Table of Contents

Volume 4, Issue 4

  April 2005

  Pages 513 - 620

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 Issue Catalogue (MARC XML)

PERSPECTIVES

Open Access Article

DNA Repair in the Context of Chromatin

Ashby J. Morrison and Xuetong Shen

http://dx.doi.org/10.4161/cc.4.4.1612
Abstract | PDF

Modulation of chromatin is essential to nuclear processes that utilize DNA, such as transcription, replication, and repair. For example, transcription is assisted by histone post-translational modifications, as well as chromatin-remodeling complexes, which alter the structure of chromatin. Furthermore, recent advancements in the fields of DNA repair and chromatin reveal that both histone modifications and chromatin-remodeling complexes are essential for the repair of DNA lesions. In particular, chromatin-modifying complexes, such as the INO80 chromatin-remodeling complex and the Tip60 histone acetyltransferase complex, associate with the DNA damage-induced phosphorylated H2AX, which is often referred to as ?-H2AX. In S. cerevisiae, the association of INO80 with ?-H2AX is required for the recruitment of INO80 to sites of DNA double-strand breaks. Additionally, in Drosophila, Tip60 exchanges ?-H2AX for unmodified H2A in regions of DNA damage. This report reviews recent studies that emphasize the intimate relationship between evolutionarily-conserved chromatin-modifying complexes and histone post-translational modifications in the repair of DNA damage.




EXTRA VIEWS

Open Access Article

Changing Cell Fate by Nuclear Reprogramming

Stina Simonsson and J. B. Gurdon
Pages 513 - 515
http://dx.doi.org/10.4161/cc.4.4.1581
Abstract | PDF

The differentiated state of somatic cells is remarkably stable, but nuclear transfer can efficiently override the stability of cell differentiation by nuclear reprogramming. Genes that were silent in the differentiated state are activated, whereas genes specific for the differentiated state are switched off during the reprogramming process. The epigenetic changes that occur after nuclear transfer to Xenopus oocytes involve chromatin remodelling and DNA demethylation. In particular, we have reported that reactivation of the mouse stem cell specific gene oct-4 depends on demethylation of CpG:s in the proximal oct-4 promoter. Here we discuss molecular mechanisms of nuclear reprogramming, with special emphasis on DNA demethylation.

Open Access Article

HuntING4 New Tumor Suppressors

Suwon Kim
Pages 516 - 517
http://dx.doi.org/10.4161/cc.4.4.1584
Abstract | PDF

The well-characterized function of many tumor suppressors is to directly regulate cell cycle events. We recently reported a genetic screen to identify genes that suppress loss of contact inhibition but do not inhibit normal cell proliferation. ING4, one of the genes identified by the screen, is frequently mutated in human cancer, suggesting a role for ING4 as a tumor suppressor. ING4 suppresses tumor cell features such as loss of contact inhibition, cell growth in soft agar, and tumor growth in nude mice. ING4 may belong to a class of tumor suppressors whose function is specific to a tumorigenic event that does not directly involve deregulation of the cell cycle. Studies of these tumor suppressors may provide insight into the molecular mechanisms underlying such tumorigenic events.

Open Access Article

A Mouse Model of Activating Met Mutations

Carrie R. Graveel, Cheryl A. London and George F. Vande Woude
Pages 518 - 520
http://dx.doi.org/10.4161/cc.4.4.1590
Abstract | PDF

Tyrosine kinase–activating mutations in Met have been observed in hereditary papillary renal carcinomas (HPRC) and their transforming potential has been examined both in vitro and in tumor xenografts. To study the influence of these mutations in the mouse germline, we generated mice with targeted mutations in the murine met locus. Five mouse lines with mutant Met were created: WT, D1226N, Y1228C, M1248T, and M1248T/L1193V. Unexpectedly, the different mutant Met lines developed unique tumor profiles including carcinomas, sarcomas, and lymphomas. More surprising was that we observed nonrandom duplication of the mutant met allele in a majority of tumors from the mutant mouse lines. This selective chromosomal amplification has been observed in patients with HPRC. This study illustrates the importance of activating Met mutations in tumorigenesis and indicates that mutations within the kinase domain distinctly affect downstream signaling. Our Met mutant mice will provide a valuable model for testing Met inhibitors on tumors containing activating mutations present in human cancers and for understanding the molecular events critical for Met-mediated tumorigenesis.

