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

1996 ◽  
Vol 16 (12) ◽  
pp. 7173-7181 ◽  
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.

scholarly journals Domains A and B in the Rb pocket interact to form a transcriptional repressor motif.

1996 ◽  
Vol 16 (9) ◽  
pp. 4862-4868 ◽  
Author(s):  
K N Chow ◽  
D C Dean
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Transcriptional Repressor ◽  
Inhibitory Effect ◽  
Binding Assays ◽  
Adenovirus E1a ◽  
Tumor Viruses ◽  
A Site ◽  
Repressor Activity

The retinoblastoma protein (Rb) is a tumor suppressor that regulates progression from the G1 phase to the S phase of the cell cycle. Previously, we found that Rb is a transcriptional repressor that is selectively targeted to promoters through an interaction with the E2F family of cell cycle transcription factors--when Rb is tethered to a promoter through E2F, it not only blocks E2F activity, it also binds surrounding transcription factors, preventing their interaction with the basal transcription complex, thus resulting in a dominant inhibitory effect on transcription of cell cycle genes. Here we examine the repressor motif of Rb. The two domains in the Rb pocket, A and B, which are conserved across species and in the Rb-related proteins p107 and p130, are both required for repressor activity. The nonconserved spacer separating A and B is not required. Although neither A nor B alone had any repressor activity, surprisingly, repressor activity was observed when the domains were coexpressed on separate proteins. Transfection assays suggest that one domain can recruit the other to the promoter to form a repressor motif that can both interact with E2F and have a dominant inhibitory effect on transcription. Using coimmunoprecipitation and in vitro binding assays, we show that A and B interact directly and that mutations which disrupt this interaction inhibit repressor activity. The Rb pocket was originally defined as the binding site for oncoproteins from DNA tumor viruses such as adenovirus E1a. We present evidence that E1a interacts with a site formed by the interaction of A and B and that this interaction with A and B induces or stabilizes the A-B interaction.

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

2010 ◽  
Vol 21 (19) ◽  
pp. 3421-3432 ◽  
Author(s):  
Donna Garvey Brickner ◽  
Jason H. Brickner
Keyword(s):  
Cell Cycle ◽  
Nuclear Periphery ◽  
S Phase ◽  
Nuclear Pore ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Initiation Of Dna Replication ◽  
Cdk Phosphorylation ◽  
Inducible Genes ◽  
Active Genes

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.

scholarly journals Structure-function relationships of the yeast cyclin-dependent kinase Pho85.

1995 ◽  
Vol 15 (10) ◽  
pp. 5482-5491 ◽  
Author(s):  
R C Santos ◽  
N C Waters ◽  
C L Creasy ◽  
L W Bergman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Structure Function ◽  
Cell Cycle Progression ◽  
Substrate Recognition ◽  
The Other ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Induced Mutagenesis

The PHO85 gene of Saccharomyces cerevisiae encodes a cyclin-dependent kinase involved in both transcriptional regulation and cell cycle progression. Although a great deal is known concerning the structure, function, and regulation of the highly homologous Cdc28 protein kinase, little is known concerning these relationships in regard to Pho85. In this study, we constructed a series of Pho85-Cdc28 chimeras to map the region(s) of the Pho85 molecule that is critical for function of Pho85 in repression of acid phosphatase (PHO5) expression. Using a combination of site-directed and ethyl methanesulfonate-induced mutagenesis, we have identified numerous residues critical for either activation of the Pho85 kinase, interaction of Pho85 with the cyclin-like molecule Pho80, or substrate recognition. Finally, analysis of mutations analogous to those previously identified in either Cdc28 or cdc2 of Schizosaccharomyces pombe suggested that the inhibition of Pho85-Pho80 activity in mechanistically different from that seen in the other cyclin-dependent kinases.

scholarly journals Expression of p27Kip1 in Osteoblast-Like Cells during Differentiation with Parathyroid Hormone*

Endocrinology ◽  
1997 ◽  
Vol 138 (5) ◽  
pp. 1995-2004 ◽  
Author(s):  
Takehisa Onishi ◽  
Keith Hruska
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Osteogenic Sarcoma ◽  
S Phase ◽  
G1 Phase ◽  
Osteoblastic Cell ◽  
Cyclin Dependent Kinases ◽  
Kinase A

Abstract PTH is a major systemic regulator of bone metabolism and plays an important role in both bone formation and resorption. PTH either inhibits or stimulates osteoblastic cell proliferation depending on the model that is studied. We analyzed the cell cycle of the UMR-106 cell line, a relatively differentiated osteoblastic osteogenic sarcoma line in which PTH is known to inhibit proliferation but the mechanism of action is unknown. PTH decreased the proportion of cells in S phase and increased the number of G1 phase cells. We examined the effect of PTH on the regulators of the G1 phase cyclin-dependent kinases and found that PTH increased p27Kip1, but not p21Cip1, levels. This effect was mimicked by 8-bromo-cAMP, but not by phorbol 12-myristate 13-acetate. The protein kinase A inhibitor KT5720 abolished the effect of PTH on the increase in p27Kip1 expression. PTH increased CDK2-associated p27Kip1 without affecting the levels of CDK2. CDK2 activity was down-regulated by both PTH and 8-bromo-cAMP treatment. These data suggest that PTH blocks entry of cells into S phase and inhibits cell proliferation as the consequence of an increase in p27Kip1, which is mediated through the protein kinase A pathway. The inhibition of G1 cyclin-dependent kinases by p27Kip1 could cause a reduction of phosphorylation of key substrates and inactivation of transcription factors essential for entry into S phase. The inhibition of cell cycle progression through PKA-mediated p27Kip1 induction might play an important role in PTH-induced differentiation of osteoblasts.

