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Results for “CDC2 Protein Kinase”

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PubMed1992

Oscillation of MPF is accompanied by periodic association between cdc25 and cdc2-cyclin B.

Activation of maturation-promoting factor at the onset of mitosis requires the tyrosine dephosphorylation of one of its components, the cdc2 protein kinase. cdc25 is the specific tyrosine phosphatase that activates cdc2. We find that Xenopus oocytes contain a relative of cdc25, p72. In Xenopus embryos the abundance of p72 does not oscillate during the cell cycle. However, p72 directly associates with cdc2-cyclin B in a cell cycle-dependent manner, reaching a peak at M phase. The M phase kinase that associates with p72 is catalytically active. These results suggest that the mechanism by which cdc25 triggers cdc2 activation involves a periodic physical association between cdc25 and the cyclin B-cdc2 complex and also that mitotic control can be affected by mechanisms other than transcriptional regulation of the cdc25 gene.

Animals↗

PubMed1999

Emodin, an anthraquinone derivative isolated from the rhizomes of Rheum palmatum, selectively inhibits the activity of casein kinase II as a competitive inhibitor.

Ser/Thr protein kinases play important roles in signal transduction pathways that control the proliferation and differentiation of eukaryotic cells. In this paper, we present evidence that emodin, an anthraquinone derivative, selectively inhibits casein kinase II (CKII), a Ser/Thr kinase, as a competitive inhibitor. The results with ethyl acetate extracts of the rhizomes of Rheum palmatum showed that emodin significantly inhibited the activity of cyclin B/cdc2 protein kinase (cdc2). We measured IC50 values for emodin on the activities of several Ser/Thr protein kinases, including cAMP-dependent protein kinase (PKA), protein kinase C (PKC), cdc2, casein kinases I (CKI) and CKII. Interestingly, emodin inhibited CKII activity with an IC50 value of 2 microM, which was two to three orders of magnitude lower than those against the other kinases. Enzyme kinetic assays showed that emodin inhibited CKII activity as a competitive inhibitor against ATP with a Ki value of 7.2 microM. Collectively, we suggest that emodin is a selective CKII inhibitor, whose action mechanism is mediated through competitively binding to the ATP binding site.

Adenosine Triphosphate↗

PubMed2006

Arabidopsis CDKA;1, a cdc2 homologue, controls proliferation of generative cells in male gametogenesis.

The protein kinase cdc2 is conserved throughout eukaryotes and acts as a key regulator of the cell cycle. In plants, A-type cyclin-dependent kinase (CDKA), a homologue of cdc2, has a role throughout the cell cycle. Here we show that a loss-of-function mutation in CDKA;1, encoding the only Arabidopsis CDKA, results in lethality of the male gametophyte. Heterozygous plants produced mature siliques containing about 50% aborted seeds, and segregation distortion was observed in paternal inheritance. Microspores normally undergo an asymmetric cell division, pollen mitosis I (PMI), to produce bicellular pollen grains. The larger vegetative cell does not divide, but the smaller generative cell undergoes mitosis, PMII, to form the two sperm cells, thereby generating tricellular pollen grains. The cdka-1 mutant, however, produces mature bicellular pollen grains, consisting of a single sperm-like cell and a vegetative cell, due to failure of PMII. The mutant sperm-like cell is fertile, and preferentially fuses with the egg cell to initiate embryogenesis. As the central cell nucleus remains unfertilized, however, double fertilization does not occur. In heterozygous plants, the embryo is arrested at the globular stage, most likely because of loss of endosperm development, whereas it is arrested at the one- or two-cell stage in presumptive homozygous plants. Thus, CDKA;1 is essential for cell division of the generative cell in male gametogenesis.

Amino Acid Sequence↗

PubMed1991

A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.

Animals↗

PubMed1995

Cross-linking of ICAM-1 on T cells induces transient tyrosine phosphorylation and inactivation of cdc2 kinase.

