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)

The genes that encode human cdc2 and cdk2 proteins are essential for cell cycle progression. In this report, we describe the purification of cyclin-associated cdc2 and cdk2 kinases as well as cyclin-free cdc2 and cdk2 protein preparations from HeLa cells. The cdc2-cyclin B kinase complex that we have isolated, consisting of two polypeptides of p60 (cyclin B) and p34 (cdc2), phosphorylated both the p34 and p70 subunits of the three-subunit human single-stranded DNA-binding protein (also called RP-A), a DNA replication and repair factor. We also partially purified a histone H1 kinase activity that is associated with the cdk2 and cyclin A proteins. Purified human cyclins A and B1, overproduced in bacteria, complemented a cellular fraction enriched in cdc2 and cdk2 proteins to reconstitute histone H1 kinase activity. Using this complementation system, human cdc2 and cdk2 proteins were purified and separated from one another. Glycerol gradient analyses demonstrated that the purified cdk2 (p33) protein co-sedimented with a cyclin A-dependent H1 kinase activity. Thus, cdk2 and cyclin A proteins are components that assemble to yield a kinase complex that catalyzes the phosphorylation of histone H1.
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PMID:Reconstitution of cyclin-dependent cdc2 and cdk2 kinase activities in vitro. 839 6

Human cyclins A and B1 were assembled with the cdk2 or cdc2 protein to reconstitute their respective kinase activities in vitro. Both cyclins complemented either cdk2 or cdc2, yielding kinase activities that supported the phosphorylation of histone H1. Activation of cdk2-catalyzed H1 kinase activity by cyclin A required a 10-min preincubation of the two components, whereas cdc2 kinase supported phosphate incorporation without a detectable time lag upon the addition of cyclin B1, suggesting a slower association rate of cdk2 with cyclin A compared with cdc2 and cyclin B1. Both cdk2 and cyclin A, as well as cdc2 and cyclin B1, formed stable complexes in the absence of ATP and substrate that could be isolated after glycerol gradient centrifugation. Incubation of the isolated complexes with ATP and histone H1 supported the phosphorylation of the substrate. Cyclin A-activated cdk2 or cdc2 phosphorylated p107, a pRB-related cellular protein, 10 times more effectively than the cyclin B1-complexed kinases. This was most likely due to a direct association of cyclin A with p107 (Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. (1992) Science 255, 85-87; Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D., and Harlow, E. (1992) Science 255, 87-90). The reconstituted cdc2-cyclin B1 complex incorporated 4-5-fold more phosphate into the p34 subunit of the three-subunit (p70, p34, and p14) human single-stranded DNA-binding protein (also called RP-A), a DNA replication and DNA repair factor, than cdc2-cyclin A. No detectable phosphorylation of the p34 protein was observed with cdk2 complexed with either cyclin B1 or A. These data indicate that both cyclins as well as the catalytic subunits are important factors in controlling the rate of phosphorylation of a given substrate. The cyclin-activated cdc2 family kinases may target their cellular substrates through cyclin-mediated protein-protein interactions.
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PMID:Characterization of the in vitro reconstituted cyclin A or B1-dependent cdk2 and cdc2 kinase activities. 839 7

A serine/threonine kinase from bovine brain has been purified (Lew, J., Beaudette, K. N., Litwin, C. M. E., and Wang, J. H. (1992) J. Biol. Chem. 267, 13383-13390) and found to consist of a 33-kDa catalytic subunit having high sequence homology to p34cdc2 and cdk2 (Lew, J., Winkfein, R. J., Paudel, H., and Wang, J. H. (1992) J. Biol. Chem. 267, 25922-25926). Substrate specificity determinants for this cdc2-like kinase were examined using synthetic peptide substrates derived from the in vitro p34cdc2 phosphorylation sites of histone H1. The peptide P-K-T-P-K-K-A-K-K-L was found to be an excellent substrate for the bovine cdc2-like kinase, having a Km value in the micromolar range. Important determinants for efficient substrate phosphorylation of this peptide were found both within the proposed substrate consensus motif (S/T-P-X-K/R) of p34cdc2 kinase and outside of this sequence. In addition to the absolute requirement for a proline residue immediately COOH-terminal to the phosphorylatable residue (+1) and a basic residue at the +3 position, a basic amino acid at the +2 position was greatly preferred over an acidic amino acid. A proline residue at the -2 position and a cluster of basic amino acids further COOH-terminal to the consensus motif were also found to be important for substrate binding. HeLa cell p34cdc2 kinase displays similar specificity to that of the bovine cdc2-like kinase, as the additional determinants outside of the consensus motif that contribute to the efficient phosphorylation of the histone peptide by this novel enzyme also appear to be important for p34cdc2-catalyzed phosphorylation.
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PMID:Substrate specificity characterization of a cdc2-like protein kinase purified from bovine brain. 840 12

