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)

Two different fractions of cdk2 and cdc2 have been found in the nucleus of HeLa cells. One, which can be extracted by nuclease treatment, possibly associated with DNA- or RNA-containing structures and another one, which is bound to the nuclear matrix. Nuclear cdk2 forms high molecular weight complexes which migrate at the same position as DNA polymerase alpha and proliferating cell nuclear antigen in sucrose gradient centrifugation experiments. These results suggest that nuclear cdk2 complexes could be associated with the replication factories. Immunoprecipitation experiments reveal that nuclear cdk2 complexes display histone H1-kinase activity and phosphorylate a protein of 18 kDa which is present in these complexes.
Biochem Biophys Res Commun 1994 Sep 30
PMID:Cyclin/cdk2 complexes in the nucleus of HeLa cells. 794 2

We examined the effect of suramin, an anticancer agent and a functional analog of naturally occuring glycosaminoglycans, on p34cdc2 kinase. We find that suramin strongly inhibits the catalytic activity of purified p34cdc2 kinase (IC50 approximately 4 microM), whereas it only weakly inhibits the p13-agarose precipitated kinase activity from nuclear and cytoplasmic extracts of the asynchronous H69 human small cell lung cancer cells. We also find that the tyrosine phosphorylation of p34cdc2 kinase in the nuclear extract is increased about twice when the extracts are preincubated with 50 microM of suramin prior to the p13-agarose precipitation. We propose that this increase might result from the inhibitory effect of suramin towards p34cdc2-specific tyrosine phosphatases. These results suggest both a direct and an indirect effect of suramin on p34cdc2 kinase. We also find that heparin is a potent inhibitor of purified cdc2 kinase (IC50 approximately 3.5 micrograms/ml). Therefore, glycosaminoglycans might be physiological regulators of p34cdc2 kinase in vivo.
Biochem Biophys Res Commun 1994 Sep 30
PMID:Effect of suramin on p34cdc2 kinase in vitro and in extracts from human H69 cells: evidence for a double mechanism of action. 794 7

The Fos family of transcription factors, c-Fos, FosB, Fra-1 and Fra-2, are rapidly induced in quiescent fibroblasts following serum or growth factor stimulation. The Fos proteins show distinct patterns of expression during cell growth with only Fra-1 and Fra-2 maintained at significant levels in growing cells, suggesting that the different family members direct unique functions for cell growth. Post-translational modification of Fos proteins has been observed following serum stimulation, which may allow an additional level of regulation. Our studies show that the synthesis and post-translational modification of Fra-1 and Fra-2 in Swiss 3T3 cells is serum-dependent during G1 following the transition from G0 and during asynchronous growth but is serum-independent during S phase and mitosis. Post-translational modification of Fra-1 and Fra-2 causes a significant shift in their gel mobility which is eliminated by alkaline phosphatase treatment. Several kinases can phosphorylate Fra-1 and Fra-2 in vitro, including cAMP-dependent kinase (PKA), protein kinase C (PKC), cyclin-dependent kinase 1-cdc2 (cdc2), and mitogen activated protein (MAP) kinase. From these, MAP kinase is the only one that causes a shift in gel mobility similar to that observed in vivo. One dimensional phosphopeptide maps of Fra-1 and Fra-2 phosphorylated by MAP kinase in vitro are similar to those of in vivo labeled Fra-1 and Fra-2, suggesting that MAP kinase may also phosphorylate Fra-1 and Fra-2 in vivo. We have also determined that phosphorylation of Fra-1 and Fra-2 by MAP kinase increases their DNA binding activity.
Oncogene 1994 Sep
PMID:Regulation of Fra-1 and Fra-2 phosphorylation differs during the cell cycle of fibroblasts and phosphorylation in vitro by MAP kinase affects DNA binding activity. 805 17

Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [3H]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression. tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
Oncogene 1994 Sep
PMID:A cyclin-dependent kinase inhibitor, butyrolactone I, inhibits phosphorylation of RB protein and cell cycle progression. 805 18

