P34cdc2 Protein Kinase Research Articles

Regulation of p34cdc2 protein kinase activity by phosphorylation and cyclin binding.

Activation of the protein kinase p34cdc2 is required for entry into meiotic or mitotic M phase in all eukaryotic cells. One important mechanism regulating the activity of p34cdc2 during the cell cycle is based on phosphorylation/dephosphorylation. Avian p34cdc2 is phosphorylated on threonine 14 (Thr14), tyrosine 15 (Tyr15), threonine 161 (Thr161) and serine 277 (Ser277). Dephosphorylation of both Thr14 and Tyr15 is required for activation of p34cdc2 at the G2/M transition, indicating that phosphorylation of these residues negatively regulates p34cdc2 activity. Conversely, phosphorylation of Thr161 is required for kinase activity. Whether modification of this residue is due to intramolecular autophosphorylation or to the action of an as yet unidentified kinase remains unresolved. Likewise, the role of phosphorylation of p34cdc2 on Ser277 during G1 phase of the cell cycle remains to be determined. The function of p34cdc2 is regulated also by cell cycle-dependent complex formation with cyclin proteins. We found that chicken cyclin B2 undergoes a striking redistribution from the cytoplasm to the nucleus just prior to the onset of mitosis. Expression of a non-destructible cyclin B2 mutant causes HeLa cells to arrest in mitosis. Frequently, arrested cells displayed multiple mitotic spindles.

Hela l-CaD undergoes a DNA replication-associated switch in localization from the cytoplasm to the nuclei of endothelial cells/endothelial progenitor cells in human tumor vasculature

Caldesmon (CaD) is a major actin-binding protein distributed in a variety of cell types. So far no diversity in functions of the different isoforms were found in in vitro studies. The low molecular weight isoform (Hela l-CaD) was detected in the vasculature of a variety of tumor types in our previous study. Proliferation of endothelial cells/endothelial progenitor cells is a crucial event for formation of new blood vessels. Here we report the intranuclear translocation of Hela l-CaD in cell cycle activated ECs/EPCs in the vasculature of human tumors. The nuclear translocation coincides with phosphorylation of the molecule and the activation of intranuclear protein kinase p34cdc2. These findings point to a function of this molecule relating to DNA synthesis which is triggered by cell-cycle signalling pathways. The data challenge and update the generally accepted concept that CaD is a pure cytoplasmic protein in vitro study. It suggests that nuclear translocation of Hela l-CaD serves as an additional regulatory step in the control of mitotic initiation and triggers further investigations in the role of this protein in the regulation of nuclear functions.

Involvement of Ca 2+-dependent proteasome in the degradation of both cyclin B1 and Mos during spontaneous activation of matured rat oocytes

In matured rat oocytes, spontaneous activation from the metaphase-II (MII) stage occurred after collection from the oviducts. It is well known that the mitogen-activated protein kinase (MAPK) pathway and p34 cdc2 kinase play an important role in the arrest at MII in other species. However, there is no information about the difference in these factors among strains of rats. In the present study, in spontaneously activated oocytes from the Wistar rat, the Mos protein level and the activity of MAPK kinase (MEK)/MAPK were decreased at 120 min (13.8, 25.7, and 19.3, respectively, P < 0.05), whereas Sprague–Dawley (SD) oocytes, which were not spontaneously activated, had a high level of Mos protein and MEK/MAPK activity (75.9, 76.2, and 87.9, respectively, P < 0.05). Phosphorylation of MAPK in the SD oocytes was significantly suppressed by MEK inhibitor, U0126 at 60 min; this treatment decreased p34 cdc2 kinase activity via cyclin B1 degradation in a time-dependent manner. The treatment with proteasome inhibitor, MG132 or Ca 2+-chelator, BAPTA-AM, overcame the spontaneous degradation of both Mos and cyclin B1 in a dose-dependent manner in Wistar oocytes. More than 90% of Wistar oocytes treated with BAPTA-AM were arrested at MII until 120 min. In conclusion, SD oocytes carrying Mos/MEK/MAPK, maintained a high activity of p34 cdc2 kinase by stabilizing cyclin B1, thus involved in their meiotic arrest. In contrast, Wistar oocytes had a relatively low cytostatic factor activity; rapid decrease of Mos/MEK/MAPK failed to stabilize both cyclin B1 and Mos, and these oocytes were likely to spontaneously activate.

