Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The transcription factor E2F-1 interacts stably with cyclin A via a small domain near its amino terminus and is negatively regulated by the cyclin A-dependent kinases. Thus, the activities of E2F, a family of transcription factors involved in cell proliferation, are regulated by at least two types of cell growth regulators: the retinoblastoma protein family and the cyclin-dependent kinase family. To investigate further the regulation of E2F by cyclin-dependent kinases, we have extended our studies to include additional cyclins and E2F family members. Using purified components in an in vitro system, we show that the E2F-1-DP-1 heterodimer, the functionally active form of the E2F activity, is not a substrate for the active cyclin D-dependent kinases but is efficiently phosphorylated by the cyclin B-dependent kinases, which do not form stable complexes with the E2F-1-DP-1 heterodimer. Phosphorylation of the E2F-1-DP-1 heterodimer by cyclin B-dependent kinases, however, did not result in down-regulation of its DNA-binding activity, as is readily seen after phosphorylation by cyclin A-dependent kinases, suggesting that phosphorylation per se is not sufficient to regulate E2F DNA-binding activity. Furthermore, heterodimers containing E2F-4, a family member lacking the cyclin A binding domain found in E2F-1, are not efficiently phosphorylated or functionally down-regulated by cyclin A-dependent kinases. However, addition of the E2F-1 cyclin A binding domain to E2F-4 conferred cyclin A-dependent kinase-mediated down-regulation of the E2F-4-DP-1 heterodimer. Thus, both enzymatic phosphorylation and stable physical interaction are necessary for the specific regulation of E2F family members by cyclin-dependent kinases.
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PMID:Specific regulation of E2F family members by cyclin-dependent kinases. 919 21

The p16INK4a gene product acts as a negative regulator of the cell cycle by binding to cyclin-dependent kinases (CDKs) 4 and 6, thereby inhibiting the formation of an active CDK/cyclin D complex. Deletion of the p16 locus has been observed in tumor cell lines and, less frequently, in primary human neoplasms. We analyzed 31 glioblastomas and identified 6 cases with hemizygous and 6 with homozygous deletions of the p16 locus. Eight of these cases showed a concurrent amplification of the EGFR gene (epidermal growth factor receptor) while the overall frequency was 35%. This close correlation suggests that deletion of the p16 chromosomal region constitutes another genetic hallmark of the primary glioblastoma, which rapidly develops de novo, without a less malignant precursor lesion and for which EGFR amplification is a characteristic genetic change. The p16 protein was not detectable in 15 of 22 glioblastomas but only 4 of these showed homozygous deletion of the gene. The alternative transcript p16 beta, for which a growth-suppressing function has been suggested, was co-expressed with p16 alpha mRNA in most cases. Hypermethylation of CpG islands in the 5' region of the p16 gene was identified in only 1 case, suggesting that this alternative mechanism of gene silencing is rarely responsible for loss of p16 expression in glioblastomas. Likewise, only 1 glioblastoma carried a p16 mutation and in addition, unexpectedly, a homozygous deletion of p16 in approximately 80% of tumor cells. This mutation, Arg24Pro, has previously been identified in a melanoma kindred.
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PMID:Hemizygous or homozygous deletion of the chromosomal region containing the p16INK4a gene is associated with amplification of the EGF receptor gene in glioblastomas. 933 10

