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)

The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation. We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase II (CK-II) site S111/112. In this study purified p34cdc2 kinase was used to phosphorylate T antigen proteins specifically at T124 and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import. T124 phosphorylation, which could be functionally simulated by a T-to-D124 substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import rate. The inhibition of nuclear import depended on the stoichiometry of phosphorylation. T124 and S111/112 could be phosphorylated independently of one another. Two alternative mechanisms were considered to explain the inhibition of nuclear import by T124 phosphorylation: inactivation of the NLS and cytoplasmic retention, respectively. Furthermore, we speculate that in vivo T124 phosphorylation may regulate the small but functionally significant amount of cytoplasmic SV-40 T antigen. A sequence comparison showed that many transcription regulatory proteins contain domains comprising potential CK-II-sites, cdc2-sites, and NLS. This raises the possibility that the three elements represent a functional unit regulating nuclear protein import.
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PMID:p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins. 165 75

Cyclins, as subunits of the protein kinase encoded by the cdc2 gene are major controlling elements of the eukaryotic cell cycle. The fission yeast Schizosaccharomyces pombe has a B-type cyclin, which is a nuclear protein encoded by the cdc13 gene. Here we demonstrate the presence of two spatially distinct cdc13 cyclin populations in the nucleus of S. pombe, one of which is associated with the mitotic spindle poles. Both populations colocalize with the product of the cdc2 gene (p34cdc2). Treatment of cells with the antimicrotubule drug thiabendazole prevents cyclin degradation and blocks the tyrosine dephosphorylation and activation of cdc2. These results suggest a key regulatory role of the cdc2-cyclin complex in the initiation of mitotic spindle formation and also that mitotic microtubule function is required for cdc2 activation.
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PMID:Distinct nuclear and spindle pole body population of cyclin-cdc2 in fission yeast. 169 36

Numatrin is a nuclear matrix phosphoprotein whose synthesis and abundance were shown to be regulated during the cell cycle in mitogen-stimulated lymphocytes (Feuerstein, N., and Mond, J. (1987) J. Biol. Chem. 262, 11389-11397). We examined the effect of (a) CTD-kinase, which contains the cdc2 catalytic component (p34) in a complex with a p58 subunit (cdc2/p58) and (b) the M phase-specific histone H1 kinase, which contains the cdc2 kinase in association with a p62 subunit (cdc2/p62), on phosphorylation of numatrin. We show that both cdc2 kinase complexes can phosphorylate numatrin. However, cdc2/p58 at conditions that caused a similar effect to cdc2/p62 on phosphorylation of histone H1 (dpm/micrograms of substrate/micrograms of enzyme) was found to have a 5-25-fold higher catalytic activity in the phosphorylation of numatrin. Analysis of the tryptic phosphopeptide map of numatrin phosphorylated by these cdc2 kinase complexes showed that both kinase complexes phosphorylated two major identical peptides, but minor additional peptides were differentially phosphorylated by each of these kinases. This indicates that under certain experimental conditions cdc2/p58 and cdc2/p62 may express some differences in their catalytic activity. In vitro phosphorylation by CTD kinase of a whole nuclear protein extract from murine fibroblasts showed that numatrin is the most prominent substrate for CTD kinase in this nuclear extract. CTD kinase cdc2/p58 was found to induce significantly the phosphorylation of five other discrete nuclear substrates. Particularly, two nuclear proteins at 75 kDa/pI approximately 6.5 and 85 kDa/pI approximately 5.3, which were not Coomassie Blue stainable, were found to be markedly phosphorylated by CTD kinase. The results of this study call for further study of the role of CTD kinase cdc2/p58 in the phosphorylation of numatrin under physiological conditions and to further characterization of the other nuclear substrates for CTD kinase.
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PMID:Phosphorylation of numatrin and other nuclear proteins by cdc2 containing CTD kinase cdc2/p58. 187 52

The retinoblastoma gene product (RB) is a nuclear protein which has been shown to function as a tumor suppressor. It is phosphorylated from S to M phase of the cell cycle and dephosphorylated in G1. This suggests that the function of RB is regulated by its phosphorylation in the cell cycle. Ten phosphotryptic peptides are found in human RB proteins. The pattern of RB phosphorylation does not change from S to M phases of the cell cycle. Hypophosphorylated RB prepared from insect cells infected with an RB-recombinant baculovirus is used as a substrate for in vitro phosphorylation reactions. Of several protein kinases tested, only cdc2 kinase phosphorylates RB efficiently and all 10 peptides can be phosphorylated by cdc2 in vitro. Removal of cdc2 from mitotic cell extracts by immunoprecipitation causes a concomitant depletion of RB kinase activity. These results indicate that cdc2 or a kinase with similar substrate specificity is involved in the cell cycle-dependent phosphorylation of the RB protein.
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PMID:Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase. 200 61

Type beta transforming growth factors represent a family of polypeptides that modulate growth and differentiation. They exert their effect on target cells through interaction with multiple cell surface receptors. Transforming growth factor-beta 1 has a strong inhibitory action on cell division in mink lung CC164 cells, a process that is initiated by immediate induction of junB and phosphorylation of nuclear protein followed by a reduced expression of cdk4. However, its signal transduction pathways are still unresolved. In this study we report a detailed analysis of cell kinetic events following addition of transforming growth factor-beta 1 to mink lung CCL64 cells. We show that transforming growth factor-beta 1 reduces [3H]thymidine incorporation after a delay of 8 hours, which reaches its nadir at 16 hours. The reduced growth rate is maintained for at least 48 hours as shown by flow cytometric analysis of DNA content. Using time-lapse video microscopy it was shown that control cells double on average every 14.4 hours, whereas the transforming growth factor-beta 1-treated cells have a doubling time of on average 20.3 hours. The difference in intermitotic time is a consequence of a prolonged G1 phase (a shift from 7.5 to 13.5 hours on average). However, changes in intermitotic times occur only after cells have undergone division in the presence of transforming growth factor-beta 1 and treated cells finish the ongoing cell cycle exactly like control cells. From these findings we conclude that transforming growth factor-beta 1 may change cell cycle parameters by interfering with cellular events prior to G1.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Initiation of growth inhibition by TGF beta 1 is unlikely to occur in G1. 770 98

