Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase

1994 ◽  
Vol 14 (11) ◽  
pp. 7265-7275
Author(s):  
M Matsuoka ◽  
J Y Kato ◽  
R P Fisher ◽  
D O Morgan ◽  
C J Sherr
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Regulatory Protein ◽  
Cyclin A ◽  
Catalytic Subunit ◽  
Regulatory Control ◽  
Cyclin D ◽  
Enzymatic Activation ◽  
Rate Limiting

The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.

scholarly journals Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase.

1994 ◽  
Vol 14 (11) ◽  
pp. 7265-7275 ◽  
Author(s):  
M Matsuoka ◽  
J Y Kato ◽  
R P Fisher ◽  
D O Morgan ◽  
C J Sherr
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Regulatory Protein ◽  
Cyclin A ◽  
Catalytic Subunit ◽  
Regulatory Control ◽  
Cyclin D ◽  
Enzymatic Activation ◽  
Rate Limiting

The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.

scholarly journals Cell cycle regulation of cyclin A gene expression by the cyclic AMP-responsive transcription factors CREB and CREM.

1995 ◽  
Vol 15 (6) ◽  
pp. 3301-3309 ◽  
Author(s):  
C Desdouets ◽  
G Matesic ◽  
C A Molina ◽  
N S Foulkes ◽  
P Sassone-Corsi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cyclic Amp ◽  
Transcription Initiation ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Regulatory Protein ◽  
Cyclin A ◽  
S Phase ◽  
Signalling Pathway

Cyclin A is a pivotal regulatory protein which, in mammalian cells, is involved in the S phase of the cell cycle. Transcription of the human cyclin A gene is cell cycle regulated. We have investigated the role of the cyclic AMP (cAMP)-dependent signalling pathway in this cell cycle-dependent control. In human diploid fibroblasts (Hs 27), induction of cyclin A gene expression at G1/S is stimulated by 8-bromo-cAMP and suppressed by the protein kinase A inhibitor H89, which was found to delay S phase entry. Transfection experiments showed that the cyclin A promoter is inducible by activation of the adenylyl cyclase signalling pathway. Stimulation is mediated predominantly via a cAMP response element (CRE) located at positions -80 to -73 with respect to the transcription initiation site and is able to bind CRE-binding proteins and CRE modulators. Moreover, activation by phosphorylation of the activators CRE-binding proteins and CRE modulator tau and levels of the inducible cAMP early repressor are cell cycle regulated, which is consistent with the pattern of cyclin A inducibility by cAMP during the cell cycle. These results suggest that the CRE is, at least partly, implicated in stimulation of cyclin A transcription at G1/S.

scholarly journals Spy1 Interacts with p27Kip1 to Allow G1/S Progression

2003 ◽  
Vol 14 (9) ◽  
pp. 3664-3674 ◽  
Author(s):  
Lisa A. Porter ◽  
Monica Kong-Beltran ◽  
Daniel J. Donoghue
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Regulatory Protein ◽  
Cell Cycle Regulatory Protein ◽  
Proliferative Effect ◽  
Cycle Arrest ◽  
Mammalian Cell Cycle

Progression through the G1/S transition commits cells to synthesize DNA. Cyclin dependent kinase 2 (CDK2) is the major kinase that allows progression through G1/S phase and subsequent replication events. p27 is a CDK inhibitor (CKI) that binds to CDK2 to prevent premature activation of this kinase. Speedy (Spy1), a novel cell cycle regulatory protein, has been found to prematurely activate CDK2 when microinjected into Xenopus oocytes and when expressed in mammalian cells. To determine the mechanism underlying Spy1-induced proliferation in mammalian cell cycle regulation, we used human Spy1 as bait in a yeast two-hybrid screen to identify interacting proteins. One of the proteins isolated was p27; this novel interaction was confirmed both in vitro, using bacterially expressed and in vitro translated proteins, and in vivo, through the examination of endogenous and transfected proteins in mammalian cells. We demonstrate that Spy1 expression can overcome a p27-induced cell cycle arrest to allow for DNA synthesis and CDK2 histone H1 kinase activity. In addition, we utilized p27-null cells to demonstrate that the proliferative effect of Spy1 depends on the presence of endogenous p27. Our data suggest that Spy1 associates with p27 to promote cell cycle progression through the G1/S transition.

scholarly journals Regulation of Cyclin A-Cdk2 by SCF Component Skp1 and F-Box Protein Skp2