The cyclin-dependent kinases (CDKs) promote cell cycle transitions in mammalian cells by phosphorylation of key substrates. To characterize substrates of the G1 and S phase cyclin-CDK complexes, including cyclin D1-CDK4, cyclin D3-CDK4, cyclin D3-CDK6, cyclin E-CDK2, and cyclin A-CDK2, which are largely undefined, we phosphorylated T-47D breast cancer cell nuclear lysates partially purified by ion-exchange chromatography with purified baculovirus expressed cyclin-CDK complexes. A comparison of the substrates that were phosphorylated by the different cyclin D-CDKs revealed some common as well as specific substrates. Hence, cyclin D1-CDK4 specifically phosphorylated a 38-kDa protein while cyclin D3-CDK4 specifically phosphorylated proteins of 105, 102, and 42 kDa. A 24-kDa protein was phosphorylated by both complexes. Cyclin D3-CDK6 exhibited similar substrate preferences to cyclin D3-CDK4, phosphorylating the 105- and 102-kDa proteins but not the 24-kDa protein. Hence, both the cyclin D1 and D3 as well as CDK4 and CDK6 subunits can confer substrate specificity on the overall cyclin D-CDK complex. Cyclin E-CDK2 and cyclin A-CDK2 phosphorylated a greater number of substrates than the cyclin D-CDKs, ranging in size from 10 kDa to over 200 kDa. Twenty-two substrates were common to both complexes, while six were specific for cyclin A-CDK2 and only one protein of 34 kDa was specific for cyclin E-CDK2. These studies indicate that cyclins E and A modulate the specificity of CDK2 and have demonstrated substrates that may be important for the specific roles of these cyclin-CDKs during G1 and S phase progression. Protein sequencing of one of the cyclin-CDK substrates characterized in this study identified this protein as nucleolin, a previously characterized CDC2 (CDK1) substrate, thus indicating the utility of this approach in identifying cyclin-CDK targets. These results show that both the cyclin and CDK subunits can regulate the substrate specificity of the overall cyclin-CDK complex and have demonstrated numerous substrates of D-, E-, and A-type cyclin-CDK complexes potentially involved in regulating transit through the G1 and S phases of the cell cycle.
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PMID:Differential phosphorylation of T-47D human breast cancer cell substrates by D1-, D3-, E-, and A-type cyclin-CDK complexes. 940 25

G1 cyclin, as a candidate proto-oncogene, plays a key role as a cellular regulator through direct interaction with retinoblastoma gene product (pRB), p107, and cyclin-dependent kinase. Human papillomavirus (HPV) 16/18 E6/7 proteins may bind the unphosphorylated pRB and act as a down-regulator of cyclin D. However, this theory has not been proven and the relationship between cyclin E and HPV also remains unclear. Using formalin-fixed and paraffin-embedded cervical tissues, we examined for HPV types 16 and 18 by nested polymerase chain reaction (PCR), and for the accumulation of cyclin D1 and E by immunohistochemistry in 22 cases of normal cervix, 23 cases of cervical intraepithelial neoplasia (CIN), 39 cases of invasive squamous cell carcinoma (SCC), and 6 cases of adenocarcinoma. Cyclin index (CI) was defined as the percentage of positively labeled nuclei per 1000 cells. The accumulation of cyclin D1 was detected in the normal basal and parabasal epithelium and was markedly decreased in neoplastic epithelium (0.87% CI in CIN, 5.88% in SCC). However, cyclin E was absent in the normal cervix but increased in neoplastic epithelium (7.17% CI in CIN, 10.32% in SCC). Cyclin D1 expression was significantly low (p = 0.004), but cyclin E expression was significantly high (p = 0.026) in HPV-positive cases. Cyclin E plays a leading role in neoplastic transformation whereas cyclin D is down-regulated, especially when associated with HPV.
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PMID:Correlation between G1 cyclins and HPV in the uterine cervix. 942 Oct 73

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

Ectopic expression of the c-Myc oncoprotein prevents cell cycle arrest in response to growth-inhibitory signals, differentiation stimuli, or mitogen withdrawal. Moreover, Myc activation in quiescent cells is sufficient to induce cell cycle entry in the absence of growth factors. Thus, Myc transduces a potent mitogenic stimulus but, concomitantly, induces apoptosis in the absence of survival factors. We review here recent progress in our understanding of the molecular mechanisms linking Myc activity to cell cycle control. Myc is a positive regulator of G1-specific cyclin-dependent kinases (CDKs) and, in particular, of cyclin E/CDK2 complexes. Cyclin D/CDK4 and CDK6 may conceivably also be activated by Myc, but the circumstances in which this occurs remain to be explored. Myc acts via at least three distinct pathways which can enhance CDK function: (1) functional inactivation of the CDK inhibitor p27Kip1 and probably also of p21Cip1 and p57Kip2, (2) induction of the CDK-activating phosphatase Cdc25A and (3) - in an ill understood and most likely indirect way - deregulation of cyclin E expression. Constitutive expression of either Myc or cyclin E can prevent growth arrest by p16INK4a (an inhibitor of cyclin D/CDK4, but not of cyclin E/CDK2). In cells, p16INK4a inhibits phosphorylation, and thus induces activation of the Retinoblastoma-family proteins (pRb, p107 and p130). Surprisingly, this effect of p16 is not altered in the presence of Myc or cyclin E. Thus, Myc and cyclin E/CDK2 activity unlink activation of p16 and pRb from growth arrest. Finally, Myc may itself be a functional target of cyclin D/CDK4 through its direct interaction with p107. We discuss how the effects of Myc on cell cycle control may relate to its oncogenic activity, and in particular to its ability to cooperate with activated Ras oncoproteins.
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PMID:Myc and the cell cycle. 946 63

