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:2.7.11.22 (cdc2)
8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Senescent human diploid fibroblasts are unable to enter S phase in response to mitogenic stimulation. One of the key deficiencies in mitogen-stimulated senescent cells is their failure to phosphorylate the retinoblastoma protein, which acts as an inhibitor of entry into S phase in its unphosphorylated form. Recent data suggest that cyclin-dependent kinases (Cdks) regulated by G1 cyclins (D type and E) are responsible for the primary phosphorylation of the retinoblastoma protein prior to the G1/S boundary. Surprisingly, we found 10- to 15-fold higher constitutive amounts of both cyclin E and cyclin D1 in senescent cells compared to quiescent early-passage cells. Nevertheless, cyclin E-associated kinase activity in senescent cells was very low and did not increase significantly upon mitogenic stimulation even though cyclin E-Cdk2 complexes were abundant. In contrast to early-passage cells in late G1 phase, senescent cells contained mainly underphosphorylated cyclin E and proportionally more unphosphorylated and inactive Cdk2, perhaps accounting for the low kinase activity. We also show that a majority of the Cdk2 in senescent cells, but not in early-passage cells, was complexed with cyclin D1. Cyclin D1-Cdk2 complexes, severalfold enriched in senescent cells, contained exclusively unphosphorylated Cdk2. Amounts of cyclin A, which ordinarily accumulates in S and G2 phases, were extremely low in stimulated senescent cells. We suggest that the failure to activate cyclin E-Cdk2 kinase activity in senescent cells may account for the inability of these cells to phosphorylate the retinoblastoma protein in late G1 phase, which in turn may block the expression of late G1 genes such as cyclin A that are required for entry into S phase.
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PMID:Altered regulation of G1 cyclins in senescent human diploid fibroblasts: accumulation of inactive cyclin E-Cdk2 and cyclin D1-Cdk2 complexes. 824 8

By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes. (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide interchanged these complexes with similar kinetics. (v) When MT2-shifted E2F.G0/G1, E2F.S, and free E2F were eluted and analyzed by Western blot assay using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositions of these MT2-bound complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene.
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PMID:G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter. 828 95

The transcription factor E2F activates the expression of multiple genes involved in cell proliferation, such as c-myc and the dihydrofolate reductase gene. Regulation of E2F involves its interactions with other cellular proteins, including the retinoblastoma protein (Rb), the Rb-related protein p107, cyclin A, and cdk2. We undertook a detailed analysis of E2F DNA-binding activities and their cell cycle behavior in primary human T cells. Three E2F DNA-binding activities were identified in resting (G0) T cells with mobilities in gel shift assays distinct from those of previously defined E2F complexes. One of these activities was found to be a novel, less abundant, Rb-E2F complex. The most prominent E2F activity in resting T cells (termed complex X) was abundant in both G0 and G1 but disappeared as cells entered S phase, suggesting a possible role in negatively regulating E2F function. Complex X could be dissociated by adenovirus E1A with a requirement for an intact E1A conserved region 2. However, X failed to react with a variety of antibodies against Rb or p107, implicating the involvement of an E1A-binding protein other than Rb or p107. In addition to these novel E2F complexes, three distinct forms of unbound (free) E2F were resolved in gel shift experiments. These species showed different cell cycle kinetics. UV cross-linking experiments suggested that a distinct E2F DNA-binding protein is uniquely associated with the S-phase p107 complex and is not associated with Rb. Together, these results suggest that E2F consists of multiple, biochemically distinct DNA-binding proteins which function at different points in the cell cycle.
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PMID:Cell cycle analysis of E2F in primary human T cells reveals novel E2F complexes and biochemically distinct forms of free E2F. 832 Dec 4

The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G1 cyclins (D2 and D3) in vitro. Binding depended upon the minimal regions of Rb necessary for its growth-suppressive activity, as well as upon the D-type cyclin sequence motif shared with Rb-binding DNA tumor virus oncoproteins. Coexpression of the three D-type cyclins with the cyclin-dependent kinase (cdk4) in insect cells generated Rb kinase activity. By contrast, cyclins D2 and D3, but not D1, activated another such kinase, cdk2. Introduction of cyclin D2 and Rb into the Rb-deficient cell line SAOS-2 led to overt Rb hyperphosphorylation, whereas Rb, expressed alone or together with cyclin D1, remained unphosphorylated. Cyclin D2-dependent phosphorylation inhibited its binding to the transcription factor E2F and reversed the Rb G1 exit block in the cell cycle. Thus, all D-type cyclins do not function equivalently, and one of them plays a major role in reversing the cycle-blocking function of a known tumor suppressor.
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PMID:Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. 834 2

Decreased affinity of the retinoblastoma protein (RB) for the nuclear compartment has been correlated with cell cycle-dependent phosphorylation of the RB protein during the G1/S phase of the cell cycle. We examined the effects of microinjected protein-serine/threonine phosphatases types 1 (PP1) and 2A (PP2A) on nuclear association of RB monitored as the resistance of RB to extraction at the G1/S transition. Microinjection of PP1 into either the nucleus or the cytoplasm of cells synchronized in G1 increased the amount of RB that was resistant to extraction from the nucleus. Microinjection of PP2A, however, required direct injection into the nucleus to generate this effect. In addition, we found that nuclear injection of only the PP2A catalytic subunit (PP2AC) and not the complex containing the A and C subunits inhibited RB extraction. Microinjection of either PP1 or PP2A and the resultant increased affinity of RB for the nucleus corresponded with the inhibition of cell cycle progression into S phase. Injection of either phosphatase into cells that had entered S phase did not block DNA synthesis, suggesting that the effect of the injected phosphatases on cell cycle arrest was specific. In vitro biochemical studies with purified PP1 and PP2A showed that intact RB protein phosphorylated by cdc2 kinase served as a substrate for both protein phosphatases. Our results suggest that protein phosphatases may be important regulators of RB function and support the idea that cell cycle progression is regulated by the phosphorylation state of the RB protein.
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PMID:Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases. 838 Jun 37

