The PP2A-B56 Phosphatase Opposes Cyclin E Autocatalytic Degradation via Site-Specific Dephosphorylation

ABSTRACT Cyclin E, in conjunction with its catalytic partner cyclin-dependent kinase 2 (CDK2), regulates cell cycle progression as cells exit quiescence and enter S-phase. Multiple mechanisms control cyclin E periodicity during the cell cycle, including phosphorylation-dependent cyclin E ubiquitylation by the SCFFbw7 ubiquitin ligase. Serine 384 (S384) is the critical cyclin E phosphorylation site that stimulates Fbw7 binding and cyclin E ubiquitylation and degradation. Because S384 is autophosphorylated by bound CDK2, this presents a paradox as to how cyclin E can evade autocatalytically induced degradation in order to phosphorylate its other substrates. We found that S384 phosphorylation is dynamically regulated in cells and that cyclin E is specifically dephosphorylated at S384 by the PP2A-B56 phosphatase, thereby uncoupling cyclin E degradation from cyclin E-CDK2 activity. Furthermore, the rate of S384 dephosphorylation is high in interphase but low in mitosis. This provides a mechanism whereby interphase cells can oppose autocatalytic cyclin E degradation and maintain cyclin E-CDK2 activity while also enabling cyclin E destruction in mitosis, when inappropriate cyclin E expression is genotoxic.

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Phospholipase Cδ1 regulates cell proliferation and cell-cycle progression from G1- to S-phase by control of cyclin E–CDK2 activity

Biochemical Journal ◽

10.1042/bj20080233 ◽

2008 ◽

Vol 415 (3) ◽

pp. 439-448 ◽

Cited By ~ 14

Author(s):

Katherine A. Kaproth-Joslin ◽

Xiangquan Li ◽

Sarah E. Reks ◽

Grant G. Kelley

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Cellular Proliferation ◽

Cyclin E ◽

Critical Role ◽

S Phase ◽

G1 Phase ◽

Serum Starvation ◽

Cycle Progression ◽

Cdk2 Activity

In the present study, we examined the role of PLCδ1 (phospholipase C δ1) in the regulation of cellular proliferation. We demonstrate that RNAi (RNA interference)-mediated knockdown of endogenous PLCδ1, but not PLCβ3 or PLCϵ, induces a proliferation defect in Rat-1 and NIH 3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approx. 60% inhibition of [3H]thymidine incorporation. Analysis of the cell cycle with BrdU (bromodeoxyuridine)/propidium iodide-labelled FACS (fluorescence-activated cell sorting) demonstrated an accumulation of cells in G0/G1-phase and a corresponding decrease in cells in S-phase. Further examination of the cell cycle after synchronization by serum-starvation demonstrated normal movement through G1-phase but delayed entry into S-phase. Consistent with these findings, G1 cyclin (D2 and D3) and CDK4 (cyclin-dependent kinase 4) levels and associated kinase activity were not affected. However, cyclin E-associated CDK2 activity, responsible for G1-to-S-phase progression, was inhibited. This decreased activity was accompanied by unchanged CDK2 protein levels and paradoxically elevated cyclin E and cyclin E-associated CDK2 levels, suggesting inhibition of the cyclin E–CDK2 complex. This inhibition was not due to altered stimulatory or inhibitory phosphorylation of CDK2. However, p27, a Cip/Kip family CKI (CDK inhibitor)-binding partner, was elevated and showed increased association with CDK2 in PLCδ1-knockdown cells. The result of the present study demonstrate a novel and critical role for PLCδ1 in cell-cycle progression from G1-to-S-phase through regulation of cyclin E–CDK2 activity and p27 levels.

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HTLV-I p30 inhibits multiple S phase entry checkpoints, decreases cyclin E-CDK2 interactions and delays cell cycle progression

Molecular Cancer ◽

10.1186/1476-4598-9-302 ◽

2010 ◽

Vol 9 (1) ◽

pp. 302 ◽

Cited By ~ 19

Author(s):

Hicham H Baydoun ◽

Joanna Pancewicz ◽

XueTao Bai ◽

Christophe Nicot

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Cyclin E ◽

S Phase ◽

Cycle Progression ◽

Phase Entry

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The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

Journal of Virology ◽

10.1128/jvi.76.24.12543-12552.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 12543-12552 ◽

Cited By ~ 37

Author(s):