Open Access Article

Rare Variant Hypothesis for Multifactorial Inheritance: Susceptibility to Colorectal Adenomas as a Model

Nicola S. Fearnhead, Bruce Winney and Walter F. Bodmer
Pages 521 - 525
http://dx.doi.org/10.4161/cc.4.4.1591
Abstract | PDF

The rare variant hypothesis postulates that genetic susceptibility to colorectal neoplasia within the general population is due to a number of low frequency variants in a variety of different genes. Each variant confers a moderate, but detectable, increase in relative risk of developing the disease. Recent evidence suggests that a quarter of patients with multiple adenomatous polyps are due to rare but functionally important variants in just five genes.

Open Access Article

Inhibition of Caspase 9 and Not Caspase 8 Mediated Apoptosis May Determine Clinical Response to Chemotherapy in Primary Nodal Diffuse Large B-Cell Lymphomas

Joost J. Oudejans, Jettie J.F. Muris and Chris J.L.M. Meijer
Pages 526 - 528
http://dx.doi.org/10.4161/cc.4.4.1595
Abstract | PDF

Clinical response to chemotherapy in patients with diffuse large B-cell lymphomas is poorly predictable. We demonstrated that an expression profile consistent with inhibition of the stress-induced, caspase 9 mediated apoptosis pathway predicts poor clinical response to chemotherapy, whereas an expression profile consistent with inhibition of only the death-receptor induced, caspase 8 mediated pathway was associated with an excellent response to chemotherapy and favorable outcome. Furthermore, functional analysis of apoptosis sensitivity showed that lymphomas with a caspase 9 inhibition profile were indeed relatively resistant to Etoposide induced apoptosis. Determining the functional status of both apoptosis signaling pathways may accurately identify patients at high risk for fatal outcome and may have implications for alternative therapies triggering the death receptor mediated apoptosis pathway.

Open Access Article

9-1-1 Complex Involvement in DNA Repair: Evidence that DNA Damage Detection Machinery Participates in DNA Repair

Christopher E. Helt, Wensheng Wang, Peter C. Keng and Robert A. Bambara
Pages 529 - 532
http://dx.doi.org/10.4161/cc.4.4.1598
Abstract | PDF

The toroidal Rad9-Rad1-Hus1 checkpoint complex (9-1-1) is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication and repair. 9-1-1 has been characterized as a sensor of DNA damage that functions in concert with the checkpoint control proteins ATM and ATR. However, recent data suggest that the 9-1-1 complex and its individual Rad9 component serve different and multiple functions in cells by sensing DNA damage, stimulating apoptosis, and regulating gene transcription. Recently it was reported that 9-1-1 interacts with and/or stimulates components of the base excision repair (BER) pathway including the S. pombe MutY homolog (MYH), human polymerase ? (Pol ?), and flap endonuclease 1 (FEN1). Furthermore, preliminary results indicate a stimulation of DNA ligase I. In this review, the likely direct participation of 9-1-1 in DNA repair is discussed.

Open Access Article

Gam1 and the SUMO Pathway

Roberto Boggio and Susanna Chiocca
Pages 533 - 535
http://dx.doi.org/10.4161/cc.4.4.1605
Abstract | PDF

Post-translational modifications of proteins have critical roles in many cellular processes because they can cause rapid changes in the functions of pre-existing proteins, multiprotein complexes and subcellular structures. Sumoylation, a ubiquitin-like dynamic and reversible post-translational modification system, is an enzymatic cascade leading to the covalent attachment of SUMO to it target proteins. This modification involves three steps and different enzymes: SUMO-activating enzyme E1 (SAE1/SAE2), SUMO-conjugating enzyme E2 (UBC9), SUMO ligases E3s, and SUMO cleaving enzymes. Although the identification of SUMO-modified substrates has progressed rapidly, the biological function of SUMO and regulation of SUMO conjugation are still not well understood. Some viral proteins have been identified as substrates for SUMO modification as well as altering the sumoylation status of host cell proteins. We have been studying an unusual adenoviral protein, Gam1, a strong and global transcriptional activator of both viral and cellular genes that inactivates HDAC1. We have recently expanded the known functions of Gam1 by demonstrating that Gam1 also inhibits the SUMO pathway by interfering with the activity of E1 heterodimer (SAE1/SAE2), leading to the accumulation of SUMO-unmodified substrates. Our data provides a clear example of the effects of a viral infection on host sumoylation and supports the idea that viruses have multifunctional protein that can target essential biochemical pathways.