Downregulation of protein phosphatase 2A activity in HeLa cells at the G2-mitosis transition and unscheduled reactivation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA)

In the cell cycle the transition from G2 phase to cell division (M) is strictly controlled by protein phosphorylation–dephosphorylation reactions effected by several protein kinases and phosphatases. Although much indirect and direct evidence point to a key role of protein phosphatase 2A (PP2A) at the G2/M transition, the control of the enzyme activity prior to and after the transition are not fully clarified. Using synchronized HeLa cells we determined the PP2A activity (i.e. the increment sensitive to inhibition by 2 nM okadaic acid) in immunoprecipitates obtained with antibodies raised against a conserved peptide sequence (residues 169–182, Ab 169/182) of the PP2A catalytic subunit (PP2A C). Two different substrates were offered: the phospho-peptide KR(p)TIRR and histone H1 phosphorylated by means of the cyclin-dependent protein kinase p34 cdc2. The results indicate that in HeLa cells the specific activity of PP2A towards both substrates goes through a minimum in late G2 phase and stays low until metaphase. Treatment of G2 cells with TPA (10 −7 M) caused a reactivation of the downregulated PP2A activity within 20 min, i.e. the same time frame within which TPA was shown earlier to block HeLa cells at the transition from G2 to mitosis [Kinzel et al., 1988. Cancer Res. 48, 1759–1762]. Activation of PP2A was also induced by TPA in mitotic cells. The low activity of PP2A in mitotic cells was accompanied by a strong reaction of mitotic PP2A C with anti-P-Tyr antibodies in Western blots, which was reversed by treatment of mitotic cells with TPA. The results suggest that the activity of cellular PP2A requires downregulation for the transition from G2 phase to mitosis. Unscheduled reactivation of PP2A induced by TPA in late G2 phase appears to inhibit the progress into mitosis.

Relationship between intracellular localization of p34cdc2 protein and differentiation of esophageal squamous cell carcinoma

G2/M cyclins including cyclins A and B can exert their biologic functions of mitosis and proliferation of the tumor cells by being combined by protein kinase p34cdc2. The aim of the current study was to elucidate the clinicopathologic significance of immunohistochemical expression of p34(cdc2) in esophageal squamous cell carcinoma (ESCC), which has not been resolved. Immunohistochemical expression of p34(cdc2) was examined for 91 cases of ESCC, and the relationship between the type of p34(cdc2) expression and the clinicopathologic features of the patients and tumors was analyzed. Forty-one ESCCs demonstrated cytoplasm dominant expression of p34(cdc2) and the other 50 ESCCs showed nuclei dominant p34(cdc2) expression. This differential expression pattern of p34(cdc2) did not reflect a prognostic aspect; however, the proportion of keratinizing tumors ESCCs with cytoplasm dominant expression of p34(cdc2) was significantly higher than that among ESCCs presenting nuclei-dominant p34(cdc2) expression (P=0.006). Cellular differentiation in squamous cell carcinoma of the esophagus may be mediated by an intracellular localization of p34(cdc2).

Expression of the viviparous 1 (Pavp1) and p34cdc2 protein kinase (cdc2Pa) genes during somatic embryogenesis in Norway spruce (Picea abies [L.] Karst).

Detailed expression analysis of the Norway spruce (Picea abies [L.] Karst) Viviparous 1 (Pavp1) and p34cdc2 (cdc2Pa) genes was carried out during somatic embryogenesis. Pavp1, a gene associated with embryo development, was expressed in proliferating embryogenic suspension cultures in the absence of exogenous ABA. When somatic embryo formation was promoting by blocking proliferation, Pavp1 expression was reduced. During maturation, exogenous ABA induced increased Pavp1 expression, which peaked at the early cotyledonary stage of somatic embryogenesis. Following partial desiccation of mature somatic embryos at high relative humidity, Pavp1 expression persisted under germination conditions. Pavp1 expression was also detected in non-dormant immature male strobili and dormant terminal buds. These data confirm the functional conservation of Pavp1 during the evolution of seed plants and extend its function beyond the embryo. Cdc2Pa, a gene associated with the cell cycle, was up-regulated when the proliferation of embryogenic cells was blocked. Expression was again up-regulated in early embryogeny and again during germination. The implications of this up-regulation of cdc2Pa are discussed.