Terminally differentiated skeletal muscle myotubes are arrested in G0 phase of the cell cycle and are unable to be released from this arrest by stimulation with mitogens including serum and growth factors. To inspect a possibility of reversing the quiescence at the G0 phase, we have exploited the mouse skeletal muscle cell line C2SVTts11, which is a clone of C2 cells transfected with the SV40 T antigen gene (encoding thermolabile large T and wild-type small t) fused to an inducible promoter. When the large T is induced in the myotubes, the terminally differentiated cells reenter the cell cycle and proceed to S and M phases. To elucidate how large T forces the myotubes to traverse each phase of the cell cycle, we examined the expression and activity of Cdk2 and Cdc2, which in complex with cyclin A and cyclin B are essential for S and M phases, respectively in undifferentiated cells. The levels of their mRNAs and proteins and histone H1 kinase activity, which was ascribed to Cdc2-cyclin B, were high in the proliferating myoblasts but gradually decreased during terminal differentiation. In contrast, they were reinduced in the myotubes reentering the cell cycle. Stimulation of the myotubes with serum failed to evoke these factors. These results indicate that large T, but not mitogens, is able to drive terminally differentiated myotubes to pass each phase of the cell cycle through eliciting these factors as do mitogens on proliferating undifferentiated cells. Since large T is a nuclear protein, signals generated by the protein in the nucleus are likely to be sufficient to induce each phase of the cell cycle in the terminally differentiated cells.
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PMID:SV40 large T antigen reinduces the cell cycle in terminally differentiated myotubes through inducing Cdk2, Cdc2, and their partner cyclins. 808 30

The mammalian nuclear protein E2F-1 has recently been cloned based on its ability to bind the retinoblastoma protein. To determine whether E2F-1 plays a role in the control of the cell proliferation, we introduced an inducible construct expressing an E2F-1 antisense RNA into the human glioblastoma T98G cell line and assessed DNA synthesis during the cell cycle. Expression of the antisense transcripts during the G1-S transition resulted in a marked delay in the completion of DNA synthesis. Band-shift analysis of bacterially produced E2F-1 showed that this protein bound to the promoters of human DNA polymerase-alpha, cyclin D1, and c-myb but not to the cdc2 gene promoter. E2F-1 also transactivated the bound promoters in transient transfection assays. These results suggest a major role for E2F-1 in the control of cell cycle progression via transcriptional regulation of proliferation-associated genes.
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PMID:Correlation between E2F-1 requirement in the S phase and E2F-1 transactivation of cell cycle-related genes in human cells. 813 37

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 wee1 tyrosine kinase and cdc25 tyrosine phosphatase of fission yeast play antagonistic roles in the induction of mitosis through cdc2 regulation. We show here that the human wee1-like tyrosine kinase is a nuclear protein that ensures the completion of DNA replication prior to mitosis in cells expressing otherwise catastrophic levels of cdc2 activators. Paradoxically, wee1-rescued cells display very high levels of mitotic cdc2 kinase activity. We account for this anomaly by our observation that the cdc2 activator, cdc25C, is a cytoplasmic protein that, like cyclin B1, enters the nucleus at the G2/M transition. Thus, cdc2 is likely to be activated in the cytoplasm and requires nuclear localization to initiate both cytoplasmic and nuclear mitotic transformations. The human wee1 kinase appears to coordinate the transition between DNA replication and mitosis by protecting the nucleus from this cytoplasmically activated cdc2 kinase.
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PMID:Human wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase. 834 13

The regulation of nuclear protein transport by phosphorylation plays a central role in gene expression in eukaryotic cells. We previously showed that nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is regulated by the CcN motif, comprising phosphorylation sites for casein kinase II and the cyclin-dependent kinase cdc2, together with the nuclear localization signal. Regulation of nuclear uptake by CcN motif kinase sites also holds true for the yeast transcription factor SWI5 and the Xenopus nuclear phosphoprotein nucleoplasmin. To test directly whether a kinase site other than those of the CcN motif could regulate nuclear import of T-ag, the CcN motif casein kinase II site, which markedly increases the rate of T-ag nuclear import, was replaced by a consensus site for the cAMP-dependent protein kinase (PK-A) using site-directed mutagenesis. The resultant fusion protein could be specifically phosphorylated by PK-A in vitro and in cell extracts. Nuclear import of the fluorescently labeled protein was analyzed in the HTC rat hepatoma cell line both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in the presence and the absence of cAMP and/or PK-A catalytic subunit using confocal laser scanning microscopy. In vitro PK-A-prephosphorylated protein was also tested. All results indicated that the rate of nuclear import was increased by phosphorylation at the PK-A site (2-5-fold), demonstrating that kinases other than those of the CcN motif can regulate nuclear import in response to stimulatory signals. The phosphorylation-regulated nuclear localization signal derived here represents an important first step toward developing a signal conferring inducible nuclear targeting of molecules of interest.
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PMID:A consensus cAMP-dependent protein kinase (PK-A) site in place of the CcN motif casein kinase II site simian virus 40 large T-antigen confers PK-A-mediated regulation of nuclear import. 862 46


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