Truncation and Optimisation of Peptide Inhibitors of Cyclin-Dependent Kinase 2-Cyclin A Through Structure-Guided Design

Abstract In humans, the molecular mechanisms underlying ovarian follicle endowment and activation, which are closely related to the control of female reproduction, occurrence of menopause, and related diseases such as premature ovarian failure, are poorly understood. In the current study, we provide several lines of genetic evidence that the cyclin-dependent kinase (Cdk) inhibitor 1B (commonly known as p27kip1 or p27) controls ovarian development in mice by suppressing follicle endowment and activation, and by promoting follicle death. In p27-deficient (p27−/−) mice, postnatal follicle assembly was accelerated, and the number of endowed follicles was doubled as compared with p27+/+ mice. Moreover, in p27−/− ovaries the primordial follicle pool was prematurely activated once it was endowed, and at the same time the massive follicular death that occurs before sexual maturity was rescued by loss of p27. In early adulthood, however, the overactivated follicular pool in p27−/− ovaries was largely depleted, causing premature ovarian failure. Furthermore, we have extensively studied the molecular mechanisms underlying the above-mentioned phenotypes seen in p27−/− ovaries and have found that p27 controls follicular development by several distinct mechanisms at different stages of development of the ovary. For example, p27 controls oocyte growth by suppressing the functions of Cdk2/Cdc2-cyclin A/E1 in oocytes that are arrested at the diplotene stage of meiosis I. This function of p27 is distinct from its well-known role as a suppressor of cell cycle progression. In addition, we have found that p27 activates the caspase-9-caspase-3-caspase-7-poly (ADP-ribose) polymeraseapoptotic cascade by inhibiting Cdk2/Cdc2-cyclin A/B1 kinase activities in follicles, thereby inducing follicle atresia. Our results suggest that the p27 gene is important in determining mammalian ovarian development. This study therefore provides insight into ovary-borne genetic aberrations that cause defects in folliculogenesis and infertility in humans.

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Crystal structure of the p27Kip1 cyclin-dependent-kinase inibitor bound to the cyclin A–Cdk2 complex

Nature ◽

10.1038/382325a0 ◽

1996 ◽

Vol 382 (6589) ◽

pp. 325-331 ◽

Cited By ~ 593

Author(s):

Alicia A. Russo ◽

Philip D. Jeffrey ◽

Andrea K. Patten ◽

Joan Massagué ◽

Nikola P. Pavletich

Keyword(s):

Crystal Structure ◽

Cyclin A ◽

Cyclin Dependent Kinase ◽

Cdk2 Complex

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Acetylation of cyclin A: a new cell cycle regulatory mechanism

Biochemical Society Transactions ◽

10.1042/bst0380083 ◽

2010 ◽

Vol 38 (1) ◽

pp. 83-86 ◽

Cited By ~ 13

Author(s):

Francesca Mateo ◽

Miriam Vidal-Laliena ◽

Maria Jesus Pujol ◽

Oriol Bachs

Keyword(s):

Binding Protein ◽

Cyclin A ◽

Camp Response Element Binding ◽

Cyclin Dependent Kinase ◽

General Control ◽

Camp Response Element ◽

Lysine Residues ◽

Subsequent Degradation ◽

The Status ◽

Element Binding Protein

Cyclin A must be degraded at prometaphase in order to allow mitosis progression. Nevertheless, the signals that trigger cyclin A degradation at mitosis have been largely elusive. In the present paper, we review the status of cyclin A degradation in the light of recent evidence indicating that acetylation plays a role in cyclin A stability. The emerging model proposes that the acetyltransferase PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] [perhaps also its homologue GCN5 (general control non-derepressible 5)] acetylates cyclin A at Lys54, Lys68, Lys95 and Lys112 during mitosis, leading to its ubiquitylation by the anaphase-promoting factor/cyclosome and its subsequent degradation via proteasome. Interestingly, these four lysine residues in cyclin A also participate in the regulation of cyclin A–Cdk (cyclin-dependent kinase) activity by modulating its interaction with Cdks.

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Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors.

Molecular and Cellular Biology ◽

10.1128/mcb.16.12.6623 ◽

1996 ◽

Vol 16 (12) ◽

pp. 6623-6633 ◽

Cited By ~ 247

Author(s):

P D Adams ◽

W R Sellers ◽

S K Sharma ◽

A D Wu ◽

C M Nalin ◽

...

Keyword(s):

Kinase Inhibitors ◽

Cyclin A ◽

Cdk Inhibitors ◽

Cyclin Dependent Kinase ◽

Binding Motifs ◽

Recognition Motif ◽

Binding Inhibition ◽

In Vivo Growth

Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A-cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphorylation of the retinoblastoma protein by cyclin A- but not cyclin B-associated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 and E2F1 likewise prevented their phosphorylation by cyclin A-associated kinases in vitro. The cdk inhibitor p21 was found to contain two functional copies of this recognition motif, as determined by in vitro kinase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate recognition motif. Furthermore, these data suggest that p21-like cdk inhibitors function, at least in part, by blocking the interaction of substrates with cyclin-cdk2 complexes.

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Human Cyclin a Is Required for Mitosis until Mid Prophase

The Journal of Cell Biology ◽

10.1083/jcb.147.2.295 ◽

1999 ◽

Vol 147 (2) ◽

pp. 295-306 ◽

Cited By ~ 148

Author(s):

Nobuaki Furuno ◽

Nicole den Elzen ◽

Jonathon Pines

Keyword(s):

Cyclin A ◽

Time Lapse ◽

S Phase ◽

Early Prophase ◽

Cyclin Dependent Kinase ◽

Late Prophase ◽

Daughter Cells ◽

G2 Phase ◽

Rate Limiting

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.4843 ◽

1999 ◽

Vol 19 (7) ◽

pp. 4843-4854 ◽

Cited By ~ 66

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.

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p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes.

Molecular Biology of the Cell ◽

10.1091/mbc.8.9.1815 ◽

1997 ◽

Vol 8 (9) ◽

pp. 1815-1827 ◽

Cited By ~ 14

Author(s):

P Shiyanov ◽

S Hayes ◽

N Chen ◽

D G Pestov ◽

L F Lau ◽

...

Keyword(s):

Cell Cycle ◽

Binding Sites ◽

Culture Medium ◽

Cyclin E ◽

Cyclin A ◽

Cyclin Dependent Kinase ◽

Binding Motifs ◽

Cyclin Dependent Kinases ◽

Cell Cycle Inhibition ◽

A Cell

p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.

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Noncatalytic Requirement for Cyclin A-cdk2 in p27 Turnover

Molecular and Cellular Biology ◽

10.1128/mcb.24.13.6058-6066.2004 ◽

2004 ◽

Vol 24 (13) ◽

pp. 6058-6066 ◽

Cited By ~ 28

Author(s):

Xin-Hua Zhu ◽

Hoang Nguyen ◽

H. Dorota Halicka ◽

Frank Traganos ◽

Andrew Koff

Keyword(s):

Ubiquitin Ligase ◽

Cyclin E ◽

Flow Cytometric Analysis ◽

E3 Ubiquitin Ligase ◽

Cyclin A ◽

Cyclin B1 ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Flow Cytometric

ABSTRACT Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27kip1 is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G1 cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCFskp2 complex.

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