Publications by authors named "Steven I Reed"

56 Publications

Cyclin E Overexpression in Human Mammary Epithelial Cells Promotes Epithelial Cancer-Specific Copy Number Alterations.

iScience 2019 Sep 27;19:850-859. Epub 2019 Aug 27.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

Cyclin E, a key cell cycle regulatory protein, has been linked to oncogenesis when dysregulated. We have previously shown that overexpression of cyclin E causes replication stress, leading to failure to complete replication at specific chromosomal loci during S phase of the cell cycle. This in turn promotes chromosomal damage during anaphase. Here we show that non-transformed human mammary epithelial cell clones that survive such aberrant mitoses have a specific and reproducible pattern of chromosomal Copy Number Alterations (CNAs) that we have characterized and termed the cyclin E CNA signature. Using a number of computational approaches, we show that this signature resembles one specific CNA pattern enriched in differentiated epithelial-like tumors of the breast and ovary. Analysis of the CNA profile of these clones provides a potential mechanism for cyclin E-mediated oncogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.isci.2019.08.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739637PMC
September 2019

SCF(FBXW7)-mediated degradation of p53 promotes cell recovery after UV-induced DNA damage.

FASEB J 2019 10 23;33(10):11420-11430. Epub 2019 Jul 23.

Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Seville, Spain.

Eukaryotic cells have developed sophisticated mechanisms to ensure the integrity of the genome and prevent the transmission of altered genetic information to daughter cells. If this control system fails, accumulation of mutations would increase risk of diseases such as cancer. Ubiquitylation, an essential process for protein degradation and signal transduction, is critical for ensuring genome integrity as well as almost all cellular functions. Here, we investigated the role of the SKP1-Cullin-1-F-box protein (SCF)-[F-box and tryptophan-aspartic acid (WD) repeat domain containing 7 (FBXW7)] ubiquitin ligase in cell proliferation by searching for targets implicated in this process. We identified a hitherto-unknown FBXW7-interacting protein, p53, which is phosphorylated by glycogen synthase kinase 3 at serine 33 and then ubiquitylated by SCF(FBXW7) and degraded. This ubiquitylation is carried out in normally growing cells but primarily after DNA damage. Specifically, we found that SCF(FBXW7)-specific targeting of p53 is crucial for the recovery of cell proliferation after UV-induced DNA damage. Furthermore, we observed that amplification of FBXW7 in wild-type p53 tumors reduced the survival of patients with breast cancer. These results provide a rationale for using SCF(FBXW7) inhibitors in the treatment of this subset of tumors.-Galindo-Moreno, M., Giráldez, S., Limón-Mortés, M. C., Belmonte-Fernández, A., Reed, S. I., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. SCF(FBXW7)-mediated degradation of p53 promotes cell recovery after UV-induced DNA damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201900885RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766643PMC
October 2019

Reducing gene dosage induces dopaminergic neuronal loss and motor impairments in knockout mice.

Commun Biol 2019 4;2:125. Epub 2019 Apr 4.

Department of Molecular Medicine, Scripps Research, 10550 North Torrey Pines Road, La Jolla, CA 92037 USA.

Mutations in the gene are associated with early onset Parkinsonism. The mouse, however, does not exhibit neurodegeneration or other Parkinson's disease (PD) phenotypes. Previously, we discovered that translation of Mcl-1, a pro-survival factor, is upregulated in the mouse, suggesting a compensatory mechanism during development. Here we generated the mouse and show that by reducing gene dosage by 50%, the genotype is sensitized, conferring both dopaminergic neuron loss and motor impairments. We propose that this murine model could be a useful tool for dissecting PD etiology and developing treatment strategies against this neurodegenerative disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-019-0366-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449387PMC
May 2020

CKS protein overexpression renders tumors susceptible to a chemotherapeutic strategy that protects normal tissues.

Oncotarget 2017 Dec 4;8(70):114911-114923. Epub 2017 Dec 4.

Department of Molecular Medicine, The Scripps Research Institute, CA 92037, La Jolla, San Diego, USA.