Amy Mauser ◽

Elizabeth Holley-Guthrie ◽

Adam Zanation ◽

Wendall Yarborough ◽

William Kaufmann ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Cyclin E ◽

Human Fibroblasts ◽

Cell Types ◽

S Phase ◽

Cycle Progression ◽

Stem Loop ◽

Barr Virus ◽

Early Protein

ABSTRACT The Epstein-Barr virus (EBV) immediate-early protein BZLF1 mediates the switch between the latent and lytic forms of EBV infection and has been previously shown to induce a G1/S block in cell cycle progression in some cell types. To examine the effect of BZLF1 on cellular gene expression, we performed microarray analysis on telomerase-immortalized human keratinocytes that were mock infected or infected with a control adenovirus vector (AdLacZ) or a vector expressing the EBV BZLF1 protein (AdBZLF1). Cellular genes activated by BZLF1 expression included E2F-1, cyclin E, Cdc25A, and a number of other genes involved in cell cycle progression. Immunoblot analysis confirmed that BZLF1 induced expression of E2F-1, cyclin E, Cdc25A, and stem loop binding protein (a protein known to be primarily expressed during S phase) in telomerase-immortalized keratinocytes. Similarly, BZLF1 increased expression of E2F-1, cyclin E, and stem loop binding protein (SLBP) in primary tonsil keratinocytes. In contrast, BZLF1 did not induce E2F-1 expression in normal human fibroblasts. Cell cycle analysis revealed that while BZLF1 dramatically blocked G1/S progression in normal human fibroblasts, it did not significantly affect cell cycle progression in primary human tonsil keratinocytes. Furthermore, in EBV-infected gastric carcinoma cells, the BZLF1-positive cells had an increased number of cells in S phase compared to the BZLF1-negative cells. Thus, in certain cell types (but not others), BZLF1 enhances expression of cellular proteins associated with cell cycle progression, which suggests that an S-phase-like environment may be advantageous for efficient lytic EBV replication in some cell types.

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A Protein Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 Is Expressed in Trigeminal Ganglionic Neurons of Latently Infected Cattle and Interacts with Cyclin-Dependent Kinase 2 during Productive Infection

Journal of Virology ◽

10.1128/jvi.72.10.8133-8142.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 8133-8142 ◽

Cited By ~ 43

Author(s):

Yunquan Jiang ◽

Ashfaque Hossain ◽

Maria Teresa Winkler ◽

Todd Holt ◽

Alan Doster ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Acute Infection ◽

Cyclin A ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Bovine Herpesvirus 1 ◽

Cycle Progression ◽

Latently Infected ◽

Herpesvirus 1

ABSTRACT Despite productive viral gene expression in the peripheral nervous system during acute infection, the bovine herpesvirus 1 (BHV-1) infection cycle is blocked in sensory ganglionic neurons and consequently latency is established. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA. LR gene products inhibit S-phase entry, and binding of the LR protein (LRP) to cyclin A was hypothesized to block cell cycle progression. This study demonstrates LRP is a nuclear protein which is expressed in neurons of latently infected cattle. Affinity chromatography indicated that LRP interacts with cyclin-dependent kinase 2 (cdk2)-cyclin complexes or cdc2-cyclin complexes in transfected human cells or infected bovine cells. After partial purification using three different columns (DEAE-Sepharose, Econo S, and heparin-agarose), LRP was primarily associated with cdk2-cyclin E complexes, an enzyme which is necessary for G1-to-S-phase cell cycle progression. During acute infection of trigeminal ganglia or following dexamethasone-induced reactivation, BHV-1 induces expression of cyclin A in neurons (L. M. Schang, A. Hossain, and C. Jones, J. Virol. 70:3807–3814, 1996). Expression of S-phase regulatory proteins (cyclin A, for example) leads to neuronal apoptosis. Consequently, we hypothesize that interactions between LRP and cell cycle regulatory proteins promote survival of postmitotic neurons during acute infection and/or reactivation.

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Cortactin Modulates RhoA Activation and Expression of Cip/Kip Cyclin-Dependent Kinase Inhibitors To Promote Cell Cycle Progression in 11q13-Amplified Head and Neck Squamous Cell Carcinoma Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00249-10 ◽

2010 ◽

Vol 30 (21) ◽

pp. 5057-5070 ◽

Cited By ~ 17

Author(s):

David R. Croucher ◽

Danny Rickwood ◽

Carole M. Tactacan ◽

Elizabeth A. Musgrove ◽

Roger J. Daly

Keyword(s):

Cell Cycle ◽

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Cell Cycle Progression ◽

Kinase Inhibitors ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Cycle Progression ◽

Fadu Cells ◽

Phase Entry

ABSTRACT The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.

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Activity of the APCCdh1 form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p

The Journal of Cell Biology ◽

10.1083/jcb.200102007 ◽

2001 ◽

Vol 154 (1) ◽

pp. 85-94 ◽

Cited By ~ 70

Author(s):

James N. Huang ◽

Iha Park ◽

Eric Ellingson ◽

Laurie E. Littlepage ◽

David Pellman

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Anaphase Promoting Complex ◽

Cycle Progression ◽

Essential Step ◽

Complete Inactivation ◽

Regulatory Loop ◽

Normal Cell Cycle

Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APCCdh1 was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APCCdh1 is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APCCdh1 requires S phase cyclins. Further, persistent APCCdh1 activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APCCdh1, and APCCdc20.