Open Access Article

DNA Damage-Induced Cohesion

Lena Ström and Camilla Sjögren
Pages 536 - 539
http://dx.doi.org/10.4161/cc.4.4.1613
Abstract | PDF

The protein complex Cohesin, forming protein-links that hold sister chromatids together, is at the heart of chromatid cohesion. Cohesion is important both for correct chromosome segregation and double-strand break (DSB) repair, making Cohesin central for the maintenance of genome stability. Until now, establishment of Cohesin links between chromatids has been shown to occur during DNA replication only. Recently it was however observed that in cells arrested in G2/M, DSB induction not only elicits chromosomal recruitment of Cohesin, but also formation of chromatid cohesion. The establishment of cohesion outside the period of replication opens a new field of investigation. Here we present results further supporting the formation of sister chromatid cohesion in response to DNA damage, and propose a model of how damage-induced cohesion could contribute to the linkage of chromatids during normal cell cycle progression.

Open Access Article

PTEN: A Novel Anti-oncogenic Function Independent of Phosphatase Activity

Koichi Okumura, Mujun Zhao, Ronald A. DePinho, Frank B. Furnari and Webster K. Cavenee
Pages 540 - 542
http://dx.doi.org/10.4161/cc.4.4.1614
Abstract | PDF

The PTEN gene is an important tumor suppressor mutated in a number of cancers. To date, its growth regulatory properties have been intimately linked to its ability to act as a protein and phosphoinositol phosphatase. Inactivation of the enzymatic activity of PTEN is primarily due to direct mutation of its amino-terminal catalytic domain but ~20% of mutations are in the carboxy-terminus, which regulates membrane localization, protein stability, cellular migration and p53 function. We identified a novel protein that interacts with this domain, the v-jun transcriptional target, MSP58. Binding of MSP58 to PTEN results in the suppression of MSP58-mediated transformation. However, this PTEN effect does not require its catalytic activity, suggesting additional mechanisms of PTEN action.

Open Access Article

Chromatin Remodelling by WSTF-ISWI at the Replication Site: Opening a Window of Opportunity for Epigenetic Inheritance?

Raymond A. Poot, Ludmila Bozhenok, Debbie L.C. van den Berg, Nicola Hawkes and Patrick D. Varga-Weisz
Pages 543 - 546
http://dx.doi.org/10.4161/cc.4.4.1624
Abstract | PDF

During DNA replication, chromatin states have to be accurately transmitted from the parental to the daughter strands for faithful epigenetic inheritance. Chromatin remodelling factors at the replication site are thought to be involved in this process. Recent work adds ATP-dependent nucleosome remodelling factors to this category of enzymes. The WICH complex, consisting of the ISWI-type ATPase SNF2H and the Williams Syndrome Transcription Factor (WSTF), binds to replication foci using PCNA, a key factor in DNA- and chromatin replication and DNA repair, as an interaction platform. Depletion of WSTF results in decreased chromatin accessibility, which is evident already in newly replicated DNA. This leads to heterochromatin formation on a global scale and a decrease in overall transcriptional activity. Here, we propose that WICH, by keeping nucleosomes mobile, provides access to the newly replicated DNA and may thereby create a window of opportunity after DNA replication for rebinding of factors that maintain the epigenetic state, and thus prevents aberrant heterochromatin formation. Our model may provide an explanation for the long-standing observation of a delay in chromatin “maturation” on newly replicated DNA, by connecting this delay with the action of PCNA-bound WSTF-ISWI, and highlights chromatin remodeling shortly after DNA replication as a critical point for regulation.