The cyclin-dependent kinase-interacting proteins Cyclin-dependent Kinase Subunit 1 and 2 (CKS1 and 2) are frequently overexpressed in cancer and linked to increased aggressiveness and poor prognoses. We previously showed that CKS protein overexpression overrides the replication stress checkpoint activated by oncoproteins. Since CKS overexpression and oncoprotein activation/overexpression are often observed in the same tumors, we have hypothesized that CKS-mediated checkpoint override could enhance the ability of premalignant cells experiencing oncoprotein-induced replication stress to expand. This tumor advantage, however, could represent a vulnerability to exploit therapeutically. Here, we first show that CKS protein overexpression selectively sensitizes tumor-derived cell lines to nucleoside analog-mediated toxicity under replication stress conditions. A treatment combination of the nucleoside analog gemcitabine and an agent that induces replication stress (thymidine or methotrexate) resulted in selective targeting of CKS protein-overexpressing tumor-derived cells while protecting proliferative cells with low CKS protein levels from gemcitabine toxicity. We validated this strategy and observed that Cks2-overexpressing mammary tumors in nude mice were selectively sensitized to gemcitabine under conditions of methotrexate-induced replication stress. These results suggest that high CKS expression might be useful as a biomarker to identify subgroups of cancer patients who might benefit from the described therapeutic approach.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.22931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777741PMC
December 2017

Cyclin E Deregulation and Genomic Instability.

Adv Exp Med Biol 2017 ;1042:527-547

Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.

Precise replication of genetic material and its equal distribution to daughter cells are essential to maintain genome stability. In eukaryotes, chromosome replication and segregation are temporally uncoupled, occurring in distinct intervals of the cell cycle, S and M phases, respectively. Cyclin E accumulates at the G1/S transition, where it promotes S phase entry and progression by binding to and activating CDK2. Several lines of evidence from different models indicate that cyclin E/CDK2 deregulation causes replication stress in S phase and chromosome segregation errors in M phase, leading to genomic instability and cancer. In this chapter, we will discuss the main findings that link cyclin E/CDK2 deregulation to genomic instability and the molecular mechanisms by which cyclin E/CDK2 induces replication stress and chromosome aberrations during carcinogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-981-10-6955-0_22DOI Listing
July 2018

CKS Proteins Promote Checkpoint Recovery by Stimulating Phosphorylation of Treslin.

Mol Cell Biol 2017 Oct 26;37(20). Epub 2017 Sep 26.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, USA

CKS proteins are small (9-kDa) polypeptides that bind to a subset of the cyclin-dependent kinases. The two paralogs expressed in mammals, Cks1 and Cks2, share an overlapping function that is essential for early development. However, both proteins are frequently overexpressed in human malignancy. It has been shown that CKS protein overexpression overrides the replication stress checkpoint, promoting continued origin firing. This finding has led to the proposal that CKS protein-dependent checkpoint override allows premalignant cells to evade oncogene stress barriers, providing a causal link to oncogenesis. Here, we provide mechanistic insight into how overexpression of CKS proteins promotes override of the replication stress checkpoint. We show that CKS proteins greatly enhance the ability of Cdk2 to phosphorylate the key replication initiation protein treslin Furthermore, stimulation of treslin phosphorylation does not occur by the canonical adapter mechanism demonstrated for other substrates, as cyclin-dependent kinase (CDK) binding-defective mutants are capable of stimulating treslin phosphorylation. This effect is recapitulated , where silencing of Cks1 and Cks2 decreases treslin phosphorylation, and overexpression of wild-type or CDK binding-defective Cks2 prevents checkpoint-dependent dephosphorylation of treslin. Finally, we provide evidence that the role of CKS protein-dependent checkpoint override involves recovery from checkpoint-mediated arrest of DNA replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00344-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615187PMC
October 2017

Mechanism of ubiquitin chain synthesis employed by a HECT domain ubiquitin ligase.

J Biol Chem 2017 06 1;292(25):10398-10413. Epub 2017 May 1.

From the Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037,

omologous to 6AP -erminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing Lys-63, Lys-48, and Lys-11 linkages (Lys-63 > Lys-48 > Lys-11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and are linked exclusively through Lys-63 of Ub. In the second phase, chains are elongated in a multidirectional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M117.789479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481553PMC
June 2017

Cdc6: Skin in the carcinogenesis game.

