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PubMed1995

Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2.

The cyclin-dependent protein kinases (CDKs) are activated by association with cyclins and by phosphorylation at a conserved threonine residue by the CDK-activating kinase (CAK). We have studied the binding of various human CDK and cyclin subunits in vitro, using purified proteins derived from baculovirus-infected insect cells. We find that most CDK-cyclin complexes known to exist in human cells (CDC2-cyclin B, CDK2-cyclin A, and CDK2-cyclin E) form with high affinity in the absence of phosphorylation or other cellular components. One complex (CDC2-cyclin A) forms with high affinity only after CAK-mediated phosphorylation of CDC2 at the activating threonine residue. CDC2 does not bind with high affinity to cyclin E in vitro, even after phosphorylation of the CDC2 subunit. Thus, phosphorylation is of varying importance in the formation of high-affinity CDK-cyclin complexes.

CDC2 Protein Kinase↗
PubMed1994

Histone H1 kinase activity, germinal vesicle breakdown and M phase entry in mouse oocytes.

Meiotic reinitiation of the mouse oocyte is characterized by a slow entry into metaphase I, beginning with germinal vesicle breakdown and ending with spindle formation. It is accompanied by a cascade of protein kinases and phosphatases increasing protein phosphorylation. The activation of histone H1 kinase and that of the mitogen-activated protein kinase p42 have been compared during spontaneous or okadaic acid-induced meiotic reinitiation. In spontaneously maturing oocytes, histone H1 kinase activity increases before germinal vesicle breakdown (2-fold), in a protein synthesis-independent manner. It is associated with the disappearance of the upper migrating form of p34cdc2, which, in our system, seems to represent the tyrosine phosphorylated form. Following germinal vesicle breakdown, histone H1 kinase activity culminates (8-fold) in metaphase I and requires protein synthesis. Activation by phosphorylation of p42MAPK is observed as a permanent shift upward-migrating form and by its myelin basic protein kinase activity. It occurs after germinal vesicle breakdown and depends on protein synthesis. In contrast, no increase of histone H1 kinase is detectable in oocytes induced to reinitiate meiosis by a transient inhibition of okadaic acid-sensitive phosphatase(s), either before germinal vesicle breakdown or during the following 7 hours of culture. A slight increase is nevertheless evident after 17 hours, when oocytes are arrested with an abnormal metaphase I spindle. The upper migrating form of p34cdc2 is present for 8 hours. The activation of p42MAPK begins before germinal vesicle breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Butyrolactone I, a selective inhibitor of cdk2 and cdc2 kinase.

We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.

4-Butyrolactone↗
PubMed1993

Reconstitution of cyclin-dependent cdc2 and cdk2 kinase activities in vitro.

The genes that encode human cdc2 and cdk2 proteins are essential for cell cycle progression. In this report, we describe the purification of cyclin-associated cdc2 and cdk2 kinases as well as cyclin-free cdc2 and cdk2 protein preparations from HeLa cells. The cdc2-cyclin B kinase complex that we have isolated, consisting of two polypeptides of p60 (cyclin B) and p34 (cdc2), phosphorylated both the p34 and p70 subunits of the three-subunit human single-stranded DNA-binding protein (also called RP-A), a DNA replication and repair factor. We also partially purified a histone H1 kinase activity that is associated with the cdk2 and cyclin A proteins. Purified human cyclins A and B1, overproduced in bacteria, complemented a cellular fraction enriched in cdc2 and cdk2 proteins to reconstitute histone H1 kinase activity. Using this complementation system, human cdc2 and cdk2 proteins were purified and separated from one another. Glycerol gradient analyses demonstrated that the purified cdk2 (p33) protein co-sedimented with a cyclin A-dependent H1 kinase activity. Thus, cdk2 and cyclin A proteins are components that assemble to yield a kinase complex that catalyzes the phosphorylation of histone H1.

CDC2 Protein Kinase↗
PubMedDate not supplied

Treatment of human glioblastoma cells with the staurosporine derivative CGP 41251 inhibits CDC2 and CDK2 kinase activity and increases radiation sensitivity.

CGP, 41251, a staurosporine derivative, is a potent inhibitor of protein kinase C (PKC). In recent studies we found that this compound causes growth inhibition and induces apoptosis in human glioblastoma cell lines and also inhibits the growth of xenografts of a human astrocytoma. In this study we investigate its effects on cell cycle control. Treatment of glioblastoma or gliosarcoma cells with CGP 41251 lead to a time and dose dependent increase of the percentage of cells in the G2-M phase of the cell cycle. This correlated with a decrease of CDC2- and CDK2-associated histone H1 kinase activities as well as a decrease in the cellular level of the CDC2 protein. The decrease of CDC2- associated histone H1 kinase activity was detected within 5 hours, and there was complete inhibition after 24 hours. Assays of mixtures of cell extracts obtained from cultures treated with CGP 41251, the inactive analog CGP 42700, or untreated cultures indicated that this decrease was due to a decrease in the CDC2 kinase itself rather than the accumulation of an inhibitor of this kinase. In vitro assays in which CGP 41251 was added directly to the in vitro assay system revealed marked inhibition of both CDC2- and CDK2-associated kinase activity at about 1 microM. Thus CGP 41251 inhibits CDC2- and CDK2-associated kinase activities both in vivo and in vitro. Its biologic effects may, therefore, not be due simply to inhibition of PKC. Since cells in the G2-M phase of the cell cycle are relatively more sensitive to killing by gamma- radiation than cells in other phases of the cell cycle, we carried out radiosensitization studies. We found that CGP 41251 was a radiation sensitizer in two glioblastoma cell lines. Therefore, this compound may be useful in the treatment of glioblastomas, possibly in combination with radiation therapy.

Antineoplastic Agents↗
PubMed1994

Suppression of neointimal smooth muscle cell accumulation in vivo by antisense cdc2 and cdk2 oligonucleotides in rat carotid artery.

Deendothelializing balloon injury of rat carotid artery results in progressive intimal smooth muscle cell accumulation and luminal stenosis over 14 days after injury. We have found transient rises (approximately 3-fold maximal increases over the uninjured control value) of the kinase activities of both cdc2 and cdk2, key molecules for cell cycle progression, in the injured carotid artery along with the development of intimal proliferation. The topical application of the antisense, but not the sense, cdc2 and cdk2 phosphorothioate oligodeoxynucleotides dissolved in F127 pluronic gel around the freshly injured artery resulted in reductions of the intimal smooth muscle cell accumulation by 47% and 55% respectively, as estimated by an intimal to medial cross-sectional area ratio, with concomitant decreases in cdc2 and cdk2 kinase activities. These results indicate that both cdc2 and cdk2 kinases are involved in intimal smooth muscle cell accumulation after balloon angioplasty and suggest a potential usefulness of the antisense cdc2 and cdk2 oligonucleotide therapy for arterial stenosis.

Angioplasty, Balloon↗
PubMedDate not supplied

Regulatory properties of neuronal cdc2-like kinase.

Neuronal cdc2-like kinase, nclk, is a heterodimer of cyclin dependent protein kinase 5, cdk5, and a 25 kDa