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)

Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.
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PMID:MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts. 883 8

Treatment of cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, was found to inhibit the proliferation of mouse fibroblast LP1-1 cells and the p34cdc2 kinase activity of M-phase promoting factor (MPF). However, it showed relatively little effect on expression of the cyclin B1 and cdc2 genes. On the other hand, when the nuclear extracts obtained from the cells at early G2 phase were treated with cAMP analogs, the kinase activity was significantly decreased as compared to the untreated control. Furthermore, the inhibitory effect of cAMP analogs could be reversed upon treating with okadaic acid even in the presence of the cAMP analogs, implying that cdc25 remains in an active form. In addition, the treatment of okadaic acid stimulated the cell progression. These results suggest that down-regulation of MPF activity through protein kinase A-mediated pathway is under post-translational control and cdc25 activation pathway involving okadaic acid-sensitive phosphatase play a role in the regulation of MPF activity.
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PMID:Regulation of the activity of M-phase promoting factor through protein kinase A-mediated pathway in LP1-1 cells. 886 16

EBNA-LP is a viral nuclear oncoprotein implicated in the immortalization of B lymphocytes by Epstein-Barr virus. An analysis of EBNA-LP migration on polyacrylamide gels was performed with protein derived from the X50-7 lymphoblastoid cell line blocked by hydroxyurea or aphidicolin at the G1/S phase of the cell cycle or by nocodazole at the G2/M phase. More slowly migrating species of EBNA-LP were detected in G2/M phase-arrested cell extracts. Release from nocodazole G2/M block or treatment with phosphatase caused the more slowly migrating species of EBNA-LP to disappear. Analyses of 32PO(4)(3-)-labeled EBNA-LP protein immunoprecipitated from the drug-synchronized cells showed that phosphorylated EBNA-LP was present throughout the cell cycle but that phosphorylation increased in G2 and was maximal at G2/M. Phosphoamino acid analysis revealed that all phosphorylation was on serine residues only. The ability of EBNA-LP to be phosphorylated by p34(cdc2) kinase and casein kinase II exclusively on serines implicates these enzymes as being potentially involved in EBNA-LP phosphorylation.
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PMID:Cell cycle stage-specific phosphorylation of the Epstein-Barr virus immortalization protein EBNA-LP. 889 11

The G2-M transition of the cell cycle is triggered by the p34(cdc2)/cyclin B kinase. During the prophase/metaphase transition, the inactive, Thr-14/Tyr-15 phosphorylated form of p34(cdc2) (TP-YP) is modified to an active, Thr-14/Tyr-15 dephosphorylated form (T-Y) by the cdc25 dual-specificity phosphatase. Using highly synchronized starfish oocytes as a cellular model, we show that dephosphorylation in vivo and in vitro occurs in two steps: Thr-14 dephosphorylation precedes Tyr-15 dephosphorylation. The transient intermediate form (T-YP), which can be obtained in vitro by treatment of TP-YP by protein phosphatase 2A, displays low but significant kinase activity. These results raise the possibility that the intermediate form T-YP may be involved in the autocatalytic amplification of the p34(cdc2)/cyclin B complex through phosphorylation/activation of the cdc25 phosphatase and phosphorylation/inactivation of the wee1 kinase.
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PMID:Sequential dephosphorylation of p34(cdc2) on Thr-14 and Tyr-15 at the prophase/metaphase transition. 891 Mar 83

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Using Xenopus egg extracts shifted to the mitotic state with recombinant cyclin B1 protein, we have been able to reproduce mitotic repression of transcription in vitro. Active RNA polymerase III transcription is observed in interphase extracts in the absence of added cyclin, but is strongly repressed by the induction of cdc2/cyclin B (maturation/mitosis promoting factor, MPF) kinase activity in the mitotic extract. Studies with protein kinase inhibitors show that protein phosphorylation is required for repression. Add-back experiments indicate that repression of class III gene transcription is due to inactivation of the transcription factor TFIIIB. TFIIIB is composed of the TATA-box binding protein (TBP) and TBP-associated factors of 75 and 92 kDa. In the present study, we show that TBP and a polypeptide of 92 kDa are substrates of the mitotic kinase in highly purified TF- IIIB fractions. We also show that a phosphatase present in the Xenopus egg extract can reactivate transcription after repression by the mitotic kinases. This result suggests a mechanism for reactivation of transcription after exit from mitosis into the G1 phase of the cell cycle. As for pol III genes, purified cdc2/cyclin B kinase is sufficient to inhibit transcription by RNA polymerase II in a reconstituted transcription system containing the basal transcription factors and polymerase.
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PMID:Repression of RNA polymerase II and III transcription during M phase of the cell cycle. 898 11