Cell Cycle 2016 ;15(3):313

b Department of Cell and Molecular Biology , The Scripps Research Institute , La Jolla , CA , USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15384101.2015.1131528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943738PMC
August 2016

Cyclin E deregulation promotes loss of specific genomic regions.

Curr Biol 2015 May 7;25(10):1327-33. Epub 2015 May 7.

Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

Cell-cycle progression is regulated by the cyclin-dependent kinase (Cdk) family of protein kinases, so named because their activation depends on association with regulatory subunits known as cyclins. Cyclin E normally accumulates at the G1/S boundary, where it promotes S phase entry and progression by activating Cdk2. In normal cells, cyclin E/Cdk2 activity is associated with DNA replication-related functions. However, deregulation of cyclin E leads to inefficient assembly of pre-replication complexes, replication stress, and chromosome instability. In malignant cells, cyclin E is frequently overexpressed, correlating with decreased survival in breast cancer patients. Transgenic mice deregulated for cyclin E in the mammary epithelia develop carcinoma, confirming that cyclin E is an oncoprotein. However, it remains unknown how cyclin E-mediated replication stress promotes genomic instability during carcinogenesis. Here, we show that deregulation of cyclin E causes human mammary epithelial cells to enter into mitosis with short unreplicated genomic segments at a small number of specific loci, leading to anaphase anomalies and ultimately deletions. Incompletely replicated regions are preferentially located at late-replicating domains, fragile sites, and breakpoints, including the mixed-lineage leukemia breakpoint cluster region (MLL BCR). Furthermore, these regions are characterized by a paucity of replication origins or unusual DNA structures. Analysis of a large set of breast tumors shows a significant correlation between cyclin E amplification and deletions at a number of the genomic loci identified in our study. Our results demonstrate how oncogene-induced replication stress contributes to genomic instability in human cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2015.03.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4439338PMC
May 2015

Parkin-dependent degradation of the F-box protein Fbw7β promotes neuronal survival in response to oxidative stress by stabilizing Mcl-1.

Mol Cell Biol 2013 Sep 15;33(18):3627-43. Epub 2013 Jul 15.

Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.

Parkinson's disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons resulting in motor dysfunction. While most PD is sporadic in nature, a significant subset can be linked to either dominant or recessive germ line mutations. PARK2, encoding the ubiquitin ligase parkin, is the most frequently mutated gene in hereditary Parkinson's disease. Here, we present evidence for a neuronal ubiquitin ligase cascade involving parkin and the multisubunit ubiquitin ligase SCF(Fbw7β). Specifically, parkin targets the SCF substrate adapter Fbw7β for proteasomal degradation. Furthermore, we show that the physiological role of parkin-mediated regulation of Fbw7β levels is the stabilization of the mitochondrial prosurvival factor Mcl-1, an SCF(Fbw7β) target in neurons. We show that neurons depleted of parkin become acutely sensitive to oxidative stress due to an inability to maintain adequate levels of Mcl-1. Therefore, loss of parkin function through biallelic mutation of PARK2 may lead to death of dopaminergic neurons through unregulated SCF(Fbw7β)-mediated ubiquitylation-dependent proteolysis of Mcl-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00535-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753862PMC
September 2013

Cks1 enhances transcription efficiency at the GAL1 locus by linking the Paf1 complex to the 19S proteasome.

Eukaryot Cell 2013 Sep 3;12(9):1192-201. Epub 2013 Jul 3.

Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.

Cks1 was originally identified based on genetic interactions with CDC28, the gene that encodes Cdk1 in the budding yeast Saccharomyces cerevisiae. Subsequent work has shown that Cks1 binds Cdc28 and modulates its activity against certain substrates. However, the Cks1/Cdc28 complex also has a role in transcriptional chromatin remodeling not related to kinase activity. In order to elucidate protein networks associated with Cks1 transcriptional functions, proteomic analysis was performed on immunoaffinity-purified Cks1, identifying a physical interaction with the Paf1 complex. Specifically, we found that the Paf1 complex component Rtf1 interacts directly with Cks1 and that this interaction is essential for efficient recruitment of Cks1 to chromatin in the context of GAL1 gene induction. We further found that Cks1 in this capacity serves as an adaptor allowing Rtf1 to recruit 19S proteasome particles, shown to be required for efficient RNA production from some rapidly inducible genes such as GAL1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/EC.00151-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811570PMC
September 2013

Ubiquitin ligases and cell cycle control.