The p34CDC2 protein kinase is a key component in the regulation of the eukaryotic cell cycle. We have isolated from the protozoan parasite Leishmania mexicana mexicana a CDC2-related kinase gene (Lmmcrk1) encoding a 34-kDa protein kinase (lmmCRK1) which has 56% amino acid identity with the human CDC2 and contains a PCTAIR motif in place of the highly conserved PSTAIR box. lmmCRK1 was detected in all life cycle stages at comparable levels, yet its histone H1 kinase activity was detected in only the promastigote form, indicating that its activity is stage-regulated at a post-translational level. lmmCRK1 did not bind p13suc1 beads and Lmmcrk1 was unable to complement a fission yeast temperature-sensitive cdc2 mutant. These data suggest that Lmmcrk1 is unlikely to be the functional L. mexicana cdc2 homologue. A distinct histone H1 kinase activity that binds p13suc1 beads (SBCRK) was also detected, with activity that correlated with the division status of the developmental forms of the parasite, being present in the dividing stages of the parasite and absent in nondividing metacyclic forms. SBCRK is a candidate for the functional CDC2 homologue, but it does not react with an anti-PSTAIR monoclonal antibody on Western blots when eluted from p13suc1 beads, indicating a divergent PSTAIR box. These data suggest that a family of CDC2-related protein kinases are present in Leishmania. Some share sequence and biochemical properties with CDC2, but significant differences also exist, possibly reflecting the evolutionary distance between Leishmania and higher eukaryotes.
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PMID:A novel CDC2-related protein kinase from Leishmania mexicana, LmmCRK1, is post-translationally regulated during the life cycle. 840 41

In serum-deprived human fibroblasts IMR-90 and WI-38 cells, the addition of fetal calf serum or basic fibroblast growth factor stimulates DNA synthesis in an extracellular Ca(2+)-dependent manner; the effect of serum on [3H]thymidine incorporation into DNA is 4-16-fold greater at 2.0 mM CaCl2 as compared with that at 0.03 mM CaCl2. By contrast, in SV40 virus-transformed WI-38 (SV-WI-38) cells DNA synthesis is essentially independent of the extracellular calcium concentration ([Ca]out) and serum growth factors. To explore the role of Ca2+ in mitogenic signal transduction through G1 to S phase cell cycle progression, we studied and compared the effect of [Ca]out on phosphorylation of RB protein, the product of a tumor suppressor retinoblastoma gene. In IMR-90 and WI-38 cells, serum or basic fibroblast growth factor induces an increase in the amount of hyperphosphorylated forms of RB protein in a manner strictly dependent on [Ca]out. In sharp contrast, in SV-WI-38 cells, the extent of RB phosphorylation is little affected by [Ca]out or the presence or absence of serum growth factors. In addition, potent calmodulin antagonists W-7 and calmidazolium, but not an inactive analogue W-12 or W-5, strongly inhibit serum-induced increases in DNA synthesis and RB phosphorylation in IMR-90 and WI-38 cells, whereas in SV-WI-38 cells, the inhibitory effect is much more limited. Under the same treatment conditions, we measured histone H1 kinase activity associated with anti-p34cdc2 immunoprecipitate and found that the serum-induced increase in p34cdc2 kinase activity is strongly dependent on [Ca]out and is potently inhibited by the active calmodulin antagonists in IMR-90 and WI-38 cells, but not in SV-WI-38 cells. In IMR-90 cells that have been incubated with serum in 0.03 mM [Ca]out for 24 h, restoration of [Ca]out to 2.0 mM results in initiation of DNA synthesis after 13 h and concomitant increases in RB phosphorylation and p34cdc2 histone H1 kinase activity. These results suggest that in human fibroblasts, Ca2+/calmodulin regulates the signaling cascade leading to cdc2 kinase activation, RB protein phosphorylation, and DNA synthesis and that this Ca(2+)-dependent regulation is abrogated in SV40-transformed cells.
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PMID:Ca(2+)-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts. 841 21

