Anchorage-dependent expression of cyclin A in primary cells requires a negative DNA regulatory element and a functional Rb

Philips, Alexandre; Huet, Xavier; Plet, Ariane; Rech, Jocelyne; Vié, Annick; Blanchard, Jean Marie

doi:10.1038/sj.onc.1202530

Cited by 17 publications

(16 citation statements)

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“…Pocket proteins are thought to regulate cyclin A promoter activity through the CDE/CHR site (18,28,29,32,33), but the proteins that bind to this site are very poorly defined. Some studies (32,44) suggest that the CDE is occupied by E2F4/p107 complexes, but the binding of any E2F to the cyclin A promoter is highly controversial (see the introduction).…”

Section: Discussionmentioning

confidence: 99%

“…However, another group reported that E2Fs do not bind with high affinity to the CDE site in the cyclin A promoter (18), and a third group (21) reported that E2F1 and 3, but not E2F4, bind to the CDE and act as transcriptional activators at G 1 /S rather than repressors at G 0 and early G 1 . Moreover, experiments with pRb-null and p107-null mouse embryo fibroblasts (MEFs) emphasize that pRb, and not p107, is important for cyclin A gene expression (28,29). Thus, the exact mechanism by which pocket proteins control cyclin A gene expression through the CDE/CHR is poorly understood and may well involve both indirect and direct effects.…”

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confidence: 99%

“…The upstream element is referred to as the cell cycle-dependent element (CDE), also called the cell cycle regulatory element, and the contiguous downstream element has been termed the cell cycle gene homology region (CHR) (47). The CDE site in the cyclin A promoter is required for transcriptional repression, preventing cyclin A gene expression in G 0 and early G 1 phase (18,28,29,32,47,48). The CHR is thought to act as a corepressor cis element (48).…”

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Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

Bottazzi

Buzzai

Zhu

et al. 2001

Molecular and Cellular Biology

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Soluble mitogens and adhesion-dependent organization of the actin cytoskeleton are required for cells to enter S phase in fibroblasts. The induction of cyclin A is also required for S-phase entry, and we now report that distinct effects of mitogens and the actin cytoskeleton on the phosphorylation of CREB and pocket proteins regulate the extent and timing of cyclin A promoter activity, respectively. First, we show that CREB phosphorylation and binding to the cyclic AMP response element (CRE) determines the extent, but not the timing, of cyclin A promoter activity. Second, we show that pocket protein inactivation regulates the timing, but not the extent, of cyclin A promoter activity. CREB phosphorylation and CRE occupancy are regulated by soluble mitogens alone, while the phosphorylation of pocket proteins requires both mitogens and the organized actin cytoskeleton. Mechanistically, cytoskeletal integrity controls pocket protein phosphorylation by allowing for sustained ERK signaling and, thereby, the expression of cyclin D1. Our results lead to a model of cyclin A gene regulation in which mitogens play a permissive role by stimulating early G 1 -phase phosphorylation of CREB and a distinct regulatory role by cooperating with the organized actin cytoskeleton to regulate the duration of ERK signaling, the expression of cyclin D1, and the timing of pocket protein phosphorylation.The proliferation of most cell types requires exposure to mitogenic growth factors, adhesion to the extracellular matrix and organization of the actin cytoskeleton. Growth factors, cell adhesion, and the organized actin cytoskeleton are all specifically required for cell cycle progression through the G 1 phase (2). Organization of the actin cytoskeleton is a consequence of integrin-mediated adhesion, but cell adhesion and the organized cytoskeleton have distinguishable effects on G 1 -phase progression (14,17). While many studies have focused on differences in signaling and cell cycle progression in adherent and nonadherent cells, the cell cycle consequence of disrupting the actin cytoskeleton in adherent cells has received much less attention.Cell cycle progression is mediated by the cyclin-dependent kinases (cdk's) with the formation of active cyclin D-cdk4 (or -cdk6) and cyclin E-cdk2 complexes controlling progression through G 1 phase (35). In fibroblasts, the activation of cyclin D1-cdk4 or -cdk6 is typically limited by the mid-G 1 -phase expression of cyclin D1, while the activation of cyclin E-cdk2 is controlled by the mid-to-late G 1 -phase downregulation of p21 family cdk inhibitors (typically p21 cip1 and p27 kip1 ). The G 1 -phase cyclin-cdk's are required for phosphorylation of the retinoblastoma protein (pRb) and p107 (referred to as pocket proteins); this phosphorylation results in the disruption of E2F-pocket protein complexes and the induction of pRb-regulated genes such as cyclin A (15, 40). We and others have shown that the expression of cyclin D1 and the downregulation of p21 cip1 and p27 kip1 are jointly dependent upon ...

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Section: Discussionmentioning

confidence: 99%

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Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

Bottazzi

Buzzai

Zhu

et al. 2001

Molecular and Cellular Biology

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“…Cyclin A mRNA and protein accumulations at the end of G 1 are required for progression into S phase and DNA replication (15,29,45). We have previously analyzed the expression of cyclin A in human and rodent cells (1,2,(30)(31)(32)(33) and identified functional DNA sequences present in the mouse cyclin A promoter (2,21). In vivo genomic dimethyl sulfate footprinting revealed the presence of cell cycle-regulated protein binding elements close to the major transcription initiation sites.…”