Annu Rev Biochem 2013 13;82:387-414. Epub 2013 Mar 13.

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

The ubiquitin-proteasome system plays a pivotal role in the sequence of events leading to cell division known as the cell cycle. Not only does ubiquitin-mediated proteolysis constitute a critical component of the core oscillator that drives the cell cycle in all eukaryotes, it is also central to the mechanisms that ensure that the integrity of the genome is maintained. These functions are primarily carried out by two families of E3 ubiquitin ligases, the Skp/cullin/F-box-containing and anaphase-promoting complex/cyclosome complexes. However, beyond those functions associated with regulation of central cell cycle events, many peripheral cell cycle-related processes rely on ubiquitylation for signaling, homeostasis, and dynamicity, involving additional types of ubiquitin ligases and regulators. We are only beginning to understand the diversity and complexity of this regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-biochem-060410-105307DOI Listing
November 2013

Fbw7α and Fbw7γ collaborate to shuttle cyclin E1 into the nucleolus for multiubiquitylation.

Mol Cell Biol 2013 Jan 29;33(1):85-97. Epub 2012 Oct 29.

Department of Cell and Molecular Biology, The Karolinska Institute, Stockholm, Sweden.

Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G(1)-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G(1) and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCF(Fbw7) (also known as SCF(Cdc4)), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7α is nucleoplasmic but excluded from the nucleolus, Fbw7β is cytoplasmic, and Fbw7γ is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7α and Fbw7γ, respectively. In vitro reconstitution showed that the role of SCF(Fbw7α) in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCF(Fbw7γ). Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7γ drive nucleolar localization of cyclin E, where it is ubiquitylated by SCF(Fbw7γ) prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00288-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536299PMC
January 2013

Cyclin-dependent kinases are regulators and effectors of oscillations driven by a transcription factor network.

Mol Cell 2012 Mar 2;45(5):669-79. Epub 2012 Feb 2.

Department of Biology, Duke University, Durham, NC 27708, USA.

During embryonic cell cycles, B-cyclin-CDKs function as the core component of an autonomous oscillator. Current models for the cell-cycle oscillator in nonembryonic cells are slightly more complex, incorporating multiple G1, S phase, and mitotic cyclin-CDK complexes. However, periodic events persist in yeast cells lacking all S phase and mitotic B-cyclin genes, challenging the assertion that cyclin-CDK complexes are essential for oscillations. These and other results led to the proposal that a network of sequentially activated transcription factors functions as an underlying cell-cycle oscillator. Here we examine the individual contributions of a transcription factor network and cyclin-CDKs to the maintenance of cell-cycle oscillations. Our findings suggest that while cyclin-CDKs are not required for oscillations, they do contribute to oscillation robustness. A model emerges in which cyclin expression (thereby, CDK activity) is entrained to an autonomous transcriptional oscillator. CDKs then modulate oscillator function and serve as effectors of the oscillator.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2011.12.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578314PMC
March 2012

Stacking the deck: putting your Ases where you want them.

Authors:
Steven I Reed

Cell Cycle 2011 Aug 15;10(16):2625-6. Epub 2011 Aug 15.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/cc.10.16.16547DOI Listing
August 2011

Cyclin-dependent kinase subunit (Cks) 1 or Cks2 overexpression overrides the DNA damage response barrier triggered by activated oncoproteins.

Proc Natl Acad Sci U S A 2012 Feb 22;109(8):2754-9. Epub 2011 Jun 22.

Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Cyclin-dependent kinase subunit (Cks) proteins are small cyclin-dependent kinase-interacting proteins that are frequently overexpressed in breast cancer, as well as in a broad spectrum of other human malignancies. However, the mechanistic link between Cks protein overexpression and oncogenesis is still unknown. In this work, we show that overexpression of Cks1 or Cks2 in human mammary epithelial and breast cancer-derived cells, as well as in other cell types, leads to override of the intra-S-phase checkpoint that blocks DNA replication in response to replication stress. Specifically, binding of Cks1 or Cks2 to cyclin-dependent kinase 2 confers partial resistance to the effects of inhibitory tyrosine phosphorylation mediated by the intra-S-phase checkpoint, allowing cells to continue replicating DNA even under conditions of replicative stress. Because many activated oncoproteins trigger a DNA damage checkpoint response, which serves as a barrier to proliferation and clonal expansion, Cks protein overexpression likely constitutes one mechanism whereby premalignant cells can circumvent this DNA damage response barrier, conferring a proliferative advantage under stress conditions, and therefore contributing to tumor development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1102434108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286935PMC
February 2012

Reduced spermatogonial proliferation and decreased fertility in mice overexpressing cyclin E in spermatogonia.