Resveratrol-induced G 2 arrest through the inhibition of CDK7 and p34 CDC2 kinases in colon carcinoma HT29 cells

Resveratrol (3,5,4′-trihydroxystilbene), a phytoalexin found in grapes and other food products, has been shown to have cancer chemopreventive activity. However, the mechanism of the anti-carcinogenic activity is not well understood. Here, we offer a possible explanation of its anti-tumor effect. Based on flow cytometric analysis, resveratrol inhibited the proliferation of HT29 colon cancer cells and resulted in their accumulation in the G 2 phase of the cell cycle. Western blot analysis and kinase assays demonstrated that the perturbation of G 2 phase progression by resveratrol was accompanied by the inactivation of p34 CDC2 protein kinase, and an increase in the tyrosine phosphorylated (inactive) form of p34 CDC2. Kinase assays revealed that the reduction of p34 CDC2 activity by resveratrol was mediated through the inhibition of CDK7 kinase activity, while CDC25A phosphatase activity was not affected. In addition, resveratrol-treated cells were shown to have a low level of CDK7 kinase-Thr 161-phosphorylated p34 CDC2. These results demonstrated that resveratrol induced cell cycle arrest at the G 2 phase through the inhibition of CDK7 kinase activity, suggesting that its anti-tumor activity might occur through the disruption of cell division at the G 2/M phase.

Activation of protein kinase C alters p34cdc2 phosphorylation state and kinase activity in early sea urchin embryos by abolishing intracellular Ca2+ transients

The p34(cdc2) protein kinase, a universal regulator of mitosis, is controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34(cdc2) are induced by Ca(2+) and prevented by Ca(2+) chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca(2+) transients may play an important physiological role in the control of p34(cdc2) kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca(2+) concentration ([Ca(2+)](i)) in early sea urchin embryos without altering the normal resting level of Ca(2+). We have used this finding to investigate whether [Ca(2+)](i) transients control p34(cdc2) kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34(cdc2). In the present study we show that the elimination of [Ca(2+)](i) transients during interphase blocks p34(cdc2) activation and entry into mitosis, while the elimination of mitotic [Ca(2+)](i) transients prevents p34(cdc2) inactivation and exit from mitosis. Moreover, we find that [Ca(2+)](i) transients are not required for the synthesis of cyclin B, its binding to p34(cdc2) or its destruction during anaphase. However, in the absence of interphase [Ca(2+)](i) transients p34(cdc2) does not undergo the tyrosine dephosphorylation that is required for activation, and in the absence of mitotic [Ca(2+)](i) transients p34(cdc2) does not undergo threonine dephosphorylation that is normally associated with inactivation. These results provide evidence that intracellular [Ca(2+)](i) transients trigger the dephosphorylation of p34(cdc2) at key regulatory sites, thereby controlling the timing of mitosis entry and exit.

Activation of protein kinase C alters p34cdc2 phosphorylation state and kinase activity in early sea urchin embryos by abolishing intracellular Ca2+ transients

The p34cdc2 protein kinase, a universal regulator of mitosis, is controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34cdc2 are induced by Ca2+ and prevented by Ca2+ chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca2+ transients may play an important physiological role in the control of p34cdc2 kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca2+ concentration ([Ca2+]i) in early sea urchin embryos without altering the normal resting level of Ca2+. We have used this finding to investigate whether [Ca2+]i transients control p34cdc2 kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34cdc2. In the present study we show that the elimination of [Ca2+]i transients during interphase blocks p34cdc2 activation and entry into mitosis, while the elimination of mitotic [Ca2+]i transients prevents p34cdc2 inactivation and exit from mitosis. Moreover, we find that [Ca2+]i transients are not required for the synthesis of cyclin B, its binding to p34cdc2 or its destruction during anaphase. However, in the absence of interphase [Ca2+]i transients p34cdc2 does not undergo the tyrosine dephosphorylation that is required for activation, and in the absence of mitotic [Ca2+]i transients p34cdc2 does not undergo threonine dephosphorylation that is normally associated with inactivation. These results provide evidence that intracellular [Ca2+]i transients trigger the dephosphorylation of p34cdc2 at key regulatory sites, thereby controlling the timing of mitosis entry and exit.

Antiproliferative effects of trapidil in vascular smooth muscle cells are associated by inhibition of MAPK and P34(cdc2) activity.