Intercellular adhesion molecules (ICAM)-1 and -3 coexist on T lymphocytes and are counter-receptors for the integrin LFA-1. Signaling through ICAM-3 stimulates a number of T cell functions and involves phosphorylation of Fyn, Lck, CD45, and other proteins. In contrast, this type of specific signaling event has not been described for signaling through ICAM-1. Here, tyrosine phosphorylation of cellular proteins was examined after cross-linking of ICAM-1. Tyrosine phosphorylation of the 34-kDa cdc2 protein kinase was induced transiently after stimulation of the leukemic T cell line, Molt-3, or peripheral blood T cells. Stimulation through ICAM-1 had no effect on constitutive presence of cdc2 or phosphorylation of cdc2 on threonine. cdc2 kinase activity was constitutive in peripheral blood T cells, and transient inhibition of kinase activity after ICAM-1 stimulation correlated kinetically with phosphorylation of cdc2 on tyrosine.

Antibodies, Monoclonal↗

PubMed1994

Cdc2 activation is not required for thymocyte apoptosis.

Apoptosis is a mode of cell death characterized by chromatin condensation and disassembly of the nuclear lamina, processes that are also characteristic of mitosis. The apparent similarity between the two events, together with observations that apoptosis can occur following G2 arrest, has led to the suggestion that apoptosis could be a defective form of mitosis. Further support for this idea comes from the recent description of activation of the p34cdc2 protein kinase (Cdc2), the universal M-phase promoter, during death induced by a lymphocyte granule protease. We have monitored the protein kinase activity of Cdc2 during apoptosis of primary rat thymocytes, which die from a quiescent (G0) state. We demonstrate unequivocally that activation of Cdc2 is not involved in the induction of apoptosis in thymocytes, indicating that chromatin condensation and lamina disassembly occur in this system by processes different from those that operate in mitosis.

Animals↗

PubMed1993

Identification of novel phosphorylation sites in murine A-type lamins.

We report the distribution of phosphorylation sites in murine lamins A and C (A-type lamins) in vitro and in vivo followed by reverse-phase high-performance liquid chromatography and microsequencing of peptides spanning the almost complete lamin sequence. We show that two distinct protein kinases, cell-division-cycle-2 kinase (cdc2 kinase) and protein kinase C (PKC), phosphorylate murine A-type lamins at the non-alpha-helical amino- and carboxy-terminal domains in vitro and in vivo. Cdc2 kinase, but not PKC, is capable of inducing depolymerization of the nuclear lamina in permeabilized cells. Accordingly, lamins were proposed to be direct in vivo substrates of cdc2 kinase and PKC with different effects on the lamina dynamics. Analysis of the original A-type lamins revealed phosphorylation of residues Ser5 and Ser392. Residue Ser392 was substoichiometrically phosphorylated in the substrate and by cdc2 kinase in vitro. PKC phosphorylated peptides with its kinase-specific motifs surrounding Ser5, Thr199, Thr416, Thr480 and Ser625. In vivo, a mitosis-specific phosphorylation at the cdc2-kinase-specific phosphoacceptor site Ser392 and of the N-terminal peptide was identified. An interphase-specific phosphorylation at Ser525 matching the PKC consensus sequence and of peptides phosphorylated by unknown kinases was determined. The results lead us to propose that different cyclin-dependent kinase activities act as lamin kinases in mitosis and in interphase. Other kinases may cooperate with cdc2 kinase during reversible disassembly in mitosis and may modulate the supramolecular assembly of lamin filaments.

Amino Acid Sequence↗

PubMed2001

Phosphorylation of amyloid-beta at the serine 26 residue by human cdc2 kinase.

The amyloid-beta (Abeta) peptide has been implicated in the pathology of Alzheimer's disease (AD). Using an antisense peptide approach a novel interaction between Abeta and the human cdc2 kinase was identified. The Abeta 1-42, 1-40 and 25-35 peptides were shown to be substrates for the cdc2 kinase and phosphorylated on the Serine 26 residue. Phosphorylated Abeta (pSAbeta) was found in extracts from NT-2 neurons and AD brain. In NT-2 neurons the levels of pSAbeta were increased in the presence of exogenous Abeta and this increase was prevented by a cdc2 protein kinase inhibitor, olomoucine, that also prevented Abeta cytotoxicity. The results from this study suggest that Abeta phosphorylation by cdc2 could play a role in the brain pathology of AD.

Alzheimer Disease↗

PubMed1996

Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.

The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.