Phosphorylation events are major regulatory mechanisms of signal transduction pathways that regulate gene expression and cell growth. To study the potential involvement of serine-threonine specific phosphatases in these processes we used okadaic acid (OA), an inhibitor of type 1 and type 2A protein phosphatases. Here we present evidence that OA arrests cells at defined points in the cell cycle. Concomitantly, expression and associated histone H1 kinase activity of cdc2 and cyclin A, two cell cycle regulatory proteins, are repressed by this agent. Furthermore, phosphorylation of the tumor suppressor protein retinoblastoma, an event thought to be necessary in order to permit cells to proliferate, is inhibited when OA is present. These effects are fully reversible since removal of OA restores cdc2 and cyclin A expression as well as histone H1 kinase activity, and the cells resume growth. Since cdc2 and cyclin A have previously been shown to be absolutely required for cell cycle progression it is likely that blockage of synthesis of these components contributes to the cytostatic effects of OA. Furthermore, our results suggest a positive role for OA sensitive protein phosphatases in the regulation of expression of these cell cycle regulatory proteins.
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PMID:Inhibition of histone H1 kinase expression, retinoblastoma protein phosphorylation, and cell proliferation by the phosphatase inhibitor okadaic acid. 838 Dec 21

Cyclin A associates with both the p34 cdc2 and p33 cdk2 kinases and is involved at two major check-points (G1-S and G2-M) of the cell cycle. The cyclin has been identified in multimeric protein complexes that incorporate the E2F transcription factor, the p33 cdk2 kinase, and p107, which is related to the retinoblastoma protein. Therefore, cyclin A provides a link between studies on the cell-cycle machinery and those aiming to elucidate the modulation of cell proliferation and regulation of gene expression by oncogenes and growth-suppressor proteins. The modification of cyclin A expression in a human liver cancer by the insertion of hepatitis B viral DNA into the cyclin A gene, and binding of cyclin A to the oncogenic E1A viral protein in adenovirus-infected cells suggest that the cyclin is implicated in human carcinogenesis. In addition, cyclin A might also be considered as a marker for tumor-cell proliferation in oncology. With these views in mind, it is now important to extend these observations to other types of cancer.
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PMID:Oncogenic activation of cyclin A. 838 33

We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.
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PMID:Butyrolactone I, a selective inhibitor of cdk2 and cdc2 kinase. 839 80

Transforming growth factor beta 1 (TGF beta 1) causes G1 growth arrest and the accumulation of unphosphorylated retinoblastoma protein (Rb) in responsive cells. Cdk4 (cyclin-dependent kinase), a major catalytic subunit of the mammalian D-type G1 cyclins, can phosphorylate Rb in vitro, and at least one D-type cyclin, D2, directs the phosphorylation of Rb in vivo. Here we show that TGF beta 1 induces suppression of cdk4 synthesis in G1 in mink lung epithelial cells. Constitutive cdk4 synthesis in these cells led to TGF beta 1 resistance. It also resulted in growth in low serum medium when these cells were released from contact inhibition. Cdk2 activity was also suppressed by TGF beta 1 action, but its constitutive expression failed to override a TGF beta 1-induced G1 block. Hence, the TGF beta 1 block is primarily mediated by cdk4 modulation. Further evidence suggests that TGF beta 1-induced down-modulation of cdk4 leads to inhibition of cdk2 activation and that both events might contribute to TGF beta 1 growth suppression.
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PMID:TGF beta inhibition of Cdk4 synthesis is linked to cell cycle arrest. 840 78

The transcription factor E2F has been shown to be involved in the expression of several cell cycle-regulated genes, and the activity of this factor is controlled by cellular proteins such as pRB and p107. E2F is also a target of the DNA virus oncoproteins (adenovirus E1A, simian virus 40 T antigen, and human papillomavirus [HPV] E7) (see the review by J. R. Nevins [Science 258: 424-429, 1992]). These viral oncoproteins dissociate an inactive complex between E2F and the retinoblastoma tumor suppressor protein (pRB), and this dissociation of the E2F-pRB complex correlates with a stimulation of the E2F-dependent transcription. In the S phase of the cell cycle, E2F forms a complex with p107, cyclin A, and the cdk2 kinase (E2F-cyclin A complex). The cellular function of this S-phase-specific complex is unclear. The adenovirus E1A protein dissociates the E2F-cyclin A complex. The HPV type 16 (HPV-16) E7 protein, which possesses significant sequence homology with E1A, does not dissociate the E2F-cyclin A complex. We find that the HPV-16 E7 protein associates very efficiently with the E2F-cyclin A complex. This association is dependent on the sequences that are also necessary for the transforming activity of E7. Moreover, the E7 protein of a low-risk HPV (type 6b) is much less efficient in binding to the E2F-cyclin A complex compared with that of the high-risk type. We also find that the E2F-cyclin A complex remains endogenously associated with the E7 protein in extracts of Caski cells, which express high levels of HPV-16 E7 protein. Finally, we have extensively purified the E2F-cyclin A complex from mouse L-cell extracts and show that, in cell extracts, the E2F-cyclin A complex remains associated with other cellular proteins.
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PMID:Association of the human papillomavirus type 16 E7 protein with the S-phase-specific E2F-cyclin A complex. 841 52


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