Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activated extracellular signal-regulated protein kinases (ERKs) and induced expression of cyclin D1. Newly synthesized cyclin D1 assembled with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that phosphorylated the retinoblastoma protein inefficiently. Activation of the MEK1/ERK pathway neither triggered degradation of the CDK inhibitor kinase inhibitory protein-1 (p27(Kip1)) nor led to activation of cyclin E- and A-dependent CDK2, and such cells did not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits generated levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27(Kip1) was mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes were activated; and serum-starved cells entered S phase. Thus, although the activity of p27(Kip1) normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequestered p27(Kip1) and reduced the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells completed S phase and divided, but they were unable to continuously proliferate, indicating that other serum-responsive factors ultimately became rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27(Kip1)-mediated inhibition.
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PMID:Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1). 944 90

To understand the mechanisms by which CDKs regulate cell cycle progression, it is necessary to identify and characterize the physiological substrates of these kinases. We have developed a screening method to identify novel CDK substrates. One of the cDNAs identified in the screen is identical to the recently isolated NPAT gene. Here we show that NPAT associates with cyclin E-CDK2 in vivo and can be phosphorylated by this CDK. The protein level of NPAT peaks at the G1/S boundary. Overexpression of NPAT accelerates S-phase entry, and this effect is enhanced by coexpression of cyclin E-CDK2. These results suggest that NPAT is a substrate of cyclin E-CDK2 and plays a role in S-phase entry.
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PMID:Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry. 947 14

Human colorectal tumor cell lines were established which express wildtype p21 or p21 with a mutation at codon 46 (Cys) or 140 (Gly) on IPTG treatment (LacSwitch). The IPTG-induced wildtype p21 bound to CDK2 and PCNA and inhibited CDK activity in the cells and reduced cell growth rate; whereas, both IPTG-induced mutated p21 proteins neither bound to CDK2 nor affected the CDK activity but did bind to PCNA, and they did not affect the cell growth rate. Wildtype p21 suppressed apoptosis and enhanced survival of X-ray-irradiated or adriamycin-treated cells; but, mutated p21 neither suppressed apoptosis nor affected cell survival. When cells were treated with mimosine, a p53-independent p21-inducer, or butyrolactone I, a specific inhibitor of CDK, cellular endogenous p21 was induced and X-ray or adriamycin-induced apoptosis was blocked. These results suggest that CDK-binding or CDK-inhibitory activity of p21 is required to prevent apoptosis, i.e., CDK is required for apoptosis in human tumor cells.
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PMID:Mutated p21(WAF1/CIP1/SDI1) lacking CDK-inhibitory activity fails to prevent apoptosis in human colorectal carcinoma cells. 948 34

A novel 761-amino-acid transcription factor, DMP1, contains a central DNA binding domain that includes three imperfect myb repeats flanked by acidic transactivating domains at the amino and carboxyl termini. D-type cyclins associate with a region of the DMP1 DNA binding domain immediately adjacent to the myb repeats to form heteromeric complexes which detectably interact neither with cyclin-dependent kinase 4 (CDK4) nor with DNA. The segment of D-type cyclins required for its interaction with DMP1 falls outside the "cyclin box," which contains the residues predicted to contact CDK4. Hence, D-type cyclin point mutants that do not interact with CDK4 can still bind to DMP1. Enforced coexpression of either of three D-type cyclins (D1, D2, or D3) with DMP1 in mammalian cells canceled its ability to activate gene expression. This property was not shared by cyclins A, B, C, or H; did not depend upon CDK4 or CDK2 coexpression; was not subverted by a mutation in cyclin D1 that prevents its interaction with CDK4; and was unaffected by inhibitors of CDK4 catalytic activity. Introduction of DMP1 into mouse NIH 3T3 fibroblasts inhibited entry into S phase. Cell cycle arrest depended upon the ability of DMP1 to bind to DNA and to transactivate gene expression and was specifically antagonized by coexpression of D-type cyclins, including a D1 point mutant that does not bind to CDK4. Taken together, these findings suggest that DMP1 induces genes that inhibit S phase entry and that D-type cyclins can override DMP1-mediated growth arrest in a CDK-independent manner.
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PMID:Gene expression and cell cycle arrest mediated by transcription factor DMP1 is antagonized by D-type cyclins through a cyclin-dependent-kinase-independent mechanism. 948 76