Depletion of guanine nucleotide pools after inhibition of inosine monophosphate dehydrogenase (IMPDH) potently inhibits DNA synthesis by arresting cells in G1 and has been shown to induce the differentiation of cultured myeloid and erythroid cell lines, as well as chronic granulocytic leukemic cells after blast transformation. Inhibitors of IMPDH are also highly effective as immunosuppressive agents. The mechanism underlying these pleiotropic effects of depletion of guanine nucleotides is unknown. We have examined the effects of mycophenolic acid (MPA), a potent IMPDH inhibitor, on the cell cycle progression of activated normal human T lymphocytes. MPA treatment resulted in the inhibition of pRb phosphorylation and cell entry into S phase. The expression of cyclin D3, a major component of the cyclin-dependent kinase (CDK) activity required for pRb phosphorylation, was completely abrogated by MPA treatment of T cells activated by interleukin-2 (IL-2) and leucoagglutinin (PHA-L), whereas the expression of cyclin D2, CDK6, and CDK4 was more mildly attenuated. The direct kinase activity of a complex immunoprecipitated with anti-CDK6 antibody was also inhibited. In addition, MPA prevented the IL-2-induced elimination of p27(Kip1), a CDK inhibitor, and resulted in the retention of high levels of p27(Kip1) in IL-2/PHA-L-treated T cells bound to CDK2. These results indicate that inhibition of the de novo synthesis of guanine nucleotides blocks the transition of normal peripheral blood T lymphocytes from G0 to S phase in early- to mid-G1 and that this cell cycle arrest results from inhibition of the induction of cyclin D/CDK6 kinase and the elimination of p27(Kip1) inhibitory activity.
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PMID:Effects of guanine nucleotide depletion on cell cycle progression in human T lymphocytes. 953

The pl6INK4a/MTS1 (p16) gene encodes a specific inhibitor of cyclin-dependent kinase (CDK)4 and CDK6. The p16 gene is frequently mutated or deleted in many types of cancer cell lines as well as in certain types of primary tumors. p16 knockout mice are viable but predisposed to sarcoma and B-cell lymphoma. To investigate the role of p16 in human soft-tissue sarcoma tumor progression, we examined the p16 gene by Southern blot analysis and PCR sequencing in 30 pairs of primary soft-tissue sarcomas and autologous normal tissue. Only one tumor sample showed possible rearrangement of the p16 gene. In contrast, Western blot analysis of the p16 protein in 20 pairs of samples showed decreased p16 expression in only 20% of the tumors but elevated p16 expression in 40% of the tumors when compared with the autologous normal controls. Overexpression of p16 was not concomitant with loss of the RB protein as is found in several other types of cancers, because more than one-half of the tumors with increased p16 expression also had high levels of RB protein. On the other hand, the p16 target protein CDK4 was overexpressed in at least 60% of the tumors. In the majority of cases, CDK4 overexpression accompanied elevated p16 and/or RB levels. Our results suggest that: (a) alteration of the p16 gene is infrequent in primary soft-tissue sarcoma; (b) Cdk4 may act as an oncogene in soft-tissue sarcoma; and (c) elevated p16 and RB levels might be the result of compensatory up-regulation of these proteins to counteract CDK4 overexpression in these tumors. Our results also suggest that it is more informative to examine aberrations in the "p16-CDK4/cyclin D-RB" pathway than to selectively examine individual components in this pathway when investigating genetic changes involved in human malignancy.
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PMID:Infrequent mutation of the p16/MTS1 gene and overexpression of cyclin-dependent kinase 4 in human primary soft-tissue sarcoma. 956 3

