scholarly journals p27Kip1 Inhibits Cyclin D-Cyclin-Dependent Kinase 4 by Two Independent Modes

2008 ◽  
Vol 29 (4) ◽  
pp. 986-999 ◽  
Author(s):  
Arpita Ray ◽  
Melissa K. James ◽  
Stéphane Larochelle ◽  
Robert P. Fisher ◽  
Stacy W. Blain
Keyword(s):  
Cell Cycle ◽  
Active Site ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Growth Conditions ◽  
Cyclin D ◽  
Cyclin Dependent Kinase ◽  
Proliferating Cells ◽  
Cell Cycle Reentry

ABSTRACT Cell cycle progression is regulated by cyclin-dependent kinases (cdk's), which in turn are regulated by their interactions with stoichiometric inhibitors, such as p27Kip1. Although p27 associates with cyclin D-cyclin-dependent kinase 4 (cdk4) constitutively, whether or not it inhibits this complex is dependent on the absence or presence of a specific tyrosine phosphorylation that converts p27 from a bound inhibitor to a bound noninhibitor under different growth conditions. This phosphorylation occurs within the 3-10 helix of p27 and may dislodge the helix from cdk4's active site to allow ATP binding. Here we show that the interaction of nonphosphorylated p27 with cdk4 also prevents the activating phosphorylation of the T-loop by cyclin H-cdk7, the cdk-activating kinase (CAK). Even though the cyclin H-cdk7 complex is present and active in contact-arrested cells, p27's association with cyclin D-cdk4 prevents T-loop phosphorylation. When p27 is tyrosine phosphorylated in proliferating cells or in vitro with the tyrosine Y kinase Abl, phosphorylation of cdk4 by cyclin H-cdk7 is permitted, even without dissociation of p27. This suggests that upon release from the contact-arrested state, a temporal order for the reactivation of inactive p27-cyclin D-cdk4 complexes must exist: p27 must be Y phosphorylated first, directly permitting cyclin H-cdk7 phosphorylation of residue T172 and the consequent restoration of kinase activity. The non-Y-phosphorylated p27-cyclin D-cdk4 complex could be phosphorylated by purified Csk1, a single-subunit CAK from fission yeast, but was still inactive due to p27's occlusion of the active site. Thus, the two modes by which p27 inhibits cyclin D-cdk4 are independent and may reinforce one another to inhibit kinase activity in contact-arrested cells, while maintaining a reservoir of preformed complex that can be activated rapidly upon cell cycle reentry.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

scholarly journals Cyclin-Dependent Kinase Activity Is Required for Progesterone Receptor Function: Novel Role for Cyclin A/Cdk2 as a Progesterone Receptor Coactivator

2005 ◽  
Vol 25 (1) ◽  
pp. 264-277 ◽  
Author(s):  
Ramesh Narayanan ◽  
Abayomi A. Adigun ◽  
Dean P. Edwards ◽  
Nancy L. Weigel
Keyword(s):  
Cell Cycle ◽  
Progesterone Receptor ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Kinase Activity ◽  
Cyclin A ◽  
Cyclin Dependent Kinase ◽  
Histone H4 Acetylation ◽  
Target Promoter

ABSTRACT Our studies examining the role of the cell cycle-regulated kinase cyclin A/Cdk2 in progesterone receptor (PR) action have demonstrated that cyclin-dependent kinase activity is required for PR function and that cyclin A/Cdk2 functions as a PR coactivator. Although Cdk2 can phosphorylate PR, elimination of these phosphorylation sites has little effect on the ability of cyclin A/Cdk2 to stimulate PR activity. PR interacts with cyclin A and recruits cyclin A/Cdk2 to progestin-responsive promoters, stimulating transcription. Inhibition of Cdk2 activity abolishes progesterone-dependent activation of PR target genes in part through inhibition of PR-dependent recruitment of steroid receptor coactivator 1 (SRC-1) and subsequent histone H4 acetylation at the target promoter. In vitro studies revealed that the interaction between SRC-1 and PR is dependent upon phosphorylation of SRC-1. This heretofore-unknown mechanism provides a potential means for integrating the regulation of PR activity with cell cycle progression. Moreover, the ability of PR to recruit cyclin A/Cdk2 to target promoters provides locally elevated levels of kinase, which can preferentially facilitate phosphorylation-dependent interactions and enzymatic activities of coactivators at the target promoter.

scholarly journals Cyclin A expression is under negative transcriptional control during the cell cycle.

1996 ◽  
Vol 16 (7) ◽  
pp. 3789-3798 ◽  
Author(s):  
X Huet ◽  
J Rech ◽  
A Plet ◽  
A Vié ◽  
J M Blanchard
Keyword(s):  
Cell Cycle ◽  
Transcriptional Repression ◽  
Transcriptional Control ◽  
Cell Cycle Progression ◽  
Cyclin A ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Proliferating Cells

Transcription of the gene coding for cyclin A, a protein required for S-phase transit, is cell cycle regulated and is restricted to proliferating cells. To further explore transcriptional regulation linked to cell division cycle control, a genomic clone containing 5' flanking sequences of the murine cyclin A gene was isolated. When it was fused to a luciferase reporter gene, it was shown to function as a proliferation-regulated promoter in NIH 3T3 cells. Transcription of the mouse cyclin A gene is negatively regulated by arrest of cell proliferation. A mutation of a GC-rich sequence conserved between mice and humans is sufficient to relieve transcriptional repression, resulting in a promoter with constitutively high activity. In agreement with this result, in vivo footprinting reveals a protection of the cell cycle-responsive element in G0/early G1 cells which is not observed at later stages of the cell cycle. Moreover, the footprint is present in dimethyl sulfoxide-induced differentiating and not in proliferating Friend erythroleukemia cells. Conversely, two other sites, which in vitro bind ATF-1 and NF-Y, respectively, are constitutively occupied throughout cell cycle progression.

scholarly journals D-type cyclin-dependent kinase activity in mammalian cells.