Open Access Article

BRAF Kinase Activation via Chromosomal Rearrangement in Radiation-Induced and Sporadic Thyroid Cancer

Raffaele Ciampi, Jeffrey A. Knauf, Hartmut M. Rabes, James A. Fagin and Yuri E. Nikiforov
Pages 547 - 548
http://dx.doi.org/10.4161/cc.4.4.1631
Abstract | PDF

Activating point mutations of the BRAF gene have been recently described in a variety of human tumors. In a study published in the Journal of Clinical Investigation, we reported a novel mechanism of activation of this gene via paracentric inversion of chromosome 7q. The fusion protein, AKAP9-BRAF, contains the intact kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It exhibited constitutive activation of BRAF kinase and was transforming for NIH3T3 cells. This finding represents the first demonstration of RAF activation by chromosomal rearrangement in human tumors. AKAP9-BRAF was more common in radiation-induced thyroid tumors, whereas point mutations of BRAF predominated in sporadic tumors of the same type, demonstrating the association between environmental factors and specific mechanisms of BRAF activation.




PERSPECTIVES

Open Access Article

Histone Deacetylase Inhibitors in Programmed Cell Death and Cancer Therapy

Paul A. Marks and Xuejun Jiang
Pages 549 - 551
http://dx.doi.org/10.4161/cc.4.4.1564
Abstract | PDF

Histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA), are targeted anticancer agents that have significant anticancer activity at doses well tolerated by patients.1 Recently, we found that HDAC inhibitors can trigger both mitochondria-mediated apoptosis and caspase-independent autophagic cell death, indicating potential benefit of HDAC inhibitors in treating cancers with apoptotic defects.2 We also found that thioredoxin (TRX) might play a significant role in HDAC inhibitor-induced cell death, and HDAC inhibitors increase TRX levels in normal cells but not transformed cells, which is likely to be one of the reasons why HDAC inhibitors preferentially kill cancer cells.3 In this review, we discuss the study of HDAC inhibitors in cell death and cancer research, the implications of our recent findings, and some outstanding questions that need to be addressed.

Open Access Article

Cyclic Peptides, A Chemical Genetics Tool for Biologists

Alexander R. Horswill and Stephen J. Benkovic
Pages 552 - 555
http://dx.doi.org/10.4161/cc.4.4.1585
Abstract | PDF

The genomics era has presented scientists with an overwhelming amount of sequence information that encodes for a continuously expanding array of metabolic pathways and regulatory networks. In order to unravel this elaborate maze, effective approaches need to be pioneered, and cyclic peptides represent an exciting new tool that can aid this challenging task. There are considerable benefits to cyclic peptides including enhanced protein binding affinity and metabolic stability, but perhaps most important is the ability to biologically synthesize them using intein chemistry. This capacity for genetic encoding allows biologists access to diverse, intracellular small-molecule libraries that can be tailored for numerous chemical genetics approaches. An increasing number of molecular strategies are being developed to aid in the implementation of cyclic peptides in forward and reverse genetics experiments, which will provide researchers with a powerful toolbox to complement existing genomic methodologies.

Open Access Article

Connecting Reovirus Oncolysis and Ras Signaling

Paola Marcato, Maya Shmulevitz and Patrick W. Lee
Page 556
http://dx.doi.org/10.4161/cc.4.4.1600
Abstract | PDF

Reovirus is a benign virus with innate oncolytic activity and is a potential novel therapeutic for a number of cancers. Reovirus can replicate in, and induce death of cancerous cells having an activated Ras pathway. Ras activation leads to the inactivation of cellular antiviral mechanisms, specifically removing the translation block on reovirus transcripts. This review outlines recent progress towards elucidating the molecular connection between the Ras-signaling pathway and reovirus replication.