Trapidil (Tra) is a potent coronary vasodilator. Recent studies have shown that Tra possesses antiproliferative activity in glioma cells and vascular smooth muscle cells (VSMCs). These studies were undertaken to determine the effects of Tra on the cell cycle of cultured rat VSMCs, in particular, the effects on two key enzymes responsible for cell-cycle control in cultured rat VSMCs. VSMCs were synchronized by serum deprivation and then stimulated to enter the cell cycle by serum refeeding; the cell-cycle distribution was subsequently measured by flow cytometry, and VSMCs pretreated with 5, 50, and 500 microM Tra showed a decrease in S-phase cell-cycle distribution, 13.1, 18.7, and 58.6%, respectively. In addition, the mitotic activity (S + G2/M) decreased 12.9, 18.7, and 49.6%, respectively after Tra treatment. The protein expression of p34cdc2 was determined by Western blot analysis, and Tra treatment did not affect its expression even at 500 microM. Mitogen-activated protein kinase (MAPK) activity and p34cdc2 kinase activity were assayed by phosphorylation of their specific substrates, myelin basic protein (MBP) and histone H1, after immunoprecipitation. Exposure of VSMCs to Tra resulted in a significant decrease in serum-stimulated MAPK and p34cdc2 activity. The inhibitory rates of 50 and 500 microM Tra on MAPK activity were 59.2 and 80.9%, respectively. Tra concentrations of 5, 50, and 500 microM inhibited p34cdc2 activity by 16.4, 22.6, and 40.8%, respectively. Furthermore, 500 microM Tra inhibited the basal kinase activities of MAPK and p34cdc2 in the cells that were not serum stimulated. These findings demonstrate that Tra significantly decreases the mitotic activity of cultured VSMCs, and that this effect is associated with the inhibitory role of Tra on the kinase activities of MAPK and p34cdc2.

The maize mutant polymitotic affects cell cycle events during microspore development

The maize (Zea mays L.) male-sterile mutant polymitotic (po) was analyzed utilizing in-vitro cell culture and immunocytochemistry methods to better understand the relationship between the mutant phenotype and cell cycle events during microspore development. Using a live meiocyte culture system, initiation and progression through abnormal post-meiotic cell cycles at the end of meiosis II was documented in the po mutant with a CCD camera and a computer image analysis system. Our results showed that premature chromosomal condensation precedes abnormal post-meiotic cell cycle progression in the po mutant at the end of meiosis II. Temporal analysis of the po mutant in-vitro revealed that unsynchronized post-meiotic divisions occurred immediately following the end of meiosis II and did not require interaction with the surrounding somatic tissue. Furthermore, the altered distribution of p34(cdc2) protein kinase from a nuclear to a cytoplasmic location was identified in tetrads during onset of the unsynchronized divisions in the po mutant. In contrast, a predominantly nuclear location of p34(cdc2) was observed during interphase of the wild-type tetrads at the same stage.

Clostridium botulinum C2 toxin delays entry into mitosis and activation of p34cdc2 kinase and cdc25-C phosphatase in HeLa cells.

The Clostridium botulinum C2 toxin ADP-ribosylates monomeric actin, thereby inducing disassembly of actin filaments, alteration of focal adhesions, and rounding of cells. After treatment with C2 toxin, cells stop to proliferate but remain viable for about 2 days. In view of reported correlations between the structure of the actin cytoskeleton and cell cycle transition, the effects of C2 toxin on the G(2)/M phase transition of the cell division cycle were studied. Since C2 toxin delayed entry into mitosis in HeLa cells, those enzymes which control entry into mitosis, the cyclin-dependent protein kinase mitosis-promoting factor (MPF) and the phosphatase cdc25-C were examined after treatment of synchronized cells with C2 toxin. MPF is composed of the regulatory cyclin B and the enzymatic p34cdc2 kinase subunits. For its activation at the G2/M border, p34cdc2 needs to be associated with cyclin B and additionally dephosphorylated at Tyr-15 by the specific phosphatase cdc25-C. Treatment of synchronized cells in S or G2 phase with C. botulinum C2 toxin prevented p34cdc2 protein kinase activation by inhibiting its tyrosine dephosphorylation at the G2/M border. Furthermore, the activity of cdc25-C phosphatase was decreased after treatment of cells with C2 toxin. Our results suggest that the prevented activation of the mitotic inducers p34cdc2 kinase and cdc25-C phosphatase represents the final downstream events in the action of C2 toxin resulting in a G(2) phase cell cycle delay in synchronized HeLa cells.

The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis.

The protein kinase p34cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34CDC2 protein kinase from chicken. The response of the chicken p34CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34CDC2 protein divide at reduced size at 31 degrees C. Cells are temperature sensitive at 35.5 degrees C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34CDC2 are cold sensitive. At 19 degrees C to 25 degrees C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest.

A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1.

M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF) which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. Here we show that the C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. p90(rsk) in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B.