Section: E4fmentioning

confidence: 99%

Cyclin A Is a Mediator of p120^E4F-Dependent Cell Cycle Arrest in G₁

Fajas

Paul

Vié

et al. 2001

Molecular and Cellular Biology

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E4F is a ubiquitously expressed GLI-Krüppel-related transcription factor which has been identified for its capacity to regulate transcription of the adenovirus E4 gene in response to E1A. However, cellular genes regulated by E4F are still unknown. Some of these genes are likely to be involved in cell cycle progression since ectopic p120 E4F expression induces cell cycle arrest in G 1 . Although p21 WAF1 stabilization was proposed to mediate E4F-dependent cell cycle arrest, we found that p120 E4F can induce a G 1 block in p21 ؊/؊ cells, suggesting that other proteins are essential for the p120 E4F -dependent block in G 1 . We show here that cyclin A promoter activity can be repressed by p120 E4F and that this repression correlates with p120 E4F binding to the cyclic AMP-responsive element site of the cyclin A promoter. In addition, enforced expression of cyclin A releases p120 E4F-arrested cells from the G 1 block. These data identify the cyclin A gene as a cellular target for p120 E4F and suggest a mechanism for p120 E4F -dependent cell cycle regulation.E4F is a ubiquitously expressed GLI-Krüppel-related mammalian transcription factor which was first identified as a cellular factor binding to regulatory regions of the adenovirus E4 promoter and responsible for E4 regulation of expression in the course of adenovirus infection (13,35,36). The adenovirus E4 promoter contains two ATF binding sites which are also targets for E4F, as observed in extracts from adenovirus-infected cells. E4F DNA binding specificity differs from that of CREB-ATF protein family members, as only subsets of ATF sites are recognized by E4F. For example, E4F binds to two out of the four ATF binding sites found in the E4 promoter but recognizes none of the ATF sites found in the E2 or E3 promoter (37). Divergence in binding specificities between E4F and ATF proteins was further demonstrated by point mutagenesis of their DNA recognition sequence and by methylation interference assays (37). E4F is synthesized as a 120-kDa protein (p120 E4F ) that upon proteolytical cleavage gives rise to p50 E4F , a 50-kDa amino-terminal fragment (11). Interestingly the murine homolog of E4F, termed AP3, was independently identified as the cellular factor responsible for the repression of the E1A promoter in mouse fibroblasts (13). Although p50 E4F and p120 E4F recognize the same DNA motifs in vitro, they differentially regulate gene expression in vivo. p50 E4Ftransactivates expression of the adenoviral E4 gene in a E1A-dependent fashion (34, 35). p120 E4F, on the other hand, is likely to play a key role in mammalian cell cycle control. Indeed, overexpression of p120 E4F in NIH 3T3 fibroblasts inhibits progression from G 1 to S phase (12). Moreover, it was recently reported that p120 E4F interacts directly with the key cell cycle regulators pRB (retinoblastoma tumor suppressor protein) and p53 (10, 39). One proposed mechanism to explain the cell cycle arrest mediated by p120E4F is the stabilization of p21 WAF1 through a post transcriptional mechanism (12). Howev...

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“…When deprived of an ECM anchorage, cells arrest in G 1 phase with inactive Cdk4/6 and Cdk2 due largely to repression of the cyclin D1 and cyclin A genes and induction of p27 KIP1 (15)(16)(17)(18). Inactivation of CDK4 and CDK6 activates retinoblastoma protein (RB) and its cognates, which in turn bind and thereby inactivate the E2F-DP transcription factor complexes, shutting down a subset of genes essential or important for the onset of S phase, including Cdc6, cyclin A, E2F1, and Emi1 (19 -23).…”

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Rho-associated Kinase Connects a Cell Cycle-controlling Anchorage Signal to the Mammalian Target of Rapamycin Pathway

Park

Arakawa-Takeuchi

Jinno

et al. 2011

Journal of Biological Chemistry

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When deprived of anchorage to the extracellular matrix, fibroblasts arrest in G 1 phase at least in part due to inactivation of G 1 cyclin-dependent kinases. Despite great effort, how anchorage signals control the G 1 -S transition of fibroblasts remains highly elusive. We recently found that the mammalian target of rapamycin (mTOR) cascade might convey an anchorage signal that regulates S phase entry. Here, we show that Rhoassociated kinase connects this signal to the TSC1/TSC2-RHEB-mTOR pathway. Expression of a constitutively active form of ROCK1 suppressed all of the anchorage deprivation effects suppressible by tsc2 mutation in rat embryonic fibroblasts. TSC2 contains one evolutionarily conserved ROCK target-like sequence, and an alanine substitution for Thr 1203 in this sequence severely impaired the ability of ROCK1 to counteract the anchorage loss-imposed down-regulation of both G 1 cell cycle factors and mTORC1 activity. Moreover, TSC2 Thr 1203 underwent ROCK-dependent phosphorylation in vivo and could be phosphorylated by bacterially expressed active ROCK1 in vitro, providing biochemical evidence for a direct physical interaction between ROCK and TSC2.

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Anchorage-dependent expression of cyclin A in primary cells requires a negative DNA regulatory element and a functional Rb

Cited by 17 publications

References 48 publications

Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

Cyclin A Is a Mediator of p120^E4F-Dependent Cell Cycle Arrest in G₁

Rho-associated Kinase Connects a Cell Cycle-controlling Anchorage Signal to the Mammalian Target of Rapamycin Pathway

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Anchorage-dependent expression of cyclin A in primary cells requires a negative DNA regulatory element and a functional Rb

Cited by 17 publications

References 48 publications

Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

Cyclin A Is a Mediator of p120E4F-Dependent Cell Cycle Arrest in G1

Rho-associated Kinase Connects a Cell Cycle-controlling Anchorage Signal to the Mammalian Target of Rapamycin Pathway

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Cyclin A Is a Mediator of p120^E4F-Dependent Cell Cycle Arrest in G₁