Open Access Article

The Anaphase Promoting Complex: A Critical Target for Viral Proteins and Anti-Cancer Drugs

Destin W. Heilman, Michael R. Green and Jose G. Teodoro
Pages 557 - 560
http://dx.doi.org/10.4161/cc.4.4.1606
Abstract | PDF

The study of animal viruses has provided extraordinary insights into cell cycle dynamics and tumor biology. The significance of the p53 and Rb tumor suppressor proteins, for example, was discovered due to their interactions with viral oncogenes. In the past several years, investigations with four viral proteins, human immunodeficiency virus type 1 (HIV-1) vpr, adenovirus E4orf4, chicken anemia virus (CAV) apoptin and human T lymphotropic virus type I (HTLV-I) Tax, have indicated that there are also critical viral targets involved in G2/M control. In particular, recent studies with E4orf4 and apoptin have shown that they induce G2/M arrest by targeting and inhibiting the anaphase-promoting complex/cyclosome (APC/C). Notably, these two viral proteins induce apoptosis selectively in transformed cells in a p53-independent manner; thus pathways affected by these proteins are of significant therapeutic interest. Further investigation of the underlying mechanism of G2/M arrest and subsequent apoptosis induced by viral APC/C inhibitors may shed light on the mechanisms of current cancer therapies and provide the foundation for developing novel therapeutic targets.

Open Access Article

Functional Genomic Analysis of Cell Division by Endoribonuclease-Prepared siRNAs

Ralf Kittler and Frank Buchholz
Pages 561 - 564
http://dx.doi.org/10.4161/cc.4.4.1608
Abstract | PDF

The emergence of RNA interference (RNAi) technology has revolutionized functional genomic analyses in cell biology, including the study of cell division. In particular, the introduction of short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has facilitated loss-of-function studies in mammalian cell lines. We have pioneered the rapid and cost-efficient generation of libraries of endoribonuclease-prepared short interfering RNAs (esiRNAs) for large-scale genetic screens in mammalian tissue culture cells. Our first pilot screen of 5305 genes in human HeLa cells identified novel genes required for cell division, establishing esiRNA as a sophisticated method for loss-of-function screens. Future genome-wide studies with esiRNA, using image-based assays and video microscopy, will help provide novel insights on the molecular mechanisms that govern cell division in mammalian cells.




REVIEW

Open Access Article

CAK—Cyclin-Dependent Activating Kinase: A Key Kinase in Cell Cycle Control and a Target for Drugs?

Graziano Lolli and Louise N. Johnson
Pages 565 - 570
http://dx.doi.org/10.4161/cc.4.4.1607
Abstract | PDF

The Cyclin-dependent kinase (CDK) Activating Kinase (CAK) is responsible for the activating phosphorylation of CDK1, CDK2, CDK4 and CDK6 and regulation of the cell cycle. The kinase is composed of three subunits: CDK7, Cyclin H and MAT1 (ménage a trois). Together with six other subunits, CAK is also part of the general transcription factor TFIIH where it is involved in promoter clearance and progression of transcription from the pre-initiation to the initiation stage. CAK is required for cell cycle progression, which suggests that CDK7 could be a target for cancer therapy. However its role in transcription and its ubiquitous presence raise sensible concerns about possible toxicity of its inhibitors. The recently determined structure of CDK7 allows the design of inhibitors with differential specificity for the different CDKs. We review the role of CAK in different biological processes and evaluate the biological evidence for CDK7 as a possible pharmacological target.




VIEWS AND COMMENTARIES

Open Access Article

Mutated PI 3-Kinases: Cancer Targets on a Silver Platter

Sohye Kang, Andreas G. Bader, Li Zhao and Peter K. Vogt
Pages 571 - 574
http://dx.doi.org/10.4161/cc.4.4.1593
Abstract | PDF

No abstract available.




BRIEF REPORTS

Open Access Article

The PPAR-? Agonist Pioglitazone Post-Trancriptionally Induces p21 in PC3 Prostate Cancer but Not in Other Cell Lines

Senthil K. Radhakrishnan and Andrei L. Gartel
Pages 575 - 577
http://dx.doi.org/10.4161/cc.4.4.1583
Abstract | PDF

The anti-diabetic thiozolidinedione compound pioglitazone, a peroxisome proliferator-activated receptor-? (PPAR-?) agonist, has been found to have growth inhibitory effects in some cancer cells. The mechanism underlying this growth suppression is not well understood. In this study, we evaluated the effect of pioglitazone on p53 and p21 expression in various cancer cell lines with different p53 status. The cells with wild type p53 did not show any change in p53 levels in response to pioglitazone. Also, none of the cell lines exhibited p53-dependent or independent transcriptional induction of p21^WAF1/CIP1 by pioglitazone. However, PC3, an androgen-insensitive prostate cancer cell line with deletion of p53 showed an appreciable post-transcriptional induction of p21 expression after treatment with pioglitazone. These results imply that pioglitazone generally does not modulate p21 transcription in human cancer cell lines. Running Title: Induction of p21 in PC3 cells by pioglitazone

