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256


APC/C- and Mad2-mediated degradation of Cdc20 during spindle checkpoint activation

Ge, Sheng; Skaar, Jeffrey R; Pagano, Michele
The spindle assembly checkpoint (SAC) is an important mechanism that prevents the separation of sister chromatids until the microtubules radiating from the spindle poles are correctly attached to the kinetochores. Cdc20, an activator of the Anaphase Promoting Complex/Cyclosome (APC/C), is known as a major downstream target for inhibition by the SAC through the binding of mitotic checkpoint proteins, such as Mad2 and BubR1. Here, we report that the SAC negatively regulates the stability of Cdc20 by targeting it for proteasome-dependent degradation. Once the checkpoint is activated by spindle poisons, a major population of Cdc20 is degraded via APC/C, an event that requires the binding of Cdc20 to Mad2. We propose that the degradation of Cdc20 represents a critical control mechanism to ensure inactivation of APC/C(Cdc20) in response to the SAC
PMCID:2703714
PMID: 19098431
ISSN: 1551-4005
CID: 93223

Thrombin Enhances Spontaneous Tumor Growth and Cell Cycle Activation by Downregulation of p27(Kipl) and Upregulation of Skp2 and MiR-222 [Meeting Abstract]

Hu, L; Ibrahim, S; Liu, C; Skaar, J; Pagano, M; Karpatkin, S
ISI:000262104700397
ISSN: 0006-4971
CID: 93282

KDM2A represses transcription of centromeric satellite repeats and maintains the heterochromatic state

Frescas, David; Guardavaccaro, Daniele; Kuchay, Shafi M; Kato, Hiroyuki; Poleshko, Andrey; Basrur, Venkatesha; Elenitoba-Johnson, Kojo S; Katz, Richard A; Pagano, Michele
Heterochromatin plays an essential role in the preservation of epigenetic information, the transcriptional repression of repetitive DNA elements and inactive genes, and the proper segregation of chromosomes during mitosis. Here we identify KDM2A, a JmjC-domain containing histone demethylase, as a heterochromatin-associated and HP1-interacting protein that promotes HP1 localization to chromatin. We show that KDM2A is required to maintain the heterochromatic state, as determined using a candidate-based approach coupled to an in vivo epigenetic reporter system. Remarkably, a parallel and independent siRNA screen also detected a role for KDM2A in epigenetic silencing. Moreover, we demonstrate that KDM2A associates with centromeres and represses transcription of small non-coding RNAs that are encoded by the clusters of satellite repeats at the centromere. Dissecting the relationship between heterochromatin and centromeric RNA transcription is the basis of ongoing studies. We demonstrate that forced expression of these satellite RNA transcripts compromise the heterochromatic state and HP1 localization to chromatin. Finally, we show that KDM2A is required to sustain centromeric integrity and genomic stability, particularly during mitosis. Since the disruption of epigenetic control mechanisms contributes to cellular transformation, these results, together with the low levels of KDM2A found in prostate carcinomas, suggest a role for KDM2A in cancer development
PMCID:2636745
PMID: 19001877
ISSN: 1551-4005
CID: 91978

PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex

Abbas, Tarek; Sivaprasad, Uma; Terai, Kenta; Amador, Virginia; Pagano, Michele; Dutta, Anindya
The DNA polymerase delta processivity factor Proliferating Cell Nuclear Antigen (PCNA) promotes the DNA damage-induced degradation of the replication initiation factor Cdt1 via the CRL4(Cdt2) E3 ubiquitin ligase complex. Here we demonstrate that PCNA promotes the ubiquitylation and degradation of the CDK inhibitor p21 in cells irradiated with low dose of ultraviolet (UV) by a similar mechanism. Human cells that are depleted of Cul4, DDB1 (damage-specific DNA-binding protein-1), or the DCAF Cdt2, are deficient in the UV-induced ubiquitylation and degradation of p21. Depletion of mammalian cells of PCNA by siRNA, or mutations in p21 that abrogate PCNA binding, prevent UV-induced p21 ubiquitylation and degradation, indicating that physical binding with PCNA is necessary for the efficient ubiquitylation of p21 via the CRL4(Cdt2) ubiquitin ligase. Cdt2 functions as the substrate recruiting factor for p21 to the rest of the CRL4 ubiquitin ligase complex. The CRL4(Cdt2) E3 ubiquitin ligase ubiquitylates p21 both in vivo and in vitro, and its activity is dependent on the interaction of p21 with PCNA. Finally, we show that the CRL4(Cdt2) and the SCF(Skp2) ubiquitin ligases are redundant with each other in promoting the degradation of p21 during an unperturbed S phase of the cell cycle
PMCID:2546691
PMID: 18794347
ISSN: 0890-9369
CID: 96958

