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Cyclin F-Mediated Degradation of Ribonucleotide Reductase M2 Controls Genome Integrity and DNA Repair
D'Angiolella, Vincenzo; Donato, Valerio; Forrester, Frances M; Jeong, Yeon-Tae; Pellacani, Claudia; Kudo, Yasusei; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Pagano, Michele
F-box proteins are the substrate binding subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes. Using affinity purifications and mass spectrometry, we identified RRM2 (the ribonucleotide reductase family member 2) as an interactor of the F-box protein cyclin F. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs), which are necessary for both replicative and repair DNA synthesis. We found that, during G2, following CDK-mediated phosphorylation of Thr33, RRM2 is degraded via SCF(cyclin F) to maintain balanced dNTP pools and genome stability. After DNA damage, cyclin F is downregulated in an ATR-dependent manner to allow accumulation of RRM2. Defective elimination of cyclin F delays DNA repair and sensitizes cells to DNA damage, a phenotype that is reverted by expressing a nondegradable RRM2 mutant. In summary, we have identified a biochemical pathway that controls the abundance of dNTPs and ensures efficient DNA repair in response to genotoxic stress.
PMCID:3616325
PMID: 22632967
ISSN: 0092-8674
CID: 167802
APC/C (Cdh1) controls the proteasome-mediated degradation of E2F3 during cell cycle exit
Ping, Zhen; Lim, Ratna; Bashir, Tarig; Pagano, Michele; Guardavaccaro, Daniele
E2F transcription factors regulate gene expression in concert with the retinoblastoma tumor suppressor family. These transcriptional complexes are master regulators of cell cycle progression and, in addition, control the expression of genes involved in DNA repair, G 2/M checkpoint and differentiation. E2F3 has recently attracted particular attention, because it is amplified in various human tumors. Here we show that E2F3 becomes unstable as cells exit the cell cycle. E2F3 degradation is mediated by the anaphase-promoting complex/cyclosome and its activator Cdh1 (APC/C (Cdh1) ). E2F3 interacts with Cdh1 but not Cdc20, the other APC/C activator. Enforced expression of Cdh1 results in proteasome-dependent degradation of E2F3, whereas the overexpression of Cdc20 has no effect on E2F3 turnover. Finally, silencing of Cdh1 by RNA interference stabilizes E2F3 in differentiating neuroblastoma cells. These findings indicate that the APC/C (Cdh1) ubiquitin ligase targets E2F3 for proteasome-dependent degradation during cell cycle exit and neuronal differentiation.
PMCID:3359123
PMID: 22580460
ISSN: 1551-4005
CID: 169025
Regulation of APC/C(Cdc20) activity by RASSF1A-APC/C(Cdc20) circuitry
Chow, C; Wong, N; Pagano, M; Lun, S W-M; Nakayama, K-I; Nakayama, K; Lo, K-W
RASSF1A is a key tumor-suppressor gene that is often inactivated in a wide variety of solid tumors. Studies have illustrated that RASSF1A plays vital roles in the regulation of cell-cycle progression and functions as a guardian of mitosis. Nevertheless, the precise mechanism of RASSF1A-dependent regulation of mitosis remains largely unclear. APC/C(Cdc20) is the master switch and regulator of mitosis. The activity of APC/C(Cdc20) is tightly controlled by phosphorylation and specific inhibitors to ensure the sequential ubiquitination of downstream targets. Here, we report on the novel finding of a regulated circuitry that controls the timely expression and hence activity of APC/C(Cdc20) during mitosis. Our study showed that RASSF1A and APC/C(Cdc20) form a molecular relay that regulates the APC/C(Cdc20) activity at early mitosis. We found that RASSF1A inhibits APC/C(Cdc20) function through its D-box motifs. Paradoxically, RASSF1A was also demonstrated to be ubiquitinated by APC/C(Cdc20) in vitro and degraded at prometaphase despite of active spindle checkpoint presence. The first two unique D-boxes at the N-terminal of RASSF1A served as specific degron recognized by APC/C(Cdc20). Importantly, we found that Aurora A and Aurora B directly phosphorylate RASSF1A, a critical step by which RASSF1A switches from being an inhibitor to a substrate of APC/C(Cdc20) during the course of mitotic progression. As a result of RASSF1A degradation, APC/C(Cdc20) can then partially activate the ubiquitination of Cyclin A in the presence of spindle checkpoint. This circuitry is essential for the timely degradation of Cyclin A. To conclude, our results propose a new model for RASSF1A-APC/C(Cdc20) interaction in ensuring the sequential progression of mitosis.