SCP2: a major protein component of the axial elements of synaptonemal complexes of the rat.

In the axial elements of synaptonemal complexes (SCs) of the rat, major protein components have been identified, with relative electrophoretic mobilities (M rs) of 30 000-33 000 and 190 000. Using monoclonal anti-SC antibodies, we isolated cDNA fragments which encode the 190 000 M r component of rat SCs. The translation product predicted from the nucleotide sequence of the cDNA, called SCP2 (for synaptonemal complex protein 2), is a basic protein (pI = 8.0) with a molecular mass of 173 kDa. At the C-terminus, a stretch of approximately 50 amino acid residues is predicted to be capable of forming coiled-coil structures. SCP2 contains two clusters of S/T-P motifs, which are common in DNA-binding proteins. These clusters flank the central, most basic part of the protein (pI = 9.5). Three of the S/T-P motifs are potential target sites for p34(cdc2) protein kinase. In addition, SCP2 has eight potential cAMP/cGMP-dependent protein kinase target sites. The gene encoding SCP2 is transcribed specifically in the testis, in meiotic prophase cells. At the amino acid sequence and secondary structural level, SCP2 shows some similarity to the Red1 protein, which is involved in meiotic recombination and the assembly of axial elements of SCs in yeast. We speculate that SCP2 is a DNA-binding protein involved in the structural organization of meiotic prophase chromosomes.

Role of calcineurin and Mpk1 in regulating the onset of mitosis in budding yeast.

Signalling via calcium is probably involved in regulating eukaryotic cell proliferation, but details of its mechanism of action are unknown. In Schizosaccharomyces pombe, the onset of mitosis is determined by activation of a complex of the p34cdc2 protein kinase and a cyclin protein that is specific to the G2 phase of the cell cycle. This activation requires dephosphorylation of p34cdc2. Weel, a tyrosine kinase that inhibits p34cdc2 by phosphorylating it, is needed to determine the length of G2 phase. Here we show that calcium-activated pathways in Saccharomyces cerevisiae control the onset of mitosis by regulating Swel, a Weel homologue. Zds1 (also known as Oss1 and Hst1) is important in repressing the transcription of SWE1 in G2 phase. In the presence of high calcium levels, cells lacking Zds1 are delayed in entering mitosis. Calcineurin and Mpk1 regulate Swel activation at the transcriptional and posttranslational levels, respectively, and both are required for the calcium-induced delay in G2 phase. These cellular pathways also induce a G2-phase delay in response to hypotonic shock.

Molecular evolution of cdc2 pseudogenes in spruce (Picea).

The p34cdc2 protein and other cyclin-dependent protein kinases (CDK) are important regulators of eukaryotic cell cycle progression. We have previously cloned a functional cdc2 gene from Picea abies and found it to be part of a family of related sequences, largely consisting of pseudogenes. We now report on the isolation of partial cdc2 pseudogenes from Picea engelmannii and Picea sitchensis, as well as partial functional cdc2 sequences from P. engelmannii, P. sitchensis and Pinus contorta. A high level of conservation between species was detected for these sequences. Phylogenetic analyses of pseudogene and functional cdc2 sequences, as well as the presence of shared insertions or deletions, support the division of most of the cdc2 pseudogenes into two subfamilies. New cdc2 pseudogenes appear to have been formed in Picea at a much higher rate than they have been obliterated by neutral mutations. The pattern of nucleotide changes in the cdc2 pseudogenes, as compared to a presumed ancestral functional cdc2 gene, was similar to that previously found in mammalian pseudogenes, with a strong bias for the transitions C to T and G to A, and the transversions C to A and G to T.

Regulation of the replication initiator protein p65cdc18 by CDK phosphorylation.

Cyclin-dependent kinases (CDKs) promote the initiation of DNA replication and prevent reinitiation before mitosis, presumably through phosphorylation of key substrates at origins of replication. In fission yeast, the p65cdc18 protein is required to initiate DNA replication and interacts with the origin recognition complex (ORC) and the p34cdc2 CDK. Here we report that p65cdc18 becomes highly phosphorylated as cells undergo the G1 --> S phase transition. This modification is dependent on p34cdc2 protein kinase activity, as well as six consensus CDK phosphorylation sites within the p65cdc18 polypeptide. Genetic interactions between cdc18+ and the S-phase cyclin cig2+ suggest that CDK-dependent phosphorylation antagonizes cdc18+ function in vivo. Using site-directed mutagenesis, we show that phosphorylation at CDK consensus sites directly targets p65cdc18 for rapid degradation and inhibits its replication activity, as strong expression of a constitutively hypophosphorylated mutant form of p65cdc18 results in large amounts of DNA over-replication in vivo. Furthermore, the over-replication phenotype produced by this mutant p65cdc18 is resistant to increased mitotic cyclin/CDK activity, a known inhibitor of over-replication. Therefore, p65cdc18 is the first example of a cellular initiation factor directly regulated in vivo by CDK-dependent phosphorylation and proteolysis. Regulation of p65cdc18 by CDK phosphorylation is likely to contribute to the CDK-driven "replication switch" that restricts initiation at eukaryotic origins to once per cell cycle.