Biological activities of many of the eukaryotic DNA replication proteins are modulated by protein phosphorylation. Investigations of the phosphorylation of adenovirus DNA polymerase (AdPol) have been difficult mainly because of its low level of synthesis in adenovirus-infected HeLa cells. However, when AdPol was overproduced using the recombinant vaccinia virus (RV-AdPol) and the baculovirus expression systems, or by a large scale metabolic labeling of adenovirus 2-infected HeLa cells (native AdPol), in vivo phosphorylation of AdPol could be demonstrated. Phosphoamino acid analysis of [32P]AdPol indicated the presence of phosphoserine independent of the source of AdPol. Comparison of tryptic peptide maps of native AdPol and RV-AdPol revealed that the majority of phosphopeptides were common. Fractionation by high performance liquid chromatography and sequencing of one of the major phosphopeptides revealed serine 67 as a site of phosphorylation. Interestingly, this site is located close to the nuclear localization signal of AdPol and has a consensus substrate recognition sequence for histone H1 (cdc2-related) kinases and mitogen-activated protein kinases. Dephosphorylation of AdPol with calf intestinal alkaline phosphatase resulted in significant decrease in its activity in the in vitro DNA replication initiation assay, suggesting that phosphorylation is important for its biological activity.
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PMID:Adenovirus DNA polymerase is a phosphoprotein. 841 49

RNA polymerase II is a multisubunit enzyme composed of two large subunits of molecular weight in excess of 100,000 and a collection of 8-10 smaller subunits. The largest subunit, designated IIa, contains at its carboxyl terminus a highly repetitive domain consisting of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Extensive phosphorylation within this COOH-terminal domain (CTD) gives rise to subunit IIo which has a markedly reduced mobility in SDS-polyacrylamide gel electrophoresis (PAGE) relative to subunit IIa. Recent evidence suggests that RNA polymerase IIA, containing an unphosphorylated CTD, is involved in preinitiation complex assembly, whereas RNA polymerase IIO is involved in elongation. Consequently, CTD phosphorylation is thought to occur after RNA polymerase II has bound to the promoter by a protein kinase that stably associates with the preinitiation complex. We present here the partial purification and characterization of two distinct CTD kinases from a HeLa cell transcription extract. These CTD kinases, designated CTDK1 and CTDK2, are fractionated by chromatography on Mono Q. CTDK1 catalyzes the incorporation of approximately 33 pmol of phosphate/pmol of calf thymus RNA polymerase subunit IIa, almost exclusively on serine. CTDK2 catalyzes the incorporation of approximately 50 pmol of phosphate/pmol of calf thymus subunit IIa, predominantly on serine; appreciable phosphate transfer onto threonine is also observed. Phosphorylation by CTDK2, but not CTDK1, results in a complete mobility shift in SDS-PAGE of subunit IIa to the position of IIo. CTDK1 can utilize ATP, dATP, or GTP as phosphate donor, whereas CTDK2 can utilize only ATP or dATP. The apparent Km for ATP is 30 microM for CTDK1 and 60 microM for CTDK2. CTDK1 and CTDK2 also differ in their protein substrate specificity. CTDK1 phosphorylates casein whereas CTDK2 does not. Neither kinase phosphorylates phosvitin or histone H1 to an appreciable extent. CTDK1 and CTDK2 do not appear to be related to cdc2 kinases as determined by their inability to phosphorylate H1 and their failure to react with antibodies directed against the cdc2 kinase. These results establish that a partially fractionated HeLa transcription extract contains two distinct CTD kinases that differ in their nucleotide requirements and in their patterns of CTD phosphorylation.
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PMID:Partial purification and characterization of two distinct protein kinases that differentially phosphorylate the carboxyl-terminal domain of RNA polymerase subunit IIa. 841 77

