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1.
We determined the mRNA levels and the activities in nuclear and non-nuclear fractions of protein phosphatase type 1 (PP1) and type 2A (PP2A) through the cell cycle in synchronized mouse NIH3T3 fibroblasts. The mRNA level for PP1 alpha was gradually elevated in late G1 phase, began to decrease in M phase, and reached the control level with entering into the next G1 phase. The mRNA level for PP2A was rapidly increased in early G1 phase, kept at the high level, and decreased after S phase. In nuclear fractions of cells, spontaneous activities of both PP1 and PP2A were gradually increased until M phase and rapidly decreased with entering the next G1 phase, while in non-nuclear fraction such dramatic alterations were not observed. Potential activities of PP1 in both fractions revealed by Co(2+)-trypsin treatment showed an oscillaion patterns similar to those of the spontaneous activities. These results strongly suggest that cell cycle dependent gene expressions and activities of PP1 and PP2A play roles in DNA synthesis and mitosis during the cell cycle.  相似文献   

2.
Nuclear and cytoplasmic protein kinases were measured during the traverse of synchronous CHO cultures through G1 into S phase. Cells were synchronized by selective detachment of cells blocked in metaphase using colcemid. Nuclei were isolated and the protein kinases extracted from the nuclear preparation with 0.6 M NaCl. This procedure solubilized greater than 90% of the total protein kinase activity present in the nuclear preparation. DEAE chromatography of this extract showed 5 apparently different ionic forms of nuclear protein kinases. The nuclear protein kinases preferred casein and phosvitin to histone as substrates and were cyclic AMP-independent. Nuclear protein kinase activities increased greater than two-fold, when expressed as units of activity per cell nucleus, during G1 phase traverse, concomitant with a 70% increase in nuclear non-histone proteins (those soluble in 0.6 M NaCl). This resulted in only a 40% increase in the specific activities (units/microgram protein in 0.6 M NaCl extractable nuclear fraction) of these enzymes as cells progressed through G1 into S phase. This was in contrast to cytoplasmic cyclic AMP-dependent protein kinase activities which also increased two-fold during progression through G1 phase while total cellular protein increased less than 20%. Activation of, as well as synthesis of, cyclic AMP-dependent cytoplasmic protein kinases during G1 phase suggests a regulatory mechanism for precise temporal phosphorylation, whereas the constant specific activity in nuclear kinases during cell cycle is more compatible with the maintenance of bulk phosphorylation processes in the nucleus.  相似文献   

3.
The change in activity of nuclear poly(ADP-ribose) glycohydrolase during the cell cycle of HeLa S3 cells was investigated. The poly(ADP-ribose) glycohydrolase activity was solubilized from HeLa S3 cell nuclei and chromosomes only by sonication at high ionic strength. The enzyme hydrolyzed poly(ADP-ribose) exoglycosidically, producing ADP-ribose. After release from mitosis, the activity of the solubilized nuclear poly(ADP-ribose) glycohydrolase per nucleus or per unit protein, assayed with [3H]poly(ADP-ribose) (average chain length, n = 15) as substrate, was lowest in the early G1 phase and highest in the late G1 phase. The specific activity in the late G1 phase was about two times that in the early G1 phase. The high activity remained constant during the S-G2-M phase. A similar change during the cell cycle was observed after release from hydroxyurea block. These results suggest that the activity of poly(ADP-ribose) glycohydrolase doubled during the G1 phase of the cell cycle of HeLa S3 cells.  相似文献   

4.
5.
The Leydig I-10 tumor cell line was synchronized by the double thymidine block method using 1.0 mM thymidine. Protein phosphokinase activity of subcellular fractions was determined at various times throughout the cell cycle. Microsomal cAMP-independent kinase activity increased in G2 and decreased during the S and G1 phases. Except for relatively small increases during the G1 and late S phases, microsomal cAMP-dependent kinase activity remained unchanged throughout most of the cycle. In the lysosomal-mitochondrial fraction, cAMP-dependent and cAMP-independent protein kinase activity increased during the S phase. Independent kinase activity peaked again during G1, while the dependent kinase became depressed. Phosphokinase activity increased in the nuclear fraction in late G2 and during mitosis, and was due to increases in both cAMP-independent and cAMP-dependent kinase activity. Cytosol cAMP-dependent kinase activity increased in G2 and during mitosis; cAMP-independent kinase activity showed some increased activity during late G2 and mitosis. These temporal variations in the subcellular kinase activities throughout the cell cycle may act to phosphorylate subcellular protein substrates in a cell cycle-specific fashion.  相似文献   