Mitogen-activated protein kinase (MAP) is involved in many signal transduction pathways and is activated during meiotic maturation in various species. In this study, we used the rat oocyte to identify some of the control mechanisms involved in MAP kinase activation which is triggered at resumption of meiosis. We examined the respective contribution of this kinase and maturation promoting factor (MPF), or cdc2 kinase, in the regulation of microtubule behavior and in the reorganization of chromatin during meiotic maturation. We found that the resumption of meiotic division in rat oocytes coincided with the activation of MPF and was followed 3 h later by the activation of MAP kinase. The activation of the two kinases also occurred in oocytes undergoing maturation in the presence of the protein phosphatase inhibitor okadaic acid (OA). However, the activation of cdc2 kinase was only partial, whereas activation of MAP kinase was accelerated and began 1 h after the resumption of meiosis, i.e. 2 h earlier than in control oocytes. We also showed that protein synthesis was required to activate MAP kinase, but not cdc2 kinase. However, once MAP kinase was activated, ongoing protein synthesis was not necessary to maintain its activity. These results suggest that a negative regulation of MAP kinase slows down its activation at the resumption of meiosis, mediated through the level of phosphatase activity. Moreover, MAP kinase activation requires protein synthesis, even upon phosphatase inactivation by OA, suggesting also the existence of a positive control pathway. We observed that during the first meiotic M-phase, the spindle did not form immediately after cdc2 kinase activation, but that its formation coincided with the appearance of MAP kinase activity. However, earlier activation of MAP kinase by treatment with OA did not lead to premature spindle formation, but instead a large aster formed consisting of long microtubules radiating from the condensed chromatin. In OA-treated oocytes, spindles did not form and an interphase network of microtubule developed with time. Thus, MAP kinase is unable to substitute for MPF under these conditions, its activity alone being insufficient to maintain the progression through meiotic maturation.
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PMID:Protein phosphatases control MAP kinase activation and microtubule organization during rat oocyte maturation. 901 23

Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression. In vitro, our previous studies showed that cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and that this leads to inhibition of enzyme activity. To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1C alpha phosphorylated at T320. Cell synchronization studies revealed in a variety of cell types that T320 of PP-1 was phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 was reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1/PP-2A inhibitor, calyculin A. Immunofluorescence microscopy using phospho-T320 antibody indicated that in NIH 3T3 cells the phosphorylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase. Immunostaining indicated that phospho-PP-1 was localized exclusively to nonchromosomal regions. Furthermore, in cell fractionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction. These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division.
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PMID:Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase. 912 66

Protein phosphatase-1 is phosphorylated "in vitro" by cdc2-cyclin B (E. Villa-Moruzzi, FEBS Lett. 304, 211-215, 1992). In the present study the phosphatase-1 isoforms alpha, gamma 1, and delta were analyzed in mitotic (nocodazole-blocked) HeLa cells. Phosphorylation on threonine increased in gamma 1 and delta at mitosis. alpha was phosphorylated only in mitotic cells and mainly on serine. Exposure of permeabilized mitotic cells to a peptide that inhibits cdc2 decreased the phosphorylation of the isoforms. Cell fractionation indicated that phosphatase-1 was over 90% inactivated and phosphorylated in the soluble, but not in the chromosomal fraction of mitotic cells. Immunoprecipitation from the mitotic soluble fraction indicated that only gamma 1 and delta, but not alpha, were inactivated. Altogether the data pointed to a correlation between phosphatase-1 inactivation and phosphorylation in mitotic cells. cdc2-cyclin B might be the kinase (or one of the kinases) that phosphorylates phosphatase-1. In cells released from the mitotic block, the phosphatase-1 activity in the soluble, but not in the nuclear fraction, increased progressively, reaching control values by 16 h. Immunoprecipitation indicated that the increase in activity was due to alpha and delta only. On the other hand, the activity of gamma 1 remained low, and this was also the only isoform that remained phosphorylated, though less than in mitotic cells. Also in the case of the cells released from mitosis, a correlation may exist between phosphorylation and inactivation of phosphatase-1. However, the regulation of phosphatase-1 is complex and may involve also regulatory subunits that are still unknown. Altogether, the results indicated the differential regulation of the phosphatase-1 isoforms both at mitosis and in G1 cells.
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PMID:Protein phosphatase-1 alpha, gamma 1, and delta: changes in phosphorylation and activity in mitotic HeLa cells and in cells released from the mitotic block. 914 19

The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation.
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PMID:Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes. 919 Feb 5

Protein Phosphatase-1 is phosphorylated in vitro by cdc2-cyclin B (Villa-Moruzzi, FEBS Lett 304: 211-215, 1992). In the present study we show that all the three Phosphatase-1 isoforms, alpha, gamma 1, delta, are phosphorylated by cdc2-cyclin B. Phosphorylation is specific for this kinase and involves a C-terminal Thr. This site is most likely Thr 320 in alpha (shown by others to be phosphorylated also by cdc2-cyclin A). Such Thr is conserved in gamma 1, delta and in the testis-specific gamma 2, and is the only Thr that fits the cdc2-consensus sequence in the C-terminal region. Phosphorylation of Phosphatase-1 purified from skeletal muscle, which is a mixture of the alpha, gamma 1 and delta isoforms, is up to 0.4 mol/mol and induces 30-35% enzyme inactivation. Following tryptic proteolysis each isoform yields a distinct phosphopeptide map. This is in agreement with the different sequences of the isoforms in the C-terminal regions and may be useful to distinguish the isoforms in extracts from metabolically-labelled cells. Our results suggest that all the Phosphatase-1 isoforms may be potentially regulated at M-phase.
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PMID:Phosphorylation of protein phosphatase-1 isoforms by cdc2-cyclin B in vitro. 920 3


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