Cell Cycle 2010 Oct 4;9(20):4222-7. Epub 2010 Oct 4.

Department of Molecular Biology, MB-7, The Scripps Research Institute, La Jolla, CA.

Cyclin E is a key component of the cell cycle regulatory machinery, contributing to the activation of Cdk2 and the control of cell cycle progression at several stages. Cyclin E expression is tightly regulated, by periodic transcription and ubiquitin-mediated degradation. Overexpression of cyclin E has been associated with tumor development and poor prognosis in several tumor types, including germ cell tumors and both cyclin E and its partner Cdk2 are required for normal spermatogenesis. Here we have generated and characterized transgenic mice overexpressing a cyclin E mutant protein, resistant to ubiquitin-mediated proteolysis, in testicular germ cells, under the control of the human EF-1alpha promoter. The transgenic mice develop normally and live a normal life span, with no signs of testicular tumor development. The transgenic mice display however reduced fertility and testicular atrophy, due to reduced spermatogonial proliferation as a consequence of deregulated cyclin E levels. Overall our results show that deregulation of cyclin E expression contribute to infertility, due to inability of the spermatogonial cells to start the mitotic cycles prior to entering meiosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055205PMC
http://dx.doi.org/10.4161/cc.9.20.13544DOI Listing
October 2010

Cks1, Cdk1, and the 19S proteasome collaborate to regulate gene induction-dependent nucleosome eviction in yeast.

Mol Cell Biol 2010 Nov 20;30(22):5284-94. Epub 2010 Sep 20.

Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Cks1, Cdk1 (Cdc28), and the proteasome are required for efficient transcriptional induction of GAL1 and other genes in Saccharomyces cerevisiae. We show here that one function of these proteins is to reduce nucleosome density on chromatin in a gene induction-specific manner. The transcriptional requirement for Cks1 can be bypassed if nucleosome density is reduced by an alternative pathway, indicating that this is the primary function of Cks1 in the context of gene induction. We further show that Cks1, Cdk1, and the 19S subunit of the proteasome are recruited to chromatin by binding directly to the histone H4 amino-terminal tail. However, this activity of the proteasome does not require the protease activity associated with the 20S subunit. These data suggest a model where binding of a complex consisting of Cks1, Cdk1, and the 19S proteasome to histone H4 leads to removal of nucleosomes via a nonproteolytic activity of the proteasome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00952-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2976369PMC
November 2010

Phosphorylation of Mcm2 by Cdc7 promotes pre-replication complex assembly during cell-cycle re-entry.

Mol Cell 2009 Jul;35(2):206-16

Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Cyclin E has been shown to have a role in pre-replication complex (Pre-RC) assembly in cells re-entering the cell cycle from quiescence. The assembly of the pre-RC, which involves the loading of six MCM subunits (Mcm2-7), is a prerequisite for DNA replication. We found that cyclin E, through activation of Cdk2, promotes Mcm2 loading onto chromatin. This function is mediated in part by promoting the accumulation of Cdc7 messenger RNA and protein, which then phosphorylates Mcm2. Consistent with this, a phosphomimetic mutant of Mcm2 can bypass the requirement for Cdc7 in terms of Mcm2 loading. Furthermore, ectopic expression of both Cdc6 and Cdc7 can rescue the MCM loading defect associated with expression of dominant-negative Cdk2. These results are consistent with a role for cyclin E-Cdk2 in promoting the accumulation of Cdc6 and Cdc7, which is required for Mcm2 loading when cells re-enter the cell cycle from quiescence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2009.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725784PMC
July 2009

Cyclin-dependent kinase-associated proteins Cks1 and Cks2 are essential during early embryogenesis and for cell cycle progression in somatic cells.

Mol Cell Biol 2008 Sep 14;28(18):5698-709. Epub 2008 Jul 14.

Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Cks proteins associate with cyclin-dependent kinases and have therefore been assumed to play a direct role in cell cycle regulation. Mammals have two paralogs, Cks1 and Cks2, and individually deleting the gene encoding either in the mouse has previously been shown not to impact viability. In this study we show that simultaneously disrupting CKS1 and CKS2 leads to embryonic lethality, with embryos dying at or before the morula stage after only two to four cell division cycles. RNA interference (RNAi)-mediated silencing of CKS genes in mouse embryonic fibroblasts (MEFs) or HeLa cells causes cessation of proliferation. In MEFs CKS silencing leads to cell cycle arrest in G(2), followed by rereplication and polyploidy. This phenotype can be attributed to impaired transcription of the CCNB1, CCNA2, and CDK1 genes, encoding cyclin B1, cyclin A, and Cdk1, respectively. Restoration of cyclin B1 expression rescues the cell cycle arrest phenotype conferred by RNAi-mediated Cks protein depletion. Consistent with a direct role in transcription, Cks2 is recruited to chromatin in general and to the promoter regions and open reading frames of genes requiring Cks function with a cell cycle periodicity that correlates with their transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.01833-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2546922PMC
September 2008

The protease activity of yeast separase (esp1) is required for anaphase spindle elongation independently of its role in cleavage of cohesin.

Genetics 2008 Apr;178(4):2361-72

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

Separase is a caspase-family protease required for the metaphase-anaphase transition in eukaryotes. In budding yeast, the separase ortholog, Esp1, has been shown to cleave a subunit of cohesin, Mcd1 (Scc1), thereby releasing sister chromatids from cohesion and allowing anaphase. However, whether Esp1 has other substrates required for anaphase has been controversial. Whereas it has been reported that cleavage of Mcd1 is sufficient to trigger anaphase in the absence of Esp1 activation, another study using a temperature-sensitive esp1 mutant concluded that depletion of Mcd1 was not sufficient for anaphase in the absence of Esp1 function. Here we revisit the issue and demonstrate that neither depletion of Mcd1 nor ectopic cleavage of Mcd1 by Tev1 protease is sufficient to support anaphase in an esp1 temperature-sensitive mutant. Furthermore, we demonstrate that the catalytic activity of the Esp1 protease is required for this Mcd1-independent anaphase function. These data suggest that another protein, possibly a spindle-associated protein, is cleaved by Esp1 to allow anaphase. Such a function is consistent with the previous observation that Esp1 localizes to the mitotic spindle during anaphase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1534/genetics.107.085308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323821PMC
April 2008

Both SCF(Cdc4alpha) and SCF(Cdc4gamma) are required for cyclin E turnover in cell lines that do not overexpress cyclin E.

Cell Cycle 2008 Apr 17;7(8):1075-82. Epub 2008 Jan 17.

Cancer Center Karolinska, Karolinska Sjukhuset, Department of Oncology/Pathology, Stockholm, Sweden.

The ubiquitin-mediated turnover of cyclin E is regulated by phosphorylation and the activity of the ubiquitin ligase SCF(Cdc4) (also known as SCF(Fbw7)). In 293A cells, SCF complexes containing two different Cdc4 isoforms, alpha and gamma, are required for efficient cyclin E ubiquitylation. Whereas SCF(Cdc4gamma) ubiquitylates cyclin E directly, SCF(Cdc4alpha) serves as a cofactor for Pin1-mediated prolyl isomerization of the cyclin E phosphodegron, essential to potentiate ubiquitylation. In the current study, we show that the requirement for both Cdc4alpha and gamma is general, except in cell lines where cyclin E is expressed at an elevated level. Under these circumstances, Cdc4alpha is sufficient for cyclin E turnover. Furthermore, the requirement for Cdc4gamma can be bypassed by ectopic overexpression of cyclin E.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/cc.7.8.5648DOI Listing
April 2008

Deathproof: new insights on the role of skp2 in tumorigenesis.