In order to analyze dexamethasone effects on peripheral blood lymphocyte proliferation, we defined various experimental conditions: dexamethasone introduced (i) at the time of phytohemagglutinin stimulation, (ii) 48 h after the beginning of phytohemagglutinin stimulation, and (iii) on unstimulated lymphocytes. In stimulated lymphocytes, we observed an early G1 accumulation (P < 0.005), a delayed increase in the duration of S-phase (P < 0.03), and a consequent increase in cell-cycle duration. The expression of several cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) was modified. Cyclin D3, CDK4, and CDK6 involved in G1-phase control were significantly decreased under dexamethasone treatment whatever the level of stimulation of lymphocytes (stimulated or unstimulated PBL). Cyclin E and CDK2, acting in G1/ S-phase transition and S-phase regulation, decreased in stimulated lymphocytes before any modification of S-phase (P < 0.002). The expression of CKIs, mainly of p27Kip1, appeared to vary with the degree of cell stimulation: a decrease was observed on treated unstimulated lymphocytes, while p27Kip1 increased in dexamethasone-treated cells during stimulation. Our results indicate sequential modifications of the cell-cycle regulation by dexamethasone starting with an action on G1 followed by S-phase control modifications. The protein analysis pinpoints the major complexes concerned: CDK4 and CDK6/cyclin D are mainly involved in G1-phase modifications, while CDK2 and its partner, cyclin E, might be specifically involved in the lengthening of S-phase. The variations observed for p27Kip1 might amplify the functional effects of dexamethasone on kinasic complexes.
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PMID:Glucocorticoids induce G1 as well as S-phase lengthening in normal human stimulated lymphocytes: differential effects on cell cycle regulatory proteins. 959 99

Loss of attachment to an extracellular matrix substrate arrests the growth of untransformed cells in the G1 phase. This anchorage-dependent cell cycle arrest is linked to increased expression of the p21Cip1 (p21) and p27Kip1 (p27) cyclin-dependent kinase inhibitors. The result is a loss of cdk2-associated kinase activity, especially that of cyclin E-cdk2. The levels of p21 and p27 are also upregulated in unattached transformed cells, but cyclin E-cdk2 activity remains high, and the cells are able to grow in an anchorage-independent manner. Increased expression of cyclin E and cdk2 appears to be partially responsible for the maintenance of cyclin E-cdk2 activity in transformed cells. To explore further the regulation of cyclin E-cdk2 in transformed cells, we have analysed the subcellular distribution of cyclin-cdk complexes and their inhibitors in normal human fibroblasts, their transformed counterparts, and in various human tumor cell lines. In substrate-attached normal fibroblasts, cyclin E and cdk2 were exclusively in the nuclear fraction, associated with one another. When normal fibroblasts were detached and held in suspension, cyclin E-cdk2 complexes remained nuclear, but were now found associated with the p21 and p27 cdk inhibitors and lacked histone H1 phosphorylating activity. In contrast, the transformed fibroblasts and tumor cells, which are anchorage-independent, had more than half of their cyclin E, cdk2, p21 and p27 in the cytoplasmic fraction, both in attached and suspended cultures. The cytoplasmic p21 and p27 were bound to cyclin E-cdk2, as well as to complexes containing cyclin A and cyclin D. The nuclear cyclin E-cdk2 complexes from the transformed cells grown in suspension contained only low levels of p21 and p27 and had histone H1 kinase activity. Thus, at least three mechanisms contribute to keeping cyclin E-cdk2 complexes active in suspended anchorage-independent cells: cyclin E and cdk2 are upregulated, as reported previously, cdk inhibitors are sequestered away from the nucleus by cytoplasmic cyclin-cdk complexes, and the binding of the inhibitors to nuclear cyclin E-cdk2 complexes is impaired.
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PMID:Cytoplasmic displacement of cyclin E-cdk2 inhibitors p21Cip1 and p27Kip1 in anchorage-independent cells. 963 34


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