PMCID:3325600
PMID: 21874044
ISSN: 0950-9232
CID: 165670
SCFFbxo45 controls cytokinesis through ubiquitin-mediated proteolysis of GEF-H1 [Meeting Abstract]
Chen, X; Ahn, J -Y; Szankasi, P; Chung, F; Basrur, E; Lim, M S; Miller, A L; Pagano, M; Elenitoba-Johnson, K S J
The rho guanine nucleotide exchange factor H1 (GEF-H1) is a critical regulator of cytokinesis, but the requirement and mechanism for its timely destruction for proper execution of cytokinesis are unknown. Here we show that GEF-H1 is regulated by the evolutionarily conserved E3 ligase SCFFbxo45. Fbxo45 promotes GEF-H1 ubiquitylation upon PLK1-mediated phosphorylation of GEF-H1 on serine 644, and this event is necessary for GEF-H1 degradation during mitosis. Fbxo45 silencing causes stabilization and abnormal cellular distribution of GEF-H1 with diffuse RhoA hyperactivation. A GEF-H1 mutant that is unable to bind Fbxo45 is stabilized in mitosis, and results in an uncoordinated hyperactive membrane phenotype, with abnormal aberrant cytokinesis leading to cell death or multinucleation. Our studies provide evidence that limiting activation of the RhoAGTPase via Fbxo45-mediated degradation of the GEF-H1 activator is essential for proper execution of cytokinesis
EMBASE:70852966
ISSN: 0892-6638
CID: 178106
Fbxw7alpha- and GSK3-mediated degradation of p100 is a pro-survival mechanism in multiple myeloma
Busino, Luca; Millman, Scott E; Scotto, Luigi; Kyratsous, Christos A; Basrur, Venkatesha; O'Connor, Owen; Hoffmann, Alexander; Elenitoba-Johnson, Kojo S; Pagano, Michele
Fbxw7alpha is a member of the F-box family of proteins, which function as the substrate-targeting subunits of SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complexes. Using differential purifications and mass spectrometry, we identified p100, an inhibitor of NF-kappaB signalling, as an interactor of Fbxw7alpha. p100 is constitutively targeted in the nucleus for proteasomal degradation by Fbxw7alpha, which recognizes a conserved motif phosphorylated by GSK3. Efficient activation of non-canonical NF-kappaB signalling is dependent on the elimination of nuclear p100 through either degradation by Fbxw7alpha or exclusion by a newly identified nuclear export signal in the carboxy terminus of p100. Expression of a stable p100 mutant, expression of a constitutively nuclear p100 mutant, Fbxw7alpha silencing or inhibition of GSK3 in multiple myeloma cells with constitutive non-canonical NF-kappaB activity results in apoptosis both in cell systems and xenotransplant models. Thus, in multiple myeloma, Fbxw7alpha and GSK3 function as pro-survival factors through the control of p100 degradation.
PMCID:3339029
PMID: 22388891
ISSN: 1465-7392
CID: 163573
BubR1 Is Modified by Sumoylation during Mitotic Progression
Yang, Feikun; Hu, Liyan; Chen, Cheng; Yu, Jianxiu; O'Connell, Christopher B; Khodjakov, Alexey; Pagano, Michele; Dai, Wei
BubR1 functions as a crucial component that monitors proper chromosome congression and mitotic timing during cell division. We investigated molecular regulation of BubR1 and found that BubR1 was modified by an unknown post-translation mechanism during the cell cycle, resulting in a significant mobility shift on denaturing gels. We termed it BubR1-M as the nature of modification was not characterized. Extended (>24 h) treatment of HeLa cells with a microtubule disrupting agent including nocodazole and taxol or release of mitotic shake-off cells into fresh medium induced BubR1-M. BubR1-M was derived from neither phosphorylation nor acetylation. Ectopic expression coupled with pulling down analyses showed that BubR1-M was derived from SUMO modification. Mutation analysis revealed that lysine 250 was a crucial site for sumoylation. Significantly, compared with the wild-type control, ectopic expression of a sumoylation-deficient mutant of BubR1 induced chromosomal missegregation and mitotic delay. Combined, our study identifies a new type of post-translational modification that is essential for BubR1 function during mitosis.