APE/Ref-1 makes fine-tuning of CD40-induced B cell proliferation

Merluzzi, Sonia; Gri, Giorgia; Gattei, Valter; Pagano, Michele; Pucillo, Carlo
Apurinic/apyrimidinic endonuclease-1/Redox factor-1, a multifunctional DNA base excision repair and redox regulation enzyme, plays an important role in oxidative signalling, transcription factor regulation, and cell cycle control. Recently, we have demonstrated that following the triggering of CD40 on B cells, APE/Ref-1 translocates from the cytoplasm to the nucleus and regulates the activity of B cell-specific transcription factors. In the present paper we investigate whether APE/Ref-1 plays a role in controlling CD40-mediated B cell proliferation too. We demonstrate a concurrent increase in proliferation and decrease in apoptosis of primary mouse B cells activated by CD40 cross-linking and transfected with functional APE/Ref-1 antisense oligonucleotide. Moreover, we provide evidence that a redox-mediated signalling mechanism is involved in this process and we propose that APE/Ref-1, controlling the intracellular redox state, may also affect the cell cycle by inducing nucleus-cytoplasm redistribution of p21. Together, these findings suggest that APE/Ref-1 could act as a negative regulator in an adaptive response to elevated ROS levels following CD40 cross-linking. Considering the important role of ROS and APE/Ref-1 in CD40-mediated B cell proliferation, our data will contribute to understand the mechanisms of tumor escape and suggest APE/Ref-1 as a novel target for tumor therapeutic approaches
PMCID:2692329
PMID: 18617267
ISSN: 0161-5890
CID: 96959

The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint

Bassermann, Florian; Frescas, David; Guardavaccaro, Daniele; Busino, Luca; Peschiaroli, Angelo; Pagano, Michele
In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. Here, we show that, in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/C(Cdh1), with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin, an activator of the DNA-damage checkpoint, and Wee1, an inhibitor of cell-cycle progression, and allows an efficient G2 checkpoint. As a by-product of APC/C(Cdh1) reactivation in DNA-damaged G2 cells, Claspin, which we show to be an APC/C(Cdh1) substrate in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA-damage-response checkpoint
PMCID:2591934
PMID: 18662541
ISSN: 1097-4172
CID: 80620

Cdh1: a master G0/G1 regulator [Comment]

Skaar, Jeffrey R; Pagano, Michele
APC/C(Cdh1) controls the G0 and G1 phases of the cell cycle. Using a conditional knockout of the Cdh1 coding gene Fizzy-related (Fzr), a new study demonstrates that Cdh1 is essential for viability and that it functions as a tumour suppressor by preventing genomic instability
PMCID:2730193
PMID: 18591966
ISSN: 1476-4679
CID: 80618

The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein

Zhao, Xudong; Heng, Julian Ik-Tsen; Guardavaccaro, Daniele; Jiang, Richeng; Pagano, Michele; Guillemot, Francois; Iavarone, Antonio; Lasorella, Anna
Development of the nervous system requires that timely withdrawal from the cell cycle be coupled with initiation of differentiation. Ubiquitin-mediated degradation of the N-Myc oncoprotein in neural stem/progenitor cells is thought to trigger the arrest of proliferation and begin differentiation. Here we report that the HECT-domain ubiquitin ligase Huwe1 ubiquitinates the N-Myc oncoprotein through Lys 48-mediated linkages and targets it for destruction by the proteasome. This process is physiologically implemented by embryonic stem (ES) cells differentiating along the neuronal lineage and in the mouse brain during development. Genetic and RNA interference-mediated inactivation of the Huwe1 gene impedes N-Myc degradation, prevents exit from the cell cycle by opposing the expression of Cdk inhibitors and blocks differentiation through persistent inhibition of early and late markers of neuronal differentiation. Silencing of N-myc in cells lacking Huwe1 restores neural differentiation of ES cells and rescues cell-cycle exit and differentiation of the mouse cortex, demonstrating that Huwe1 restrains proliferation and enables neuronal differentiation by mediating the degradation of N-Myc. These findings indicate that Huwe1 links destruction of N-Myc to the quiescent state that complements differentiation in the neural tissue
PMCID:2680438
PMID: 18488021
ISSN: 1476-4679
CID: 79363

Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer

Frescas, David; Pagano, Michele
The maintenance and preservation of distinct phases during the cell cycle is a highly complex and coordinated process. It is regulated by phosphorylation--through the activity of cyclin-dependent kinases (CDKs)--and protein degradation, which occurs through ubiquitin ligases such as SCF (SKP1-CUL1-F-box protein) complexes and APC/C (anaphase-promoting complex/cyclosome). Here, we explore the functionality and biology of the F-box proteins, SKP2 (S-phase kinase-associated protein 2) and beta-TrCP (beta-transducin repeat-containing protein), which are emerging as important players in cancer biogenesis owing to the deregulated proteolysis of their substrates
PMCID:2711846
PMID: 18500245
ISSN: 1474-1768
CID: 79246

Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division

Michaelson, David; Abidi, Wasif; Guardavaccaro, Daniele; Zhou, Mo; Ahearn, Ian; Pagano, Michele; Philips, Mark R
Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus functions both as a plasma membrane-targeting motif and a nuclear localization sequence (NLS). We show that a triproline N-terminal to the polybasic region contributes to the NLS, which is cryptic in the sense that it is strongly inhibited by geranylgeranylation of the adjacent cysteine. Subcellular fractionation demonstrated endogenous Rac1 in the nucleus and Triton X-114 partition revealed that this pool is prenylated. Cell cycle-blocking agents, synchronization of cells stably expressing low levels of GFP-Rac1, and time-lapse microscopy of asynchronous cells revealed Rac1 accumulation in the nucleus in late G2 and exclusion in early G1. Although constitutively active Rac1 restricted to the cytoplasm inhibited cell division, activated Rac1 expressed constitutively in the nucleus increased the mitotic rate. These results show that Rac1 cycles in and out of the nucleus during the cell cycle and thereby plays a role in promoting cell division
PMCID:2364699
PMID: 18443222
ISSN: 1540-8140
CID: 79148