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Golgi-associated RhoBTB3 targets Cyclin E for ubiquitylation and promotes cell cycle progression

The Journal of Cell Biology ◽

10.1083/jcb.201305158 ◽

2013 ◽

Vol 203 (2) ◽

pp. 233-250 ◽

Cited By ~ 28

Author(s):

Albert Lu ◽

Suzanne R. Pfeffer

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Cyclin E ◽

Cell Cycle Control ◽

S Phase ◽

Cycle Progression ◽

Ubiquitin Ligase Complex ◽

Golgi Fragmentation ◽

Ring E3 ◽

Normal Cell Cycle

Cyclin E regulates the cell cycle transition from G1 to S phase and is degraded before entry into G2 phase. Here we show that RhoBTB3, a Golgi-associated, Rho-related ATPase, regulates the S/G2 transition of the cell cycle by targeting Cyclin E for ubiquitylation. Depletion of RhoBTB3 arrested cells in S phase, triggered Golgi fragmentation, and elevated Cyclin E levels. On the Golgi, RhoBTB3 bound Cyclin E as part of a Cullin3 (CUL3)-dependent RING–E3 ubiquitin ligase complex comprised of RhoBTB3, CUL3, and RBX1. Golgi association of this complex was required for its ability to catalyze Cyclin E ubiquitylation and allow normal cell cycle progression. These experiments reveal a novel role for a Ras superfamily member in catalyzing Cyclin E turnover during S phase, as well as an unexpected, essential role for the Golgi as a ubiquitylation platform for cell cycle control.

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NEK7 is required for G1 progression and procentriole formation

Molecular Biology of the Cell ◽

10.1091/mbc.e16-09-0643 ◽

2017 ◽

Vol 28 (15) ◽

pp. 2123-2134 ◽

Cited By ~ 12

Author(s):

Akshari Gupta ◽

Yuki Tsuchiya ◽

Midori Ohta ◽

Gen Shiratsuchi ◽

Daiju Kitagawa

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Primary Cilia ◽

S Phase ◽

Cycle Progression ◽

Restriction Point ◽

Centriole Duplication ◽

Abnormal Accumulation ◽

Phase Entry

The decision to commit to the cell cycle is made during G1 through the concerted action of various cyclin–CDK complexes. Not only DNA replication, but also centriole duplication is initiated as cells enter the S-phase. The NIMA-related kinase NEK7 is one of many factors required for proper centriole duplication, as well as for timely cell cycle progression. However, its specific roles in these events are poorly understood. In this study, we find that depletion of NEK7 inhibits progression through the G1 phase in human U2OS cells via down-regulation of various cyclins and CDKs and also inhibits the earliest stages of procentriole formation. Depletion of NEK7 also induces formation of primary cilia in human RPE1 cells, suggesting that NEK7 acts at least before the restriction point during G1. G1-arrested cells in the absence of NEK7 exhibit abnormal accumulation of the APC/C cofactor Cdh1 at the vicinity of centrioles. Furthermore, the ubiquitin ligase APC/CCdh1continuously degrades the centriolar protein STIL in these cells, thus inhibiting centriole assembly. Collectively our results demonstrate that NEK7 is involved in the timely regulation of G1 progression, S-phase entry, and procentriole formation.

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The Stress-activated Protein Kinase Hog1 Mediates S Phase Delay in Response to Osmostress

Molecular Biology of the Cell ◽

10.1091/mbc.e09-02-0129 ◽

2009 ◽

Vol 20 (15) ◽

pp. 3572-3582 ◽

Cited By ~ 42

Author(s):

Gilad Yaakov ◽

Alba Duch ◽

María García-Rubio ◽

Josep Clotet ◽

Javier Jimenez ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Kinase Activity ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Cycle Progression ◽

Replication Complex ◽

Extracellular Stimuli ◽

Phase Progression ◽

Cell Cycle Machinery

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress activates the Hog1 SAPK, which modulates cell cycle progression at G1 and G2 by the phosphorylation of elements of the cell cycle machinery, such as Sic1 and Hsl1, and by down-regulation of G1 and G2 cyclins. Here, we show that upon stress, Hog1 also modulates S phase progression. The control of S phase is independent of the S phase DNA damage checkpoint and of the previously characterized Hog1 cell cycle targets Sic1 and Hsl1. Hog1 uses at least two distinct mechanisms in its control over S phase progression. At early S phase, the SAPK prevents firing of replication origins by delaying the accumulation of the S phase cyclins Clb5 and Clb6. In addition, Hog1 prevents S phase progression when activated later in S phase or cells containing a genetic bypass for cyclin-dependent kinase activity. Hog1 interacts with components of the replication complex and delays phosphorylation of the Dpb2 subunit of the DNA polymerase. The two mechanisms of Hog1 action lead to delayed firing of origins and prolonged replication, respectively. The Hog1-dependent delay of replication could be important to allow Hog1 to induce gene expression before replication.

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