Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes through the Cell Cycle

Many inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase, and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin-dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.

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The Rb family contains a conserved cyclin-dependent-kinase-regulated transcriptional repressor motif.

Molecular and Cellular Biology ◽

10.1128/mcb.16.12.7173 ◽

1996 ◽

Vol 16 (12) ◽

pp. 7173-7181 ◽

Cited By ~ 48

Author(s):

K N Chow ◽

P Starostik ◽

D C Dean

Keyword(s):

Cell Cycle ◽

Transcription Factors ◽

Transcriptional Repressor ◽

S Phase ◽

Domain Swapping ◽

Cyclin Dependent Kinase ◽

Cyclin Dependent Kinases ◽

Tumor Viruses ◽

Cdk Phosphorylation ◽

Repressor Activity

Progression through the cell cycle is dependent on the sequential expression of cyclins, which combine with cyclin-dependent kinases (cdks) to form active kinases. The transition from G1 to S phase is dependent on D cyclins in complex with cdk4 or cdk6 and cyclin E complexed with cdk2. One target of G1 cyclins is the retinoblastoma susceptibility protein (Rb). Rb is a transcriptional repressor that is selectively targeted to genes through interaction with the E2F family of cell cycle transcription factors. Rb is a member of a family of proteins that include p107 and p130. The three proteins share a region known as the pocket that is important for binding E2F and is also the binding site for oncoproteins from DNA tumor viruses that inactivate Rb. We have found that two conserved domains within the Rb pocket (A and B) interact to form a transcriptional repressor motif (K. N. B. Chow and D. C. Dean, Mol. Cell. Biol. 16:4862-4868, 1996). Here we demonstrate that p107 also has an A-B repressor motif, and using domain swapping and coimmunoprecipitation assays, we compare A and B from Rb and p107. Finally and most importantly, we demonstrate that the A-B interaction which forms the repressor motif is blocked by G1 cdk phosphorylation, thereby blocking repressor activity. This A-B repressor motif is then the first example of a cdk-regulated transcriptional repressor.

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Saccharomyces cerevisiae Ime2 phosphorylates Sic1 at multiple PXS/T sites but is insufficient to trigger Sic1 degradation

Biochemical Journal ◽

10.1042/bj20060363 ◽

2006 ◽

Vol 399 (1) ◽

pp. 151-160 ◽

Cited By ~ 27

Author(s):

Chantelle Sedgwick ◽

Matthew Rawluk ◽

James Decesare ◽

Sheetal Raithatha ◽

James Wohlschlegel ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Phase Boundary ◽

S Phase ◽

Phosphorylation Sites ◽

Cyclin Dependent Kinase ◽

Proliferating Cells ◽

Initiation Of Dna Replication ◽

Cdk Phosphorylation

The initiation of DNA replication in Saccharomyces cerevisiae depends upon the destruction of the Clb–Cdc28 inhibitor Sic1. In proliferating cells Cln–Cdc28 complexes phosphorylate Sic1, which stimulates binding of Sic1 to SCFCdc4 and triggers its proteosome mediated destruction. During sporulation cyclins are not expressed, yet Sic1 is still destroyed at the G1-/S-phase boundary. The Cdk (cyclin dependent kinase) sites are also required for Sic1 destruction during sporulation. Sic1 that is devoid of Cdk phosphorylation sites displays increased stability and decreased phosphorylation in vivo. In addition, we found that Sic1 was modified by ubiquitin in sporulating cells and that SCFCdc4 was required for this modification. The meiosis-specific kinase Ime2 has been proposed to promote Sic1 destruction by phosphorylating Sic1 in sporulating cells. We found that Ime2 phosphorylates Sic1 at multiple sites in vitro. However, only a subset of these sites corresponds to Cdk sites. The identification of multiple sites phosphorylated by Ime2 has allowed us to propose a motif for phosphorylation by Ime2 (PXS/T) where serine or threonine acts as a phospho-acceptor. Although Ime2 phosphorylates Sic1 at multiple sites in vitro, the modified Sic1 fails to bind to SCFCdc4. In addition, the expression of Ime2 in G1 arrested haploid cells does not promote the destruction of Sic1. These data support a model where Ime2 is necessary but not sufficient to promote Sic1 destruction during sporulation.

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Cell cycle inertia underlies a bifurcation in cell fates after DNA damage

Science Advances ◽

10.1126/sciadv.abe3882 ◽

2021 ◽

Vol 7 (3) ◽

pp. eabe3882

Author(s):

Jenny F. Nathans ◽

James A. Cornwell ◽

Marwa M. Afifi ◽

Debasish Paul ◽

Steven D. Cappell

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cell Tracking ◽

Time Lapse ◽

S Phase ◽

Cdk Inhibitor ◽

Cyclin Dependent Kinase ◽

Cell Fates ◽

Damage Response ◽

Time Lapse Imaging

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.