1999 ◽  
Vol 19 (1) ◽  
pp. 635-645 ◽  
Author(s):  
Cain H. Yam ◽  
Raymond W. M. Ng ◽  
Wai Yi Siu ◽  
Anita W. S. Lau ◽  
Randy Y. C. Poon
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Cyclin A ◽  
Cell Cycle Regulators ◽  
Cycle Arrest ◽  
Ubiquitin Ligase E3 ◽  
F Box Protein ◽  
Kinase Phosphorylation

ABSTRACT Cyclin A-Cdk2 complexes bind to Skp1 and Skp2 during S phase, but the function of Skp1 and Skp2 is unclear. Skp1, together with F-box proteins like Skp2, are part of ubiquitin-ligase E3 complexes that target many cell cycle regulators for ubiquitination-mediated proteolysis. In this study, we investigated the potential regulation of cyclin A-Cdk2 activity by Skp1 and Skp2. We found that Skp2 can inhibit the kinase activity of cyclin A-Cdk2 in vitro, both by direct inhibition of cyclin A-Cdk2 and by inhibition of the activation of Cdk2 by cyclin-dependent kinase (CDK)-activating kinase phosphorylation. Only the kinase activity of Cdk2, not of that of Cdc2 or Cdk5, is reduced by Skp2. Skp2 is phosphorylated by cyclin A-Cdk2 on residue Ser76, but nonphosphorylatable mutants of Skp2 can still inhibit the kinase activity of cyclin A-Cdk2 toward histone H1. The F box of Skp2 is required for binding to Skp1, and both the N-terminal and C-terminal regions of Skp2 are involved in binding to cyclin A-Cdk2. Furthermore, Skp2 and the CDK inhibitor p21 Cip1/WAF1 bind to cyclin A-Cdk2 in a mutually exclusive manner. Overexpression of Skp2, but not Skp1, in mammalian cells causes a G1/S cell cycle arrest.

scholarly journals Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4.

1995 ◽  
Vol 15 (5) ◽  
pp. 2682-2688 ◽  
Author(s):  
F K Chan ◽  
J Zhang ◽  
L Cheng ◽  
D N Shapiro ◽  
A Winoto
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Mammalian Cells ◽  
Cyclin E ◽  
G1 Arrest ◽  
Cdk Inhibitor ◽  
Cyclin D ◽  
Human And Mouse

The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G1/S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.

scholarly journals Only Akt1 Is Required for Proliferation, while Akt2 Promotes Cell Cycle Exit through p21 Binding

2006 ◽  
Vol 26 (22) ◽  
pp. 8267-8280 ◽  
Author(s):  
Lisa Héron-Milhavet ◽  
Celine Franckhauser ◽  
Vanessa Rana ◽  
Cyril Berthenet ◽  
Daniel Fisher ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Specific Interaction ◽  
Cyclin A ◽  
Cell Cycle Exit ◽  
Cycle Progression ◽  
Negative Effect

ABSTRACT Protein kinase B (PKB/Akt) is an important modulator of insulin signaling, cell proliferation, and survival. Using small interfering RNA duplexes in nontransformed mammalian cells, we show that only Akt1 is essential for cell proliferation, while Akt2 promotes cell cycle exit. Silencing Akt1 resulted in decreased cyclin A levels and inhibition of S-phase entry, effects not seen with Akt2 knockdown and specifically rescued by microinjection of Akt1, not Akt2. In differentiating myoblasts, Akt2 knockout prevented myoblasts from exiting the cell cycle and showed sustained cyclin A expression. In contrast, overexpression of Akt2 reduced cyclin A and hindered cell cycle progression in M-G1 with increased nuclear p21. p21 is a major target in the differential effects of Akt isoforms, with endogenous Akt2 and not Akt1 binding p21 in the nucleus and increasing its level. Accordingly, Akt2 knockdown cells, and not Akt1 knockdown cells, showed reduced levels of p21. A specific Akt2/p21 interaction can be reproduced in vitro, and the Akt2 binding site on p21 is similar to that in cyclin A spanning T145 to T155, since (i) prior incubation with cyclin A prevents Akt2 binding, (ii) T145 phosphorylation on p21 by Akt1 prevents Akt2 binding, and (iii) binding Akt2 prevents phosphorylation of p21 by Akt1. These data show that specific interaction of the Akt2 isoform with p21 is key to its negative effect on normal cell cycle progression.

scholarly journals Regulation of cyclin D-dependent kinase 4 (cdk4) by cdk4-activating kinase.