CDC2 Protein Kinase↗

PubMed1997

Cell cycle independent interaction of CDC2 with the centrosome, which is associated with the nuclear matrix-intermediate filament scaffold.

The cell cycle regulating Cdc2 protein kinase helps orchestrate cell cycle dependent changes in cell structure and function. This report shows that Cdc2 is localized to the centrosome region and is tightly bound to the nuclear matrix-intermediate filament scaffold. Antibodies to Cdc2 and to the centrosome-specific protein, pericentrin, label the centrosome in an apparently cell cycle independent manner. Isolated centrosomes also label similarly with both antibodies. Essentially, all cells show Cdc2 labeling of the centrosomes, implying an independence of the stage in the cell cycle, a conclusion supported by studies of synchronized cells. In contrast to the labeling of every cell with the Cdc2 monoclonal antibody, fewer centrosomes were labeled with an antibody to the PSTAIRE domain of Cdc2. Embedment-free, immunogold electron micrographs of extracted cell whole mounts show the centrioles and a pericentriolar network of filaments. Both Cdc2 and pericentrin antibodies decorate the amorphous pericentriolar material, while the Cdc2 antibodies also decorate the centrioles themselves. The constitutive presence of Cdc2 at the centrosome suggests a continuing role in the dynamics of centrosome function throughout the cell cycle.

CDC2 Protein Kinase↗

PubMed1989

Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication.

Simian virus 40 large tumour antigen (T) is a replication origin binding protein required for viral DNA synthesis. Unphosphorylated T antigen is deficient in promoting DNA replication in vitro but can be activated by phosphorylation at residue threonine 124 by the cdc2 protein kinase. This observation demonstrates that T is regulated by phosphorylation and provides a model for cdc2 function in the control of DNA replication.

Antigens, Polyomavirus Transforming↗

PubMed1997

Phosphorylation by p34cdc2 protein kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin subunit p150.

The kinesin-related motor HsEg5 is essential for centrosome separation, and its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34(cdc2) protein kinase. To identify proteins able to interact with the tail of HsEg5, we performed a yeast two-hybrid screen with a HsEg5 stalk-tail construct as bait. We isolated a cDNA coding for the central, alpha-helical region of human p150(Glued), a prominent component of the dynactin complex. The interaction between HsEg5 and p150(Glued) was enhanced upon activation of p34(CDC28), the budding yeast homolog of p34(cdc2), provided that HsEg5 had a phosphorylatable residue at position 927. Phosphorylation also enhanced the specific binding of p150(Glued) to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly. Immunofluorescence microscopy revealed co-localization of HsEg5 and p150(Glued) during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins. Taken together, these results suggest that HsEg5 and p150(Glued) may interact in mammalian cells in vivo and that p34(cdc2) may regulate this interaction. Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors.

Amino Acid Sequence↗

PubMed1994

cdc2 kinase sets a memory phosphorylation signal on elongation factor EF-1 delta during meiotic cell division, which perdures in early development.

EF-1 delta is a physiological substrate for cdc2 protein kinase in Xenopus oocytes. The protein is part of the nucleotide exchange factor EF-1 beta gamma delta, involved in the elongation step of protein synthesis. We show that EF-1 delta exists under four isoforms in the prophase oocyte, all phosphorylable by casein kinase II. Each of the prophase isoforms was further separated into a 36 and a 38 kDa form upon phosphorylation by cdc2 kinase which therefore reveals the existence of eight different isoforms. Phosphorylation by cdc2 kinase can be monitored as the electrophoretic mobility dedoublement 36/38 kDa. Developmental regulation of EF-1 delta was analyzed. The cdc2 kinase-induced change occures at meiotic division, after complete oogenesis and perdures during early development. It is therefore a phosphorylation memory signal for early development.

Animals↗

PubMed2002

Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery.

A mechanism that triggers neuronal apoptosis has been characterized. We report that the cell cycle-regulated protein kinase Cdc2 is expressed in postmitotic granule neurons of the developing rat cerebellum and that Cdc2 mediates apoptosis of cerebellar granule neurons upon the suppression of neuronal activity. Cdc2 catalyzes the phosphorylation of the BH3-only protein BAD at a distinct site, serine 128, and thereby induces BAD-mediated apoptosis in primary neurons by opposing growth factor inhibition of the apoptotic effect of BAD. The phosphorylation of BAD serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins. Our results suggest that a critical component of the cell cycle couples an apoptotic signal to the cell death machinery via a phosphorylation-dependent mechanism that may generally modulate protein-protein interactions.