We have investigated the effect of the flavonoid derivative LY 294002, a potent and selective phosphatidylinositol 3-kinase inhibitor, on cell cycle progression in human choroidal melanoma cells. We demonstrate that LY 294002 induces a specific G1 block in asynchronously growing cells leading to an almost complete inhibition of cell proliferation after three days of treatment. When melanoma cells are released from a nocodazole-induced G2/M block, LY 294002 is shown to delay and greatly restrain the G1/S transition. The inhibitor is able to exert its action as long as it is added during the G1 progression and before the cells enter in S phase. We report that the LY 294002-induced G1 arrest is closely correlated to inhibition of CDK4 and CDK2 activities leading to the impairment of pRb phosphorylation which normally occurs during G1 progression. While the inhibition of CDK4 may be attributed at least in part to the decline in CDK4 protein level, CDK2 activity reduction is rather due to the up-regulation of the CDK inhibitor p27Kip1 and to its increased association to CDK2.
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PMID:G1 phase arrest by the phosphatidylinositol 3-kinase inhibitor LY 294002 is correlated to up-regulation of p27Kip1 and inhibition of G1 CDKs in choroidal melanoma cells. 949 22

The identification of a family of proteins that stoichiometrically regulate the activation of the G1 cyclin-dependent kinases has added to our understanding of the process of commitment to the mitotic cycle. The properties of p27 as a CDK binding protein, the ability of environmental signals to regulate the expression of p27, and the observation that p27 may link the accumulation of G1 CDK complexes with activation of the CDK2 kinase, suggest it may have a critical role in establishing a threshold for G1 cyclin/CDK accumulation prior to activation of CDK2 kinase and entry into the mitotic cycle.
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PMID:p27KIP1, an inhibitor of cyclin-dependent kinases. 955 59

Recent methodological developments allow expression measurement of many genes simultaneously, thereby revealing patterns of gene expression that can be related to phenotype. We hypothesized that through the use of such methods we could identify patterns of gene expression associated with the malignant phenotype in human bronchial epithelial cells (BEC). To test this hypothesis, a recently developed quantitative reverse transcriptase polymerase chain reaction method was used to assess simultaneously expression of 15 genes mechanistically associated with cell-cycle control (c-myc, E2F-1, p21, rb, PCNA, cyclin D2, cyclin D3, cyclin E, cdc2, CDK2, CDK4, mad, max p21, max p22, and p53) in normal cell cultures from five individuals and in nine different malignant BEC lines. Relative to the mean expression levels in cultured normal cell populations, expression of c-myc, E2F-1, PCNA, cyclin E, and CDK4 messenger RNA (mRNA) were significantly increased and expression of p21 and p53 mRNA were significantly decreased in one or two, but not all three subtypes (squamous, adenocarcinoma and small cell) of carcinoma cell lines evaluated. No single cell-cycle control gene discriminated all three subtypes from normal cell populations. In contrast, the gene expression index c-myc x E2F-1/p21 separated all carcinoma cell lines from all normal cell populations initially evaluated. This malignancy index was validated in an additional three cultured normal BEC and three carcinoma cell lines, as well as three pairs of matched primary normal bronchial epithelial and primary bronchogenic carcinoma samples, and three pairs of matched primary normal lung parenchyma and primary bronchogenic carcinoma tissue. Again, the c-myc x E2F-1/ p21 index successfully discriminated all cultured and primary normal from malignant samples and thereby had a predictive value of 1 (no false positives and no false negatives). We hypothesize that because of functional mutations in cell-cycle regulatory genes (e.g., p53 and/or rb), cells lose the ability to maintain a pattern of gene expression mechanistically associated with normal, division-limited homeostatic equilibrium. Because the c-myc x E2F-1/p21 gene expression index has high specificity for malignant tissue, it will allow confirmation that there is a significant amount of tumor tissue present in small (e.g., fine-needle) biopsy specimens prior to evaluating them for expression of other genes, such as those involved in chemoresistance or radioresistance. In addition, the goal of most gene therapy efforts is to alter levels of gene expression quantitatively. This index and others derived in a similar manner may better define potential gene therapy targets as well as response of targeted genes to therapy.
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PMID:The gene expression index c-myc x E2F-1/p21 is highly predictive of malignant phenotype in human bronchial epithelial cells. 965 Nov 76