1994 ◽  
Vol 14 (4) ◽  
pp. 2713-2721 ◽  
Author(s):  
J Y Kato ◽  
M Matsuoka ◽  
D K Strom ◽  
C J Sherr
Keyword(s):  
Mammalian Cells ◽  
Insect Cells ◽  
G1 Phase ◽  
Sf9 Cells ◽  
Cyclin D ◽  
Cyclin Dependent Kinase ◽  
Active Protein ◽  
Serum Stimulation ◽  
Rate Limiting

The accumulation of assembled holoenzymes composed of regulatory D-type cyclins and their catalytic partner, cyclin-dependent kinase 4 (cdk4), is rate limiting for progression through the G1 phase of the cell cycle in mammalian fibroblasts. Both the synthesis and assembly of D-type cyclins and cdk4 depend upon serum stimulation, but even when both subunits are ectopically overproduced, they do not assemble into complexes in serum-deprived cells. When coexpressed from baculoviral vectors in intact Sf9 insect cells, cdk4 assembles with D-type cyclins to form active protein kinases. In contrast, recombinant D-type cyclin and cdk4 subunits produced in insect cells or in bacteria do not assemble as efficiently into functional holoenzymes when combined in vitro but can be activated in the presence of lysates obtained from proliferating mammalian cells. Assembly of cyclin D-cdk4 complexes in coinfected Sf9 cells facilitates phosphorylation of cdk4 on threonine 172 by a cdk-activating kinase (CAK). Assembly can proceed in the absence of this modification, but cdk4 mutants which cannot be phosphorylated by CAK remain catalytically inactive. Therefore, formation of the cyclin D-cdk4 complex and phosphorylation of the bound catalytic subunit are independently regulated, and in addition to the requirement for CAK activity, serum stimulation is required to promote assembly of the complexes in mammalian cells.

Regulation of cyclin D-dependent kinase 4 (cdk4) by cdk4-activating kinase

1994 ◽  
Vol 14 (4) ◽  
pp. 2713-2721
Author(s):  
J Y Kato ◽  
M Matsuoka ◽  
D K Strom ◽  
C J Sherr
Keyword(s):  
Mammalian Cells ◽  
Insect Cells ◽  
G1 Phase ◽  
Sf9 Cells ◽  
Cyclin D ◽  
Cyclin Dependent Kinase ◽  
Active Protein ◽  
Serum Stimulation ◽  
Rate Limiting

The accumulation of assembled holoenzymes composed of regulatory D-type cyclins and their catalytic partner, cyclin-dependent kinase 4 (cdk4), is rate limiting for progression through the G1 phase of the cell cycle in mammalian fibroblasts. Both the synthesis and assembly of D-type cyclins and cdk4 depend upon serum stimulation, but even when both subunits are ectopically overproduced, they do not assemble into complexes in serum-deprived cells. When coexpressed from baculoviral vectors in intact Sf9 insect cells, cdk4 assembles with D-type cyclins to form active protein kinases. In contrast, recombinant D-type cyclin and cdk4 subunits produced in insect cells or in bacteria do not assemble as efficiently into functional holoenzymes when combined in vitro but can be activated in the presence of lysates obtained from proliferating mammalian cells. Assembly of cyclin D-cdk4 complexes in coinfected Sf9 cells facilitates phosphorylation of cdk4 on threonine 172 by a cdk-activating kinase (CAK). Assembly can proceed in the absence of this modification, but cdk4 mutants which cannot be phosphorylated by CAK remain catalytically inactive. Therefore, formation of the cyclin D-cdk4 complex and phosphorylation of the bound catalytic subunit are independently regulated, and in addition to the requirement for CAK activity, serum stimulation is required to promote assembly of the complexes in mammalian cells.

scholarly journals Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.

1995 ◽  
Vol 15 (5) ◽  
pp. 2672-2681 ◽  
Author(s):  
H Hirai ◽  
M F Roussel ◽  
J Y Kato ◽  
R A Ashmun ◽  
C J Sherr
Keyword(s):  
Mammalian Cells ◽  
G1 Phase ◽  
Cyclin D ◽  
Cyclin Dependent Kinases ◽  
G1 Phase Arrest ◽  
Ink4 Proteins ◽  
Nih 3T3 ◽  
Rate Limiting

Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

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