6.
To ascertain the activity and substrate specificity of nuclear protein kinases during various stages of the cell cycle of HeLa S3 cells, a nuclear phospho-protein-enriched sample was extracted from synchronised cells and assayed in vitro in the presence of homologous substrates. The nuclear protein kinases increased in activity during S and G2 phase to a level that was twice that of kinases from early S phase cells. The activity was reduced during mitosis but increased again in G1 phase. When the phosphoproteins were separated into five fractions by cellulose-phosphate chromatography each fraction, though not homogenous, exhibited differences in activity. Variations in the activity of the protein kinase fractions were observed during the cell cycle, similar to those observed for the unfractionated kinases. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the proteins phosphorylated by each of the five kinase fractions demonstrated a substrate specificity. The fractions also exhibited some cell cycle stage-specific preference for substrates; kinases from G1 cells phosphorylated mainly high molecular weight polypeptides, whereas lower molecular weight species were phosphorylated by kinases from the S, G2 and mitotic stages of the cell cycle. Inhibition of DNA and histone synthesis by cytosine arabinoside had no effect on the activity or substrate specificity of S phase kinases. Some kinase fractions phosphorylated histones as well as non-histone chromosomal proteins and this phosphorylation was also cell cycle stage dependent. The presence of histones in the in vitro assay influenced the ability of some fractions to phosphorylate particular non-histone polypeptides; non-histone proteins also appeared to affect the in vitro phosphorylation of histones.  相似文献   

7.
Cell cycle-dependent regulation of the DNA-dependent protein kinase   总被引:1,自引:0,他引:1  
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8.
T Ooka  J Daillie 《Biochimie》1975,57(2):235-246
We have demonstrated the presence of two DNA polymerases in KB cells and studied the variation of their activities in a synchronous cell population. During the cell cycle we observed in nuclei, only one DNA dependent DNA polymerase, the 3.4 S or minipolymerase, and similarly in the cytoplasm only one enzyme, the 8.3 S or maxipolymerase. The former shows preference for native DNA and the latter for denatured DNA. Their Mg++ and K+ requirements are different and their pH optima are 8.5 and 7 for nuclear polymerase and cytoplasmic polymerase respectively. The cytoplasmic polymerase activity remains stable from one cell cycle to the other with each cell reconstituting its stock at the start of the following cycle (G1 and early S phases). On the contrary nuclear activity decreases in G2, M and early G1, then increases to a maximum in the middle of the S phase. This fluctuation in enzyme activity could be due to degradation, transfer to the cytoplasm or the association of the enzyme with the chromatin and/or the nuclear membrane after completion of DNA synthesis. Our results do not permit us to choose between these three hypotheses. However their significance is discussed in the light of the results obtained by some authors who, on the contrary, have tended to minimise the role of the minipolymerase in DNA duplication, whereas we, from our findings, ascribe a preponderant role to this enzyme. The cytoplasmic maxipolymerase (8.3 S) may simply be a storage form of the enzyme from which minipolymerase can be formed as needed.  相似文献   

9.
Protein kinase CK2 is a ubiquitous protein serine/threonine kinase that is involved in cell growth and proliferation as well as suppression of apoptosis. Several studies have suggested that the kinase plays a role in cell cycle progression; however, changes in enzyme activity during phases of cell cycle have not been detected. Nuclear matrix is a key locus for CK2 signaling in the nucleus. We therefore examined CK2 signaling to the nuclear matrix in distinct phases of cell cycle by employing synchronized ALVA-41 prostate cancer cells. Removal of serum from the culture medium resulted in G0/G1 arrest, and a reduction in the nuclear matrix-associated CK2 activity which was rapidly reversed on addition of serum. Arresting the cells in G(0)/G(1) phase with hydroxyurea and subsequent release to S phase by serum gave similar results. Cells arrested in the G(2)/M phase by treatment with nocodazole demonstrated an extensive reduction in the nuclear matrix-associated CK2 which was reversed rapidly on addition of serum. Changes in the immunoreactive CK2 protein were concordant with the activity data reflecting a dynamic trafficking of the kinase in distinct phases of cell cycle. Under the same conditions, CK2 activity in total cellular lysate remained essentially unaltered. These results provide the first direct evidence of discrete modulations of CK2 in the nuclear matrix during the cell cycle progression. Inducible overexpression of CK2 in CHO cells yielded only a modest increase in CK2 activity even though a significant increase in expression was apparent at the level of CK2 alpha-specific message. Stably transfected ALVA-41 cells, however, did not show a significant change in CK2 levels despite increased expression at the message level. Not surprisingly, both types of the stably transfected cells failed to show any alteration in cell cycle progression. Distribution of the CK2 activity in the cytosolic versus nuclear matrix fractions in normal cells appears to be different from that in the cancer cells such that the ratio of nuclear matrix to cytosolic activity is much higher in the latter. Considering that nuclear matrix is central to several nuclear functions, this pattern of intracellular distribution of CK2 may have implications for its role in the oncogenic process. Published 2003 Wiley-Liss, Inc.  相似文献   

10.
The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size-distributions obtained by 1g sedimentation coincided with cell-cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10?6-10?5 M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1-phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to 3H-thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug-mediated cytolysis.  相似文献   

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