Authors:
Steven I Reed

Cancer Cell 2008 Feb;13(2):88-9

Department of Molecular Biology, MB-7, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The F box protein Skp2 is frequently overexpressed in human tumors and is capable of transforming cultured cells in vitro. It has been assumed, quite reasonably, that this oncogenic property of Skp2 is directly related to its role, as part of an SCF ubiquitin ligase complex, in the ubiquitin-mediated proteolysis of negative cell cycle regulatory proteins, notably p27Kip1. However, building on earlier results indicating that silencing of Skp2 promotes apoptosis in some tumor-derived cell lines, Kitagawa and coworkers in the February 1 issue of Molecular Cell have elucidated an alternative mechanism for promotion of tumorigenesis by Skp2, specifically the suppression of p53-mediated apoptosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccr.2008.01.023DOI Listing
February 2008

SCFCdc4 acts antagonistically to the PGC-1alpha transcriptional coactivator by targeting it for ubiquitin-mediated proteolysis.

Genes Dev 2008 Jan;22(2):252-64

Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1alpha (PGC-1alpha) is a highly regulated transcriptional coactivator that coordinates energy metabolism in mammals. Misregulation of PGC-1alpha has been implicated in the pathogenesis of several human diseases, including diabetes, obesity, and neurological disorders. We identified SCF(Cdc4) as an E3 ubiquitin ligase that regulates PGC-1alpha through ubiquitin-mediated proteolysis. PGC-1alpha contains two Cdc4 phosphodegrons that bind Cdc4 when phosphorylated by Glycogen Synthase Kinase 3beta (GSK3beta) and p38 MAPK, leading to SCF(Cdc4)-dependent ubiquitylation and proteasomal degradation of PGC-1alpha. Furthermore, SCF(Cdc4) negatively regulates PGC-1alpha-dependent transcription. We demonstrate that RNAi-mediated reduction of Cdc4 in primary neurons results in an increase of endogenous PGC-1alpha protein, while ectopic expression of Cdc4 leads to a reduction of endogenous PGC-1alpha protein. Finally, under conditions of oxidative stress in neurons, Cdc4 levels are decreased, leading to an increase in PGC-1alpha protein and PGC-1alpha-dependent transcription. These results suggest that attenuation of SCF(Cdc4)-dependent proteasomal degradation of PGC-1alpha has a role in mediating the PGC-1alpha-dependent transcriptional response to oxidative stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.1624208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2192758PMC
January 2008

FBXW7/hCDC4 is a general tumor suppressor in human cancer.

Cancer Res 2007 Oct;67(19):9006-12

Cancer Center Karolinska, Karolinska Hospital, Stockholm, Sweden.

The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skp1-Cdc53/Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin E1, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of approximately 6%. The highest mutation frequencies were found in tumors of the bile duct (cholangiocarcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational "hotspots," which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-07-1320DOI Listing
October 2007

Cyclin E overexpression impairs progression through mitosis by inhibiting APC(Cdh1).

J Cell Biol 2007 Jul;178(3):371-85

Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.

Overexpression of cyclin E, an activator of cyclin-dependent kinase 2, has been linked to human cancer. In cell culture models, the forced expression of cyclin E leads to aneuploidy and polyploidy, which is consistent with a direct role of cyclin E overexpression in tumorigenesis. In this study, we show that the overexpression of cyclin E has a direct effect on progression through the latter stages of mitotic prometaphase before the complete alignment of chromosomes at the metaphase plate. In some cases, such cells fail to divide chromosomes, resulting in polyploidy. In others, cells proceed to anaphase without the complete alignment of chromosomes. These phenotypes can be explained by an ability of overexpressed cyclin E to inhibit residual anaphase-promoting complex (APC(Cdh1)) activity that persists as cells progress up to and through the early stages of mitosis, resulting in the abnormal accumulation of APC(Cdh1) substrates as cells enter mitosis. We further show that the accumulation of securin and cyclin B1 can account for the cyclin E-mediated mitotic phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200703202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064850PMC
July 2007

Myc targets Cks1 to provoke the suppression of p27Kip1, proliferation and lymphomagenesis.

EMBO J 2007 May 26;26(10):2562-74. Epub 2007 Apr 26.

Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA.