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Biochemical and morphological characterization of the nuclear matrix during the synchronous cell cycle of Physarum polycephalum

Journal of Cell Science ◽

10.1242/jcs.105.4.1121 ◽

1993 ◽

Vol 105 (4) ◽

pp. 1121-1130 ◽

Cited By ~ 1

Author(s):

S. Lang ◽

T. Decristoforo ◽

W. Waitz ◽

P. Loidl

Keyword(s):

Cell Cycle ◽

Nuclear Matrix ◽

Physarum Polycephalum ◽

Nuclear Periphery ◽

S Phase ◽

Cycle Phase ◽

Electron Microscopic ◽

Nuclear Lamins ◽

Synchronous Cell ◽

Matrix Bound

We have investigated biochemical and ultrastructural aspects of the nuclear matrix during the naturally synchronous cell cycle of Physarum polycephalum. The morphology of the in situ nuclear matrix exhibited significant cell cycle changes as revealed by electron microscopic examination, especially during the progression of nuclei through mitosis and S-phase. In mitosis the interchromatin matrix was found to be retracted to the nuclear periphery; during S-phase this interchromatin matrix gradually resembled, concomitant with the reconstruction of a nucleolar remnant structure. During the G2-period no significant changes in matrix morphology were observed. The pattern of nuclear matrix proteins was invariant during the cell cycle; no cycle phase-specific proteins could be detected. In vivo labelling of plasmodia with [35S]methionine/cysteine showed that only a few proteins are synthesized and assembled into nuclear matrix structures in a cell cycle-dependent way; the majority of proteins were synthesized almost continuously. This was also shown for nuclear lamins homologues. In contrast to bulk nuclear histones, those histones that remain tightly bound to the nuclear matrix were synthesized and assembled into nuclear structures in the very first hour of S-phase; assembly was terminated in mid-S-phase, indicating that nuclear matrix-bound chromatin is replicated early in S-phase. Comparison of the acetylation pattern of matrix-bound histone H4 with bulk nuclear H4 revealed a largely elevated acetate content of matrix H4. The percentage of acetylated subspecies was entirely different from that in bulk nuclear H4, indicating that matrix-associated histones represent a subpopulation of nuclear histones with distinct properties, reflecting specific structural requirements of matrix-attached chromatin.

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Cdc6p establishes and maintains a state of replication competence during G1 phase

Journal of Cell Science ◽

10.1242/jcs.110.6.753 ◽

1997 ◽

Vol 110 (6) ◽

pp. 753-763 ◽

Cited By ~ 1

Author(s):

C.S. Detweiler ◽

J.J. Li

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Transcriptional Repression ◽

Prolonged Exposure ◽

S Phase ◽

Restrictive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

Important Intermediate ◽

Initiation Of Dna Replication ◽

And Function

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

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Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells

eLife ◽

10.7554/elife.18020 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 16

Author(s):

Sabrina F Mansilla ◽

Agustina P Bertolin ◽

Valérie Bergoglio ◽

Marie-Jeanne Pillaire ◽

Marina A González Besteiro ◽

...

Keyword(s):

Genomic Instability ◽

Genomic Stability ◽

S Phase ◽

Fragile Sites ◽

Dna Lesions ◽

Cdk Inhibitor ◽

Cyclin Dependent Kinase ◽

Independent Manner ◽

Genomic Regions

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21’s PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.

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Functional Interactions between Herpesvirus Oncoprotein MEQ and Cell Cycle Regulator CDK2

Journal of Virology ◽

10.1128/jvi.73.5.4208-4219.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4208-4219 ◽

Cited By ~ 29

Author(s):

Juinn-Lin Liu ◽

Ying Ye ◽

Zheng Qian ◽

Yongyi Qian ◽

Dennis J. Templeton ◽

...

Keyword(s):

Cell Cycle ◽

Phosphorylation Site ◽

Fibroblast Cell ◽

S Phase ◽

Morphological Transformation ◽

Necrosis Factor Alpha ◽

Coiled Bodies ◽

Cdk Phosphorylation

ABSTRACT Marek’s disease virus, an avian alphaherpesvirus, has been used as an excellent model to study herpesvirus oncogenesis. One of its potential oncogenes, MEQ, has been demonstrated to transform a rodent fibroblast cell line, Rat-2, in vitro by inducing morphological transformation and anchorage- and serum-independent growth and by protecting cells from apoptosis induced by tumor necrosis factor alpha, C2-ceramide, UV irradiation, or serum deprivation. In this report, we show that there is a cell cycle-dependent colocalization of MEQ protein and cyclin-dependent kinase 2 (CDK2) in coiled bodies and the nucleolar periphery during the G1/S boundary and early S phase. To our knowledge, this is the first demonstration that CDK2 is found to localize to coiled bodies. Such an in vivo association and possibly subsequent phosphorylation may result in the cytoplasmic translocation of MEQ protein. Indeed, MEQ is expressed in both the nucleus and the cytoplasm during the G1/S boundary and early S phase. In addition, we were able to show in vitro phosphorylation of MEQ by CDKs. We have mapped the CDK phosphorylation site of MEQ to be serine 42, a residue in the proximity of the bZIP domain. An indirect-immunofluorescence study of the MEQ S42D mutant, in which the CDK phosphorylation site was mutated to a charged residue, reveals more prominent cytoplasmic localization. This lends further support to the notion that the translocation of MEQ is regulated by phosphorylation. Furthermore, phosphorylation of MEQ by CDKs drastically reduces the DNA binding activity of MEQ, which may in part account for the lack of retention of MEQ oncoprotein in the nucleus. Interestingly, the localization of CDK2 in coiled bodies and the nucleolar periphery is observed only in MEQ-transformed Rat-2 cells, implicating MEQ in modifying the subcellular localization of CDK2. Taken together, our data suggest that there is a novel reciprocal modulation between the herpesvirus oncoprotein MEQ and CDK2.