In this study we have surveyed by immunoblotting the protein levels of Cyclin D1, D2, D3 and their catalytic partners, Cdk4 and Cdk6 in normal and transformed human cells. We found that all these proteins were differentially expressed in diploid cells derived from different tissues, in contrast to Cyclin E, Cyclin A and Cdk2 which are ubiquitously expressed. D-type Cyclins were never dramatically overexpressed and often very poorly expressed in tumor cell lines when compared to the levels in their normal counterparts. In contrast, Cdk4 was expressed at high levels in several tumor cell lines and Cdk6 was ectopically expressed in two sarcoma lines, suggesting a possible involvement of these two Cdks in oncogenesis. Interestingly, low levels of Cyclin D1 and D3 proteins always correlated with functional inactivation of the retinoblastoma gene product (pRb). In cells displaying active pRb, Cyclin D1 was found associated with Cdk4 regardless of whether the p53 gene was wild-type or mutant. Microinjection during G1 of Cyclin D1 anti-sense cDNA or anti-Cyclin D1 antibody in these cells arrested the cell cycle in G1. In cells lacking pRb function, Cyclin D1 was dissociated from Cdk4. Microinjection during G1 of Cyclin D1 antisense cDNA or anti-Cyclin D1 antibody in these cells did not affect G1 progression. These results show that (i) in the absence of pRb, Cyclin D1 is expressed at low levels, is dissociated from Cdk4 and becomes dispensable in G1; (ii) Cyclin D1 needs to be associated with its catalytic subunit, Cdk4, to function as a positive regulator of G1 progression.
Oncogene 1994 Sep
PMID:Differential expression and regulation of Cyclin D1 protein in normal and tumor human cells: association with Cdk4 is required for Cyclin D1 function in G1 progression. 805 30

Using a new series of p53 mutants targeting the conserved regions we have analysed the relationship of various activities of the protein. Mdm-2 and human papillomavirus (HPV) E6, two proteins which interact with and abrogate p53 function, were shown to bind independently. Deletion of the conserved regions of the protein in which most of the naturally occurring mutations are found (boxes II-V) abrogated transcriptional activity and the ability to interact with E6, supporting the importance of this DNA binding domain to these activities. Nevertheless, these mutants retained the ability to interact with mdm2. One mutant, deleted of all the C-terminal sequences, showed loss of mdm2 binding, E6 binding and transcriptional activity. More subtle mutations within the C-terminus of the protein, including alterations of the cdc2 and CKII phosphorylation sites, had no effect on the transcriptional trans-activation, mdm-2 or E6 binding functions, indicating that phosphorylation of these sites is not essential for these activities. Deletion of conserved box I sequences abolished the interaction with mdm-2 without loss of transcriptional activation or transformation suppressor activity, suggesting that mdm-2 is not a downstream effector of p53 function.
Oncogene 1994 Sep
PMID:Interaction of p53 with MDM2 is independent of E6 and does not mediate wild type transformation suppressor function. 805 35

Expression of c-myc with constitutively active mutants of the ras gene results in the cooperative transformation of primary fibroblasts, although the precise mechanism by which these genes cooperate is unknown. Since c-Myc has been shown to function as a transcriptional activator, we have examined the ability of c-Myc and activated Ras (H-RasV-12) to cooperatively induce the promoter activity of cdc2, a gene which is critical for cell cycle progression. Microinjection of expression constructs encoding H-RasV-12 and c-Myc along with a cdc2 promoter-luciferase reporter plasmid into quiescent cells led to an increase in cdc2 promoter activity approximately 30 h after injection, a period which coincides with the S-to-G2/M transition in these cells. Expression of H-RasV-12 alone weakly activated the cdc2 promoter, while expression of c-Myc alone had no effect. Mutants of c-Myc lacking either the leucine zipper dimerization domain or the phosphoacceptor site Ser-62 could not cooperate with H-RasV-12 to induce the cdc2 promoter. These mutants also lacked the ability to cooperate with H-RasV-12 to stimulate DNA synthesis. Deletion analysis identified a distinct region of the cdc2 promoter which was required for c-Myc responsiveness. Taken together, these observations suggest a mechanistic link between the molecular activities of c-Myc and Ras and induction of the cell cycle regulator Cdc2.
Mol Cell Biol 1994 Sep
PMID:c-Myc cooperates with activated Ras to induce the cdc2 promoter. 806 6