Open Access Article

Telomerase RNA Mutated in Autosomal Dyskeratosis Congenita Reconstitutes a Weakly Active Telomerase Enzyme Defective in Telomere Elongation

Maria Antonietta Cerone, Ryan J Ward, J. Arturo Londoño-Vallejo and Chantal Autexier
Pages 578 - 582
http://dx.doi.org/10.4161/cc.4.4.1586
Abstract | PDF

Dyskeratosis congenita (DC) is a rare multi-system syndrome characterized by nail dystrophy, abnormal skin pigmentation and mucosal leukoplakia. The gene mutated in the Xlinked form of human DC encodes for dyskerin, a nucleolar pseudourydilase that is involved in rRNA maturation. Dyskerin is also involved in telomerase function through its interaction with the telomerase RNA (hTR). Mutations in dyskerin result in low levels of hTR, decreased telomerase activity and telomere shortening. Autosomal dominant DC is characterized by mutations in hTR, supporting the hypothesis that the DC phenotype may be caused by impaired telomere maintenance. Several mutations have been identified in different regions of hTR in patients affected by autosomal dominant DC. Recent reports have shown that co-expression of wild-type hTR with hTR harboring mutations found in the pseudoknot domain does not affect telomerase activity in vitro. However, these studies did not assess the consequences of mutant hTR expression at the telomeres. Here we provide the first direct in vivo evidence that a mutant hTR carrying the GC to AG double substitution in the pseudoknot at nucleotides 107-108 found in patients affected by autosomal dominant DC does not behave as a dominant-negative for telomere maintenance. Rather it reconstitutes a weakly active telomerase enzyme, which is defective in telomere elongation.




REPORTS

Open Access Article

Localization of Phosphorylated Forms of Bcl-2 in Mitosis : Co-Localization with Ki-67 and Nucleolin in Nuclear Structures and on Mitotic Chromosomes

Nadia Barboule, Isabelle Truchet and Annie Valette
Pages 583 - 589
http://dx.doi.org/10.4161/cc.4.4.1587
Abstract | PDF

Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or during mitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2 phosphorylation on its function are still controversial. To better understand the role of Bcl-2 phosphorylation in mitosis, we studied the subcellular localization of phosphorylated forms of Bcl-2. Immunofluorescence experiments performed in synchronized HeLa cells indicate for the first time that mitotic phosphorylated forms of Bcl-2 can be detected in nuclear structures in prophase cells together with nucleolin and Ki-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2 are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in these structures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitation experiments reveal that, in cells synchronized at the onset of mitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase. Taken together, these data further support the idea that Bcl-2 could have a new function at mitosis.

Open Access Article

p14ARF Interacts with the SUMO-Conjugating Enzyme Ubc9 and Promotes the Sumoylation of Its Binding Partners

Helen Rizos, Sarah Woodruff and Richard F. Kefford
Pages 590 - 596
http://dx.doi.org/10.4161/cc.4.4.1588
Abstract | PDF

The p14ARF tumour suppressor regulates a series of cell cycle regulatory proteins to promote cell cycle arrest in response to abnormal hyperproliferative growth stimuli. p14ARF alterations are common in human cancers and, when inherited, confer susceptibility to cutaneous melanoma. We now propose that the mechanism of p14ARF action may involve the covalent modification of its binding partners with the small ubiquitin-related protein SUMO-1. In particular, we demonstrate that p14ARF interacts with the SUMO E2 conjugating enzyme, Ubc9 and enhances the sumoylation of its binding partners, hdm2, E2F-1, HIF-1?, TBP-1 and p120E4F. Furthermore, p14ARF-induced sumoylation is abrogated by a subset of melanoma-associated p14ARF mutations. These results provide a mechanism for p14ARF action through a common modification of diverse binding partners.

