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)

Human cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis.
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PMID:Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis. 811 45

In most eukaryotic cells, a link between S and M phases of the cell cycle must be assured in order to maintain the ploidy of newly divided cells. However, in some cell l/pes, e.g. the precursors of platelets megakaryocytes, extra S-phases can occur in the absence of concomitant mitoses, resulting in polyploidy. We have used two established cell lines with megakaryoblastic characteristics (HEL and MEG-01) to investigate the molecular events that lead these cells to bypass the regular control checkpoints that govern the interdependency of S and M phases. In the presence of the phorbol ester TPA, both cell lines stopped proliferating and displayed additional megakaryocytic features, including polyploidization. Analysis of key cell cycle regulatory factors implicated in the control of G1/S and G2/M transitions revealed a number of differences compared to normally cycling cells. Differentiating megakaryocytes were found to maintain high levels of cdk2, and cyclins E and A. This was accompanied by the appearance of the retinoblastoma protein in the hyperphosphorylated, functionally inactivated form. In addition, TPA-treated cells showed high levels of cyclin B and cdc2 proteins, however no activation of cdc2 was detected. This lack of cdc2 activation which should occur for entry into M phase appeared to be related to the down regulation of cdc25C phosphatase found in both differentiated HEL and MEG-01 cells. Together, our results suggest that in differentiating megakaryoblastic cells endoreplication is accompanied by sustained levels of cyclins A and E, and a lack of cdc2 activation, which is probably mediated through down regulation of cdc25C protein phosphatase.
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PMID:Endoreplication in megakaryoblastic cell lines is accompanied by sustained expression of G1/S cyclins and downregulation of cdc25C. 876 Dec 90

Previous studies from our laboratory demonstrated that diallyl disulfide (DADS), an oil-soluble allyl sulfur compound found in processed garlic, markedly suppressed p34(cdc2) kinase activity and induced a G(2)/M phase arrest in cultured human colon tumor (HCT-15) cells. The present studies reveal that suppression of p34(cdc2) kinase activity by DADS does not result from direct interactions with the protein, but through changes in factors influencing the formation and conversion of the enzyme to its active form. Flow cytometric analyses showed that the increased proportion of cells in the G(2)/M phase following DADS treatment was accompanied by an increase in cyclin B(1) protein expression. A temporal and dose-dependent response in cyclin B(1) expression was observed in cells treated with DADS. Western blot analysis revealed that 50 microM DADS did not influence the quantity of p34(cdc2) protein expressed, but did decrease the amount associated with cyclin B(1) by 26% (P < 0.05). Exposure of unsynchronized cells to 25 or 50 microM DADS caused a trend towards increased p34(cdc2) hyperphosphorylation (17 and 22%, respectively). Exposure of synchronized cells to 100 microM DADS increased p34(cdc2) hyperphosphorylation by 15% (P < 0.05). Consistent with its ability to slightly increase the quantity of hyperphosphorylated p34(cdc2), DADS, 25 or 50 microM, decreased cdc25C protein expression by 23 and 46%, respectively (P < 0.05). The present studies suggest that the ability of DADS to inhibit p34(cdc2) kinase activation occurs because of decreased p34(cdc2)/cyclin B(1) complex formation and modest p34(cdc2) hyperphosphorylation.
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PMID:Diallyl disulfide inhibits p34(cdc2) kinase activity through changes in complex formation and phosphorylation. 1083

The ultraviolet B (UVB) portion (280-320 nm) of solar radiation is considered to be a major etiologic factor in human skin cancer and is a known cause of extensive DNA damage. In this study, we observed that UVB exposure of immortalized epidermal keratinocytes (HaCat cells) harboring mutant p53 leads to G(2)/M cell cycle arrest in both asynchronously growing and synchronized cells in a dose dependent manner. Following UVB exposure (200 mJ/cm(2)), we observed a threefold increase in G(2)/M population at 6 h, which increased to sixfold. The observed G(2)/M arrest was associated with an increase in cyclin B level whereas cdc2 protein remained unchanged. However, we observed an accumulation of tyrosine 15 hyperphosphorylated cyclin B-cdc2 complex. In addition, we observed an increase in chk1 kinase and a decrease in cdc25C protein levels. Chk1 phosphorylates cdc25C on serine 216 and inactivates it whereas cdc25C dephosphorylates tyrosine 15 phosphate of cdc2 and activates the cdc2-cyclin B complex. Therefore, the increase in chk1 and the decrease in cdc25C both participate in inhibiting the G2/M transition. Our data identifies two upstream targets leading to inhibition of cyclin B-cdc2 complexes, which explain the inhibition in cyclin B-associated cdc2 kinase following UVB exposure. The inactive phosphorylated cdc2-cyclin B complex remains sequestered in cytoplasm and may migrate to the nucleus following activation. Our data also indicate that UVB exerts unique effects in different types of skin keratinocytes having nonfunctional or mutant p53.
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PMID:Mechanism of ultraviolet B-induced cell cycle arrest in G2/M phase in immortalized skin keratinocytes with defective p53. 1102 48

We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.
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PMID:Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase. 2356 37

The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades.
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PMID:Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades. 2463 6