We used targeted homologous recombination to disrupt one c-myc gene copy in a diploid fibroblast cell line and found that a twofold reduction in Myc expression resulted in lower exponential growth rates and a lengthening of the G0-to-S-phase transition (M. Shichiri, K. D. Hanson and J. M. Sedivy, Cell Growth Differ. 4:93-104, 1993). Myc is a transcription factor, and the number of target genes whose regulation could result in differential growth rates may be very large. We have approached this problem by examining effects of reduced c-myc expression in three broad areas: (i) secretion of growth factors, (ii) expression of growth factor receptors, and (iii) intracellular signal transduction between Myc and components of the intrinsic cell cycle clock. We have found no evidence that differential medium conditioning can account for the growth phenotypes. Likewise, the expression of receptors for platelet-derived growth factor, epidermal growth factor, basic fibroblast growth factor, and insulin-like growth factor I was the same in diploid and heterozygous cells (platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, and insulin-like growth factor are the sole growth factors required by these cells for growth in serum-free medium). In contrast, expression of cyclin E, cyclin A, and Rb phosphorylation were delayed when quiescent c-myc heterozygous cells were stimulated to enter the cell cycle. Expression of cyclin D1, cyclin D3, and Cdk2 was not affected. The timing of cyclin E induction was the earliest observable effect of reduced Myc expression. Our data indicate that Myc contributes to regulation of proliferation by a cell-autonomous mechanism that involves the modulation of cyclin E expression and, consequently, progression through the restriction point of the cell cycle.
Mol Cell Biol 1994 Sep
PMID:Effects of c-myc expression on cell cycle progression. 806 9

Cytoplasmic poly(A) elongation is one mechanism that regulates translational recruitment of maternal mRNA in early development. In Xenopus laevis, poly(A) elongation is controlled by two cis elements in the 3' untranslated regions of responsive mRNAs: the hexanucleotide AAUAAA and a U-rich structure with the general sequence UUUUUAAU, which is referred to as the cytoplasmic polyadenylation element (CPE). B4 RNA, which contains these sequences, is polyadenylated during oocyte maturation and maintains a poly(A) tail in early embryos. However, cdk2 RNA, which also contains these sequences, is polyadenylated during maturation but deadenylated after fertilization. This suggests that cis-acting elements in cdk2 RNA signal the removal of the poly(A) tail at this time. By using poly(A) RNA-injected eggs, we showed that two elements which reside 5' of the CPE and 3' of the hexanucleotide act synergistically to promote embryonic deadenylation of this RNA. When an identical RNA lacking a poly(A) tail was injected, these sequences also prevented poly(A) addition. When fused to CAT RNA, the cdk2 3' untranslated region, which contains these elements, as well as the CPE and the hexanucleotide, promoted poly(A) addition and enhanced chloramphenicol acetyltransferase activity during maturation, as well as repression of these events after fertilization. Incubation of fertilized eggs with cycloheximide prevented the embryonic inhibition of cdk2 RNA polyadenylation but did not affect the robust polyadenylation of B4 RNA. This suggests that a maternal mRNA, whose translation occurs only after fertilization, is necessary for the cdk2 deadenylation or inhibition of RNA polyadenylation. This was further suggested when poly(A)+ RNA isolated from two-cell embryos was injected into oocytes that were then allowed to mature. Such oocytes became deficient for cdk2 RNA polyadenylation but remained proficient for B4 RNA polyadenylation. These data show that CPE function is developmentally regulated by multiple sequences and factors.
Mol Cell Biol 1994 Sep
PMID:Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development. 806 20

RPA is a cellular, three-subunit, single-stranded (ss) DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post-mitotically on the decondensing chromosomes into numerous subnuclear pre-replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assembly of the preRCs. In contrast, the active cdk2-cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single-strand-specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction.
EMBO J 1994 Sep 01
PMID:Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts. 807 11


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