upregulation of muscle atrophy F-box (MAFbx) mRNA and protein. This alteration in MAFbx was also identified in nephrectomy-induced CKD model. Besides, the inhibition of p-JNK was capable to attenuate IS-induced upward change in MAFbx protein expression. These findings indicated that IS-mediated myotube atrophy may manipulate through ROS-ERK axis and JNK-MAFbx regulation in C2C12 cells.

p53 and SCFFbw7 cooperatively restrain cyclin E-associated genome instability.

Cancers often exhibit high levels of cyclin E expression, and aberrant cyclin E activity causes genomic instability and increased tumorigenesis. Two tumor suppressor pathways protect cells against cyclin E deregulation. The p53 pathway is induced by excess cyclin E in primary cells and opposes cyclin E activity through induction of p21Cip1. In contrast, the Fbw7 pathway targets cyclin E for degradation, and Fbw7 mutations occur commonly in cancers. We investigated the cooperativity of these two pathways in countering cyclin E-induced genomic instability in primary human cells. We find that loss of p53 and Fbw7 synergistically unmasks cyclin E-induced instability. In normal cells, impaired cyclin E degradation produces genome instability, but this is rapidly mitigated by induction of p53 and p21. In contrast, p53 loss allows the high level of cyclin E kinase activity that results from Fbw7 loss to persist and continuously drive genome instability. Moreover, p21 plays a critical role in suppressing cyclin E when Fbw7 is disabled, and in the absence of p21, sustained cyclin E activity induces rapid cell death via apoptosis. These data directly demonstrate the cooperative roles of these Fbw7 and p53 pathways in restraining cyclin E activity and its associated genome instability.