1994 ◽  
Vol 14 (3) ◽  
pp. 2066-2076 ◽  
Author(s):  
H Matsushime ◽  
D E Quelle ◽  
S A Shurtleff ◽  
M Shibuya ◽  
C J Sherr ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Cyclin D3 ◽  
Tween 20 ◽  
Cyclin D ◽  
Cyclin Dependent Kinase ◽  
Proliferating Cells ◽  
Quiescent Cells ◽  
Serum Stimulation

D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins D1, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone H1 or casein. Virtually all cyclin D1-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin D1-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin D1-cdk4 complexes. Thus, cyclin D1-associated kinase activity was not detected during the G0-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin D1 alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.

scholarly journals Requirement of Cyclin E-Cdk2 Inhibition in p16INK4a-Mediated Growth Suppression

1998 ◽  
Vol 18 (9) ◽  
pp. 5284-5290 ◽  
Author(s):  
Hong Jiang ◽  
Hubert S. Chou ◽  
Liang Zhu
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Tumor Cells ◽  
Kinase Activity ◽  
Cyclin E ◽  
Growth Suppression ◽  
Dominant Negative ◽  
Cyclin D ◽  
Loss Of Function

ABSTRACT Loss-of-function mutations of p16 INK4a have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro, and this inhibition is believed to be the cause of the p16-mediated G1 cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4N158, designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050–2054, 1993). In this study, we determined functional differences between p16 and Cdk4N158. We show that p16 and Cdk4N158 inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociated cyclin D1-Cdk4 complexes with the release of bound p27 KIP1 , while Cdk4N158 formed complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 formed complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities were correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E became resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G1 arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression.

scholarly journals The Hematopoietic Transcription Factor AML1(RUNX1) Is Negatively Regulated by the Cell Cycle ProteinCyclinD3

2005 ◽  
Vol 25 (23) ◽  
pp. 10205-10219 ◽  
Author(s):  
Luke F. Peterson ◽  
Anita Boyapati ◽  
Velvizhi Ranganathan ◽  
Atsushi Iwama ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cell ◽  
Cell Cycle Progression ◽  
Cyclin D3 ◽  
Cyclin D ◽  
Dependent Manner ◽  
Cycle Progression ◽  
Transactivation Activity ◽  
Mammalian Cell Cycle

ABSTRACT The family of cyclin D proteins plays a crucial role in the early events of the mammalian cell cycle. Recent studies have revealed the involvement of AML1 transactivation activity in promoting cell cycle progression through the induction of cyclin D proteins. This information in combination with our previous observation that a region in AML1 between amino acids 213 and 289 is important for its function led us to investigate prospective proteins associating with this region. We identified cyclin D3 by a yeast two-hybrid screen and detected AML1 interaction with the cyclin D family by both in vitro pull-down and in vivo coimmunoprecipitation assays. Furthermore, we demonstrate that cyclin D3 negatively regulates the transactivation activity of AML1 in a dose-dependent manner by competing with CBFβ for AML1 association, leading to a decreased binding affinity of AML1 for its target DNA sequence. AML1 and its fusion protein AML1-ETO have been shown to shorten and prolong the mammalian cell cycle, respectively. In addition, AML1 promotes myeloid cell differentiation. Thus, our observations suggest that the direct association of cyclin D3 with AML1 functions as a putative feedback mechanism to regulate cell cycle progression and differentiation.

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

2009 ◽  
Vol 20 (15) ◽  
pp. 3572-3582 ◽  
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.

scholarly journals Cyclin-dependent kinase phosphorylation of RUNX1/AML1 on 3 sites increases transactivation potency and stimulates cell proliferation

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1193-1200 ◽  
Author(s):  
Linsheng Zhang ◽  
Florence B. Fried ◽  
Hong Guo ◽  
Alan D. Friedman
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Assay ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Inhibition Of Proliferation ◽  
Cdk Phosphorylation ◽  
Kinase Phosphorylation ◽  
Lineage Specific Genes

Abstract RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and stimulates G1 cell-cycle progression. Within RUNX1, S48, S303, and S424 fit the cyclin-dependent kinase (cdk) phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cdks on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now use an in vitro kinase assay, phosphopeptide-specific antiserum, and the cdk inhibitor roscovitine to demonstrate that S48 and S424 are also phosphorylated by cdk1 or cdk6 in hematopoietic cells. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell-cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation of the cdk sites to the partially phosphomimetic aspartic acid mildly reduced DNA affinity while progressively increasing transactivation of a model reporter. Mutation to alanine increased DNA affinity, suggesting that in other gene or cellular contexts phosphorylation of RUNX1 by cdks may reduce transactivation. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBFβ-SMMHC more effectively than the tripleA mutant. Together these findings indicate that cdk phosphorylation of RUNX1 potentially couples stem/progenitor proliferation and lineage progression.

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