Overexpression of p34 cdc2 Protein Kinase in Epithelial Ovarian Carcinoma

Objective To investigate the role of expression of p34cdc2 protein kinase in normal, benign, and malignant ovarian epithelium. Material and Methods Tissue sections from 24 patients with epithelial ovarian carcinoma (EOC) along with 6 normal ovarian specimens and 12 benign cystadenomas were incubated with mouse IgG monoclonal antibody to human p34cdc2 protein kinase, followed by detection with use of a standard peroxidase labeled streptavidin-biotin technique. Immunohistochemical staining was graded and compared. Clinical data were also reviewed. Results Normal surface epithelium and 10 of 12 benign cystadenomas failed to stain for p34cdc2 protein kinase. Of the 24 EOC specimens, however, 19 (79%) stained positively. The staining pattern or intensity was not associated with the histologic grade or surgical stage. Conclusion Expression of p34cdc2 protein kinase is strongly up-regulated in most cases of EOC but not in normal epithelial ovarian tissue or in most cases of benign epithelial tumors evaluated. Therefore, it may be associated with early events in careinogenesis. Redundant overexpression of cyclin-dependent kinases such as p34cdc2 may contribute to deranged cell cycle progression and proliferation of EOC. Observation of overexpression of p34cdc2 protein kinase in other malignant lesions suggests a common mechanism. To investigate the role of expression of p34cdc2 protein kinase in normal, benign, and malignant ovarian epithelium. Tissue sections from 24 patients with epithelial ovarian carcinoma (EOC) along with 6 normal ovarian specimens and 12 benign cystadenomas were incubated with mouse IgG monoclonal antibody to human p34cdc2 protein kinase, followed by detection with use of a standard peroxidase labeled streptavidin-biotin technique. Immunohistochemical staining was graded and compared. Clinical data were also reviewed. Normal surface epithelium and 10 of 12 benign cystadenomas failed to stain for p34cdc2 protein kinase. Of the 24 EOC specimens, however, 19 (79%) stained positively. The staining pattern or intensity was not associated with the histologic grade or surgical stage. Expression of p34cdc2 protein kinase is strongly up-regulated in most cases of EOC but not in normal epithelial ovarian tissue or in most cases of benign epithelial tumors evaluated. Therefore, it may be associated with early events in careinogenesis. Redundant overexpression of cyclin-dependent kinases such as p34cdc2 may contribute to deranged cell cycle progression and proliferation of EOC. Observation of overexpression of p34cdc2 protein kinase in other malignant lesions suggests a common mechanism.

Overexpression of p34 cdc2 Protein Kinase in Epithelial Ovarian Carcinoma

To investigate the role of expression of p34cdc2 protein kinase in normal, benign, and malignant ovarian epithelium. Tissue sections from 24 patients with epithelial ovarian carcinoma (EOC) along with 6 normal ovarian specimens and 12 benign cystadenomas were incubated with mouse IgG monoclonal antibody to human p34cdc2 protein kinase, followed by detection with use of a standard peroxidase-labeled streptavidin-biotin technique. Immunohistochemical staining was graded and compared. Clinical data were also reviewed. Normal surface epithelium and 10 of 12 benign cystadenomas failed to stain for p34cdc2 protein kinase. Of the 24 EOC specimens, however, 19 (79%) stained positively. The staining pattern or intensity was not associated with the histologic grade or surgical stage. Expression of p34cdc2 protein kinase is strongly up-regulated in most cases of EOC but not in normal epithelial ovarian tissue or in most cases of benign epithelial tumors evaluated. Therefore, it may be associated with early events in carcinogenesis. Redundant overexpression of cyclin-dependent kinases such as p34cdc2 may contribute to deranged cell cycle progression and proliferation of EOC. Observation of overexpression of p34cdc2 protein kinase in other malignant lesions suggests a common mechanism.