Reduced levels of the cyclin-dependent kinase inhibitor p27(Kip1) connote poor prognosis in cancer. In human Burkitt lymphoma and in precancerous B cells and lymphomas arising in Emu-Myc transgenic mice, p27(Kip1) expression is markedly reduced. We show that the transcription of the Cks1 component of the SCF(Skp2) complex that is necessary for p27(Kip1) ubiquitylation and degradation is induced by Myc. Further, Cks1 expression is elevated in precancerous Emu-Myc B cells, and high levels of Cks1 are also a hallmark of Emu-Myc lymphoma and of human Burkitt lymphoma. Finally, loss of Cks1 in Emu-Myc B cells elevates p27(Kip1) levels, reduces proliferation and markedly delays lymphoma development and dissemination of disease. Therefore, Myc suppresses p27(Kip1) expression, accelerates cell proliferation and promotes tumorigenesis at least in part through its ability to selectively induce Cks1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.emboj.7601691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1868903PMC
May 2007

Cooperation between different Cdc4/Fbw7 isoforms may be associated with 2-step inactivation of SCF(Cdc4) targets.

Authors:
Steven I Reed

Cell Cycle 2006 Sep 1;5(17):1923-4. Epub 2006 Sep 1.

Department of Molecular Biology, MB-7, The Scripps Research Institute, La Jolla, California 92037, USA.

The recent description of a role for an SCF ubiquitin ligase in prolyl isomerization rather than ubiquitin ligation has led to a 2-step model for inactivation of cyclin E and, possibly, other SCF(Cdc4) targets in mammalian cells. In this proposed model, targets of SCF(Cdc4) are first subjected to prolyl isomerization, creating a signal for translocation and sequestration in the nucleolus, where they are then multiubiquitylated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/cc.5.17.3198DOI Listing
September 2006

The ubiquitin-proteasome pathway in cell cycle control.

Authors:
Steven I Reed

Results Probl Cell Differ 2006 ;42:147-81

Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Ubiquitin-mediated proteolysis is one of the key mechanisms underlying cell cycle control. The removal of barriers posed by accumulation of negative regulators, as well as the clearance of proteins when they are no longer needed or deleterious, are carried out via the ubiquitin-proteasome system. Ubiquitin conjugating enzymes and protein-ubiquitin ligases collaborate to mark proteins destined for degradation by the proteasome by covalent attachment of multi-ubiquitin chains. Most regulated proteolysis during the cell cycle can be attributed to two families of protein-ubiquitin ligases. The anaphase promoting complex/cyclosome (APC/C) is activated during mitosis and G1 where it is responsible for eliminating proteins that impede mitotic progression and that would have deleterious consequences if allowed to accumulate during G1. SCF (Skp1/Culin/F-box protein) protein-ubiquitin ligases ubiquitylate proteins that are marked by phosphorylation at specific sequences known as phosphodegrons. Targeting of proteins for destruction by phosphorylation provides a mechanism for linking cell cycle regulation to internal and external signaling pathways via regulated protein kinase activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/b136681DOI Listing
September 2006

Detection of low molecular weight derivatives of cyclin E1 is a function of cyclin E1 protein levels in breast cancer.

Cancer Res 2006 Jul;66(14):7355-60

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

Cyclin E1 regulates the initiation of the S phase program in the mammalian cell division cycle. In normal cells, cyclin E1 protein expression is tightly controlled through a combination of transcriptional and proteolytic regulatory processes. However, in many types of human tumor, cyclin E1 expression is frequently dysregulated, including overexpression, nonperiodic expression relative to cell division, and generation of low molecular weight (LMW) derivatives. LMW derivatives of cyclin E1 have been proposed to be generated by the in vivo proteolytic cleavage of the full-length cyclin E1 protein by a yet to be identified tumor-specific protease. Recently, it was suggested that overexpression of full-length or LMW derivatives of cyclin E1 are independent variables associated with poor outcome in patients with breast cancer. However, we have extensively analyzed cyclin E1 protein expression in primary breast tumors and breast tumor-derived cell lines and found that the ability to detect LMW derivatives of cyclin E1 correlates only with the level of cyclin E1 protein. When cyclin E1 levels on Western blots are normalized, LMW derivatives of cyclin E1 were observed at roughly equal levels in all primary breast tumors, breast tumor-derived cell lines, immortalized nontransformed human mammary epithelial cells, and normal breast tissue. Therefore, the detection of LMW derivatives of cyclin E1 is likely a function of cyclin E1 protein levels, and the activity of the proteolytic machinery responsible for their generation is not a tumor-specific property.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-05-3240DOI Listing
July 2006