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Inhibition of Cell Division by the Human Cytomegalovirus IE86 Protein: Role of the p53 Pathway or Cyclin-Dependent Kinase 1/Cyclin B1

Journal of Virology ◽

10.1128/jvi.79.4.2597-2603.2005 ◽

2005 ◽

Vol 79 (4) ◽

pp. 2597-2603 ◽

Cited By ~ 44

Author(s):

Yoon-Jae Song ◽

Mark F. Stinski

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Human Cytomegalovirus ◽

Cell Cycle Progression ◽

Fibroblast Cell ◽

Cyclin B1 ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Wild Type ◽

Atm Kinase

ABSTRACT The human cytomegalovirus (HCMV) IE86 protein induces the human fibroblast cell cycle from G0/G1 to G1/S, where cell cycle progression stops. Cells with a wild-type, mutated, or null p53 or cells with null p21 protein were transduced with replication-deficient adenoviruses expressing HCMV IE86 protein or cellular p53 or p21. Even though S-phase genes were activated in a p53 wild-type cell, IE86 protein also induced phospho-Ser15 p53 and p21 independent of p14ARF but dependent on ATM kinase. These cells did not enter the S phase. In human p53 mutant, p53 null, or p21 null cells, IE86 protein did not up-regulate p21, cellular DNA synthesis was not inhibited, but cell division was inhibited. Cells accumulated in the G2/M phase, and there was increased cyclin-dependent kinase 1/cyclin B1 activity. Although the HCMV IE86 protein increases cellular E2F activity, it also blocks cell division in both p53+/+ and p53−/− cells.

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Periodic Expression of the Cyclin-dependent Kinase Inhibitor p57Kip2 in Trophoblast Giant Cells Defines a G2-like Gap Phase of the Endocycle

Molecular Biology of the Cell ◽

10.1091/mbc.11.3.1037 ◽

2000 ◽

Vol 11 (3) ◽

pp. 1037-1045 ◽

Cited By ~ 67

Author(s):

Naka Hattori ◽

Tyler C. Davies ◽

Lynn Anson-Cartwright ◽

James C. Cross

Keyword(s):

Dna Replication ◽

Protein Targeting ◽

Kinase Inhibitor ◽

Phosphorylation Site ◽

Giant Cells ◽

S Phase ◽

Stable Form ◽

Cdk Inhibitor ◽

Cyclin Dependent Kinase ◽

Trophoblast Giant Cells

Endoreduplication is an unusual form of cell cycle in which rounds of DNA synthesis repeat in the absence of intervening mitoses. How G1/S cyclin-dependent kinase (Cdk) activity is regulated during the mammalian endocycle is poorly understood. We show here that expression of the G1/S Cdk inhibitor p57Kip2 is induced coincidentally with the transition to the endocycle in trophoblast giant cells.Kip2 mRNA is constitutively expressed during subsequent endocycles, but the protein level fluctuates. In trophoblast giant cells synchronized for the first few endocycles, the p57Kip2 protein accumulates only at the end of S-phase and then rapidly disappears a few hours before the onset of the next S-phase. The protein becomes stabilized by mutation of a C-terminal Cdk phosphorylation site. As a consequence, introduction of this stable form of p57Kip2 into giant cells blocks S-phase entry. These data imply that p57Kip2 is subject to phosphorylation-dependent turnover. Surprisingly, although this occurs in endoreduplicating giant cells, p57Kip2 is stable when ectopically expressed in proliferating trophoblast cells, indicating that these cells lack the mechanism for protein targeting and/or degradation. These data show that the appearance of p57Kip2punctuates the completion of DNA replication, whereas its turnover is subsequently required to initiate the next round of endoreduplication in trophoblast giant cells. Cyclical expression of a Cdk inhibitor, by terminating G1/S Cdk activity, may help promote the resetting of DNA replication machinery.

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Cell Cycle Regulation by Light inProchlorococcus Strains

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.782-790.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 782-790 ◽

Cited By ~ 45

Author(s):

Stéphan Jacquet ◽

Frédéric Partensky ◽

Dominique Marie ◽

Raffaella Casotti ◽

Daniel Vaulot

Keyword(s):

Cell Cycle ◽

High Irradiance ◽

S Phase ◽

Low Light ◽

Close Coupling ◽

Cell Cycles ◽

Flow Cytometric ◽

Initiation Of Dna Replication ◽

Cell Cycling ◽

Cycle Regulation

ABSTRACT The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 μmol of quanta · m−2 s−1, the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the “light on” signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G1 phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. ShiftingProchlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G2 phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.

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