Activation of the p53-mediated DNA damage response induces either G1 cell cycle arrest or apoptosis. The G1 cell cycle arrest is in part caused by the p53-dependent transcriptional activation of the CDK inhibitor, p21(Cip1/Waf1). We report here that human p21 protein is rapidly induced but selectively cleaved during the apoptotic response to gamma-irradiation. Such an event occurred early, well before the morphological appearance of apoptosis. Ectopical expression of p53 in tumor cells alone could induce p21 expression, followed by p21 cleavage and apoptosis. The cleavage of p21 could be reproduced in extracts prepared from irradiated cells or by recombinant caspase-3, suggesting that a caspase-like activity is responsible for this cleavage. p21 binds independently to both CDK2 and proliferation cell nuclear antigen (PCNA). Our studies indicated that p21 cleavage by the caspase-like activity specifically abolished its interaction with PCNA, suggesting that p21 cleavage may interfere with normal PCNA-dependent repair. Our data suggest that p21 may serve as a critical checkpoint regulator for both cell cycle arrest and apoptosis during the p53-mediated DNA damage response. Manipulation of the checkpoint regulators involved in cell cycle arrest and apoptosis may thus provide a novel strategy to cancer therapy.
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PMID:Cleavage of CDK inhibitor p21(Cip1/Waf1) by caspases is an early event during DNA damage-induced apoptosis. 966 8

Normal human diploid fibroblasts (HDF) have a finite proliferative life-span at the end of which they are arrested with a G1 phase DNA content regardless of the culture conditions. Serum stimulated senescent HDF fail to phosphorylate their retinoblastoma protein (pRb) and consequently do not express a large cohort of late G1 phase genes whose products are necessary for entry into S phase. Because pRb is believed to be phosphorylated sequentially in G1 phase by cyclin D-CDK4/6 and cyclin E-CDK2 complexes, we and others have investigated the status of these complexes in senescent HDF. There is little or no cyclin E-associated kinase activity in senescent IMR90 even though potentially active cyclin E-CDK2 complexes are present, suggesting the presence of an inhibitor. Likewise, cyclin D is complexed with its catalytic partners CDK4 and CDK6 in senescent HDF, but it is not known whether these complexes are active. p21Sdi1,Cip1,Waf1, a ubiquitous inhibitor of the activity of cyclin-CDK complexes, increases progressively throughout the life-span of HDF, but then declines again after the cells become senescent. In contrast, p16Ink4a, which binds monomeric CDK4 and CDK6 thereby preventing their binding to cyclin D, is increased dramatically at the time of senescence and remains high for at least 2 mo. Thus, it is possible that increased p21 initiates the senescent cell cycle arrest in normal cells, but p16 is important for the long-term maintenance of that arrest.
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PMID:Molecular mechanisms for the senescent cell cycle arrest. 973 51

We show here that the adenovirus E1A oncoprotein prevents growth arrest by the CDK2 inhibitor p27(Kip1) (p27) in rodent fibroblasts. However, E1A neither binds p27 nor prevents inhibition of CDK2 complexes in vivo. In contrast, the amount of free p27 available to inhibit cyclin E/CDK2 is increased in E1A-expressing cells, owing to reduced expression of cyclins D1 and D3. Moreover, E1A allows cell proliferation in the presence of supraphysiological p27 levels, while c-Myc, known to induce a cellular p27-inhibitory activity, is only effective against physiological p27 concentrations. E1A also bypasses G1 arrest by roscovitine, a chemical inhibitor of CDK2. Altogether, these findings imply that E1A can act downstream of p27 and CDK2. Retinoblastoma (pRb)-family proteins are known CDK substrates; as expected, association of E1A with these proteins (but not with p300/CBP) is required for E1A to prevent growth arrest by either p27 or the CDK4/6 inhibitor p16(INK4a). Bypassing CDK2 inhibition requires an additional function of E1A: the mutant E1A Delta26-35 does not overcome p27-induced arrest, while it binds pRb-family proteins, prevents p16-induced arrest, and alleviates pRb-mediated repression of E2F-1 transcriptional activity (although E1A Delta26-35 fails to restore expression of E2F-regulated genes in p27-arrested cells). We propose that besides the pRb family, E1A targets specific effector(s) of CDK2 in G1-S control.
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PMID:A novel function of adenovirus E1A is required to overcome growth arrest by the CDK2 inhibitor p27(Kip1). 977 42


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