PMCID:3281637
PMID: 22167194
ISSN: 0021-9258
CID: 157470
SCF ubiquitin ligases in the maintenance of genome stability
Silverman, Joshua S; Skaar, Jeffrey R; Pagano, Michele
In response to genotoxic stress, eukaryotic cells activate the DNA damage response (DDR), a series of pathways that coordinate cell cycle arrest and DNA repair to prevent deleterious mutations. In addition, cells possess checkpoint mechanisms that prevent aneuploidy by regulating the number of centrosomes and spindle assembly. Among these mechanisms, ubiquitin-mediated degradation of key proteins has an important role in the regulation of the DDR, centrosome duplication and chromosome segregation. This review discusses the functions of a group of ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) family, in the maintenance of genome stability. Given that general proteasome inhibitors are currently used as anticancer agents, a better understanding of the ubiquitylation of specific targets by specific ubiquitin ligases may result in improved cancer therapeutics.
PMCID:3278546
PMID: 22099186
ISSN: 0968-0004
CID: 155863
FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas
Duan, Shanshan; Cermak, Lukas; Pagan, Julia K; Rossi, Mario; Martinengo, Cinzia; di Celle, Paola Francia; Chapuy, Bjoern; Shipp, Margaret; Chiarle, Roberto; Pagano, Michele
BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas. By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease. In many DLBCL patients, BCL6 overexpression is achieved through translocation (~40%) or hypermutation of its promoter (~15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11 (refs 5, 6). The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene
PMCID:3344385
PMID: 22113614
ISSN: 1476-4687
CID: 149798
Linking metabolism and cell cycle progression via the APC/CCdh1 and SCFbetaTrCP ubiquitin ligases
Duan, Shanshan; Pagano, Michele
PMCID:3248501
PMID: 22173637
ISSN: 1091-6490
CID: 149805
Loss of p27KIP1 Expression in Fully-staged Node-negative Breast Cancer: Association with Lack of Hormone Receptors in T1a/b, but not T1c Infiltrative Ductal Carcinoma
Mirchandani, Deepu; Roses, Daniel F; Inghirami, Giorgio; Zeleniuch-Jacquotte, Anne; Cangiarella, Joan; Guth, Amber; Safyan, Rachael Ann; Formenti, Silvia C; Pagano, Michele; Muggia, Franco
Nuclear expression of the cell cycle inhibitor p27(KIP1) is reduced in a variety of human malignancies, including breast cancer. Loss of nuclear p27(KIP1) during tumor progression, documented by immunohistochemistry (IHC), has been studied for its potential prognostic implication. We examined by IHC the association between nuclear p27(KIP1) expression and hormone receptor status in T1N0M0 breast cancer. PATIENTS AND METHODS: The correlation between nuclear p27(KIP1) expression and estrogen (ER) and progesterone (PR) hormone receptor status was analyzed in 122 human T1N0M0 (68 T1a/b, 54 T1c) breast cancer specimens. All patients were staged as N0 by axillary node dissection. RESULTS: A statistically significant reduction in p27(KIP1) expression was observed as tumor size increased from T1a/b (7%) to T1c (22%). The proportion of tumors with low nuclear p27(KIP1) expression was higher in the ER-negative/PR-negative group compared to the ER-positive/PR-positive group, but this difference was only statistically significant in the T1a/b subgroup (p=0.0007). CONCLUSION: Further investigations into causes of p27(KIP1) deregulation and their relationship to hormone receptor expression in T1N0M0 breast ductal carcinomas are warranted. Such studies may help identify prognostic, as well as predictive, markers of therapy resistance
PMCID:3339028
PMID: 22199306
ISSN: 1791-7530
CID: 149934