Open Access Article

MdmX Inhibits ARF Mediated Mdm2 Sumoylation

Mithua Ghosh, Karen Weghorst and Steven J. Berberich
Pages 597 - 601
http://dx.doi.org/10.4161/cc.4.4.1597
Abstract | PDF

Mdm2, by virtue of an intrinsic E3 ubiquitin ligase activity, is capable of autoubiquitination and the ubiquitination of the p53 tumor suppressor protein. Additionally, Hdm2 has been reported to undergo a p14ARF-dependent sumoylationwith concurrent Hdm2 stabilization. In this present work, we report that MdmX can undergo ARF-mediated sumoylation similar to that reported for Mdm2. When coexpressed, MdmX overexpression results in a dose-dependent inhibition of Mdm2 sumoylation and a concurrent increase in Mdm2 ubiquitination. This switch from Mdm2 sumoylation to Mdm2 ubiquitination may explain the destablization of Mdm2 previously observed in cells overexpressing both ARF and MdmX. Given that MdmX can heterodimerize with Mdm2 and separately associate with ARF we employed a series of MdmX mutants to examine how MdmX blocks Mdm2 sumoylation. A MdmX miniprotein capable of binding to ARF, but not p53 or Mdm2 was able to competitively inhibit Mdm2 sumoylation and reverse ARF mediated activation of p53 transactivation. Taken together, these results demonstrate that MdmX can affect post-translational modification and stability of Mdm2 and p53 activity through interaction with ARF.

Open Access Article

The Plk1 Polo Box Domain Mediates a Cell Cycle and DNA Damage Regulated Interaction with Chk2

Lyuben M. Tsvetkov, Rumiana T. Tsekova, Xingzhi Xu and David F. Stern
Pages 602 - 610
http://dx.doi.org/10.4161/cc.4.4.1599
Abstract | PDF

Polo-like kinase 1 (Plk1) regulates multiple processes during mitosis. Chk2 is a tumor suppressor that participates in DNA damage checkpoint signaling cascades. Plk1 phosphorylates, co-localizes with, and interacts with Chk2, suggesting interconnection of DNA damage checkpoints and mitotic regulation. However, the function of their association is unknown. Here, we show that the interaction between Chk2 and Plk1 is cell cycle-regulated, with a peak in mitosis. DNA damage in G2 and M phases but not in S phase induces dissociation of Plk1 and Chk2. In vitro, the Plk1 PBD binds phosphorylated Chk2, and mediates an interaction independent of other eukaryotic proteins. Additionally, a phosphopeptide encompassing phosphoT68 of Chk2 binds Plk1 in a PBD-dependent manner, and stimulates Plk1 activity. These results identify potential mechanisms for interaction and inter-regulation of these two protein kinases.

Open Access Article

The Regulation of S Phase Initiation by p27Kip1 in NIH3T3 Cells

Gaurisankar Sa, Yang Guo and Dennis W. Stacey
Pages 611 - 620
http://dx.doi.org/10.4161/cc.4.4.1632
Abstract | PDF

Most eukaryotic cells become committed to divide once DNA synthesis begins. This critical event is controlled in yeast and Xenopus oocytes by the degradation of cyclin inhibitory proteins, while in mammalian cells over-expression of cyclin E or cyclin D1 promotes rapid entry into DNA synthesis. Curiously, however, this over-expression also lengthens S phase and promotes DNA damage. To directly assess the roles of the cyclin inhibitory protein p27Kip1 (p27) and of cyclin D1 in the regulation of DNA synthesis initiation in mammalian cells, we have utilized a quantitative cytometric approach for the study of cell cycle control in actively proliferating cultures. As evidence that p27 plays a direct role in regulating entry into S phase, we find that its levels fall at the time of DNA synthesis initiation in NIH3T3 cells, and that its suppression shortens G1 phase and shortens the length of the entire cell cycle. In this function, however, the action of p27 appears to be linked with cyclin D1. G1-S phase transition is efficiently blocked by p27 unless excess cyclin D1 is present. These two proteins are coordinately regulated by the cell, and are maintained at a nearly constant ratio throughout an actively proliferating culture. We propose that p27 directly regulates the initiation of DNA synthesis in NIH3T3 cells, and that cyclin D1 serves to modulate this activity.