scholarly journals Phosphorylation-induced unfolding regulates p19INK4dduring the human cell cycle

2018 ◽  
Vol 115 (13) ◽  
pp. 3344-3349 ◽  
Author(s):  
Amit Kumar ◽  
Mohanraj Gopalswamy ◽  
Annika Wolf ◽  
David J. Brockwell ◽  
Mechthild Hatzfeld ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Structural Biology ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Ankyrin Repeat Protein ◽  
Dependent Protein ◽  
Local Unfolding ◽  
Molecular Mode

Cell cycle progression is tightly regulated by cyclin-dependent kinases (CDKs). The ankyrin-repeat protein p19INK4dfunctions as a key regulator of G1/S transition; however, its molecular mode of action is unknown. Here, we combine cell and structural biology methods to unravel the mechanism by which p19INK4dcontrols cell cycle progression. We delineate how the stepwise phosphorylation of p19INK4dSer66 and Ser76 by cell cycle-independent (p38) and -dependent protein kinases (CDK1), respectively, leads to local unfolding of the three N-terminal ankyrin repeats of p19INK4d. This dissociates the CDK6–p19INK4dinhibitory complex and, thereby, activates CDK6. CDK6 triggers entry into S-phase, whereas p19INK4dis ubiquitinated and degraded. Our findings reveal how signaling-dependent p19INK4dunfolding contributes to the irreversibility of G1/S transition.

scholarly journals Functional Interactions between Herpesvirus Oncoprotein MEQ and Cell Cycle Regulator CDK2

1999 ◽  
Vol 73 (5) ◽  
pp. 4208-4219 ◽  
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.

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

2005 ◽  
Vol 79 (4) ◽  
pp. 2597-2603 ◽  
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.

Cyclin Dependent Kinases and Regulation of the Fission Yeast Cell Cycle

1999 ◽  
Vol 380 (7-8) ◽  
pp. 729-733 ◽  
Author(s):  
P. Nurse
Keyword(s):  
Cell Cycle ◽  
Yeast Cell ◽  
Fission Yeast ◽  
S Phase ◽  
Yeast Cell Cycle ◽  
Cyclin Dependent Kinases ◽  
Fission Yeast Cell ◽  
Nutritional Deprivation ◽  
Orderly Fashion ◽  
A Cell

AbstractThe cyclin dependent kinases (CDKs), formed by complexes between Cdc2p and the B-cyclins Cig2p and Cdc13p, have a central role in regulating the fission yeast cell cycle and maintaining genomic stability. The CDK Cig2p/Cdc2p controls the onset of S-phase and the CDK Cdc13p/Cdc2p controls the onset of mitosis and ensures that there is only one S-phase in each cell. Cdc13p/Cdc2p can replace Cig2p/Cdc2p for the onset of S-phase, suggesting that the increasing activity of a single CDK during the cell cycle is sufficient to drive a cell in an orderly fashion into S-phase and into mitosis. If S-phase is incomplete, then inhibition of Cdc13p/Cdc2p prevents cells with unreplicated DNA from undergoing a catastrophic entry into mitosis. Control of CDK activity is also important to allow cells to exit the cell cycle and accumulate in G1 in response to nutritional deprivation and the presence of pheromone.

An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3985-3987 ◽  
Author(s):  
Mu-Shui Dai ◽  
Charlie R. Mantel ◽  
Zhen-Biao Xia ◽  
Hal E. Broxmeyer ◽  
Li Lu
Keyword(s):  
Cell Cycle ◽  
Cord Blood ◽  
Cyclin E ◽  
Erythroid Differentiation ◽  
S Phase ◽  
Cyclin D3 ◽  
G1 Phase ◽  
Cyclin Dependent Kinase ◽  
Terminal Erythroid Differentiation

The dynamics of cell cycle regulation were investigated during in vitro erythroid proliferation and differentiation of CD34+cord blood cells. An unusual cell cycle profile with a majority of cells in S phase (70.2%) and minority of cells in G1 phase (27.4%) was observed in burst-forming unit-erythrocytes (BFU-E)–derived erythroblasts from a 7-day culture of CD34+ cells stimulated with interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor, and Epo. Terminal erythroid differentiation was accompanied by a rapid increase of G0/G1 phase cells. Expression of cyclin E and cyclin-dependent kinase 2 (cdk2) correlated with the proportion of S phase cells. Cyclin D3 was moderately up-regulated during the proliferation phase, and both cyclin E and D3 were rapidly down-regulated during terminal differentiation. This suggests that the high proliferation potential of erythroblasts is associated with temporal up-regulation of cyclin E and cdk2.

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