In the cell cycle of fission and budding yeast, the p34cdc2/CDC28 kinase is required for both the G1-to-S and G2-to-M phase transitions. In vertebrates, the homologous p34cdc2 kinase is required for G2-to-M phase transitions but appears to be dispensable for DNA synthesis. We have investigated the function of a related kinase, p33cdk2, using serum-stimulated quiescent human fibroblasts. While the p33cdk2 protein was expressed at constant levels throughout the cell cycle, p33cdk2 kinase activity was first detected a few hours prior to the onset of DNA synthesis. Microinjection of anti-p33cdk2 antibodies blocked cells from entering S phase. Pre-adsorption of these antibodies with cdk2 protein abrogated their blocking effect suggesting that the G1 arrest caused by these antibodies is cdk2-specific. These results indicate that p33cdk2 is required for the G1-to-S phase transition in mammalian cells. We also show evidence to suggest that the cyclin E/p33cdk2 complex is likely to be required for entry into S phase since the timing of the cyclin E-associated kinase activity was coincident with that of p33cdk2 and preclearing of either component abolished the majority of the histone H1 kinase activity present in the lysates harvested from the late G1.
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PMID:The cdk2 kinase is required for the G1-to-S transition in mammalian cells. 850 82

Mammalian cultures primarily regulate cell cycle traverse during G1. For progression through G1 and commitment to DNA synthesis, the activity of a family of proteins, the cyclin-dependent kinases (cdks), is required. There are two primary regulatory portions of G1: (a) the G0-G1 transition, which allows entry into G1; and (b) the G1-S transition, promoting entry to DNA synthesis and commitment to cell division. In the present manuscript, we provide evidence for cross-talk between these two cell cycle transitions. Extracts prepared from quiescent mouse mammary epithelial cells are shown to act in a dominant manner to specifically inhibit the histone H1 kinase activity of preformed/active cdk2, cdk4, cyclin A, or cyclin E complexes from G1-S cell extracts. The inhibitory activity arises as cells enter quiescence and decreases once cultures are stimulated to begin G1 traverse and endogenous cdk activity becomes evident. This activity is associated with the regulated binding of the cdk inhibitor p27Kip1 to cyclin A/cdk2 kinase complexes upon mixing of the extracts. Removal of p27Kip1 from the quiescent cell extract specifically abolishes the inhibitory effect. The inhibitory activity and p27Kip1 binding in vitro depend on incubation of the extracts at physiological temperature or the presence of a reducing agent. The results suggest an interplay between the acquisition of quiescence, cdk activity, and G1 traverse.
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PMID:Conditional binding to and cell cycle-regulated inhibition of cyclin-dependent kinase complexes by p27Kip1. 854 20

7-Hydroxystaurosporine (UCN-01) is a potent inhibitor of protein kinase C (PKC) isozymes alpha, beta, and gamma [Seynaeve et al., Mol. Pharmacol, 45: 1207-1214, 1994] that also has antitumor effects in vivo. To determine whether inhibition of PKC can be related to inhibition of cell growth with induction of apoptosis, we compared the effects of UCN-01 to those of the highly selective bisindolylmaleimide PKC antagonist GF 109203X in leukemic T-cell lines. Both compounds potently inhibited PKC activity when added to T-cell membrane preparations and reversed phorbol ester-induced c-fos gene expression in intact cells. However, whereas UCN-01 potently inhibited growth of Jurkat, Molt-3, Molt-4, and Hut-78 cells (IC50 = 20-65 nM, irreversible after 24 h of exposure), GF 109203X had IC50s for cell growth of 3.6-5.0 muM. Less than 3 h after addition, UCN-01 but not GF 109203X-treated cells displayed loss of cells with G2-M DNA content, appearance of a hypodiploid DNA fraction, and evidence of internucleosomal DNA fragmentation. Six h after treatment, cells appeared to accumulate with S-phase DNA content. These effects correlated with selective UCN-01 but not GF 109203X-induced decrease in total and tyrosine phosphorylation of cyclin-dependent kinases (cdks) 1 and 2, and with increases in the histone H1 kinase activities of cdk1 and cdk2. UCN-01 was relatively less potent in inhibition of properly activated cdk1 and cdk2 when added in vitro to H1 kinase assays (IC50 = 1000 and 600 nM, respectively). We conclude that inhibition of PKC alone is not sufficient to account for the actions of UCN-01 and are led to the hypothesis that inappropriate cdk activation either correlates with or actually mediates cell growth inhibition with apoptosis in T lymphoblasts exposed to UCN-01.
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PMID:Apoptosis in 7-hydroxystaurosporine-treated T lymphoblasts correlates with activation of cyclin-dependent kinases 1 and 2. 854 21


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