14-3-3 Proteins↗

PubMed1991

cdc2 phosphorylation is required for its interaction with cyclin.

Activation of the cdc2 protein kinase at different stages of the cell cycle is regulated by post-translational modifications and interactions with cyclins. We show that in vitro translated human cdc2 binds very poorly to A and B cyclins, unless it has been preincubated with a Xenopus egg extract. This results in the phosphorylation of cdc2 which allows binding to cyclins. The replacement of Thr161, a residue conserved and phosphorylated in other protein kinases, with valine inhibits cdc2 association with A and B cyclins. In addition, mutations in the amino-terminus of cdc2 and within the conserved 'PSTAIR' region strongly inhibit binding. The Thr161Val mutation causes a lethal phenotype in the fission yeast Schizosaccharomyces pombe, while replacement of Thr161 with glutamic acid, potentially mimicking phosphorylation, causes uncoordination of mitosis and multiple cytokinesis. These results suggest that a threonine phosphorylation/dephosphorylation cycle is involved in regulating cdc2 function.

Amino Acid Sequence↗

PubMed1996

A quantitative model for the cdc2 control of S phase and mitosis in fission yeast.

In this article we consider the role of the cyclin-dependent protein kinase cdc2 in regulating progression through the fission yeast cell cycle. The onset of mitosis is governed by cdc2 in partnership with the B-type cyclin, cdc13. Recent evidence shows that the cdc2-cdc13 complex can also control the onset of S phase and, in addition, ensures that there is only one S phase per cell cycle. This leads us to propose a novel quantitative model in which different levels of cdc2 activity regulate cell-cycle progression: S phase is initiated when protein kinase activity increases from a very low to a moderate level; maintenance of this moderate level prevents re-initiation of S phase, and a further increase of activity to a high level initiates mitosis. Inactivation of the kinase activity at the end of mitosis resets the cell for a new cell cycle.

Animals↗

PubMed1995

Inactivation of Cdc2 increases the level of apoptosis induced by DNA damage.

A number of lines of evidence have suggested a possible involvement of the mitosis-promoting protein kinase Cdc2 in the process of apoptotic cell death, and one recent study concluded that premature activation of Cdc2 is required for apoptosis. Here we have used a temperature-sensitive murine Cdc2 mutant cell line and Cdc2 inhibitor compounds to study the effect of inhibition of this protein kinase on apoptosis induced by DNA-damaging drugs. Inhibition of Cdc2 activity before or during exposure to DNA strand break-inducing drugs had the effect of increasing the level of subsequent apoptosis, as assessed by electron microscopy and flow cytometry. We conclude that, far from being required for cell death, a form of mammalian Cdc2 suppresses apoptosis induced by DNA damage. This form of Cdc2 appears to be active in G2-arrested cells and is therefore presumably distinct from the mitosis-promoting Cdc2-cyclin B heterodimer.

Animals↗

PubMed1991

Mutations at sites involved in Suc1 binding inactivate Cdc2.

suc1+ encodes an essential cell cycle regulator of the fission yeast Schizosaccharomyces pombe. Its product, a 13-kDa protein, interacts with the Cdc2 protein kinase. Both positive and negative effects on cell cycle progression have been attributed to Suc1. To date, the exact mechanisms and the physiological role of the interaction between Suc1 and Cdc2 remain unclear. Here we have studied the molecular basis of this association. We show that Cdc2 can bind Suc1 or its mammalian homolog directly in the absence of any additional protein component. Using an alanine scanning mutagenesis method, we analyzed the interaction between Cdc2 and Suc1. We show that the integrity of several domains on the Cdc2 protein, including sites directly involved in catalytic activity, is required for binding to Suc1. Furthermore, Cdc2 mutant proteins unable to bind Suc1 (but able to bind cyclins) are nonfunctional when overexpressed in S. pombe, indicating that a specific interaction with Suc1 is required for Cdc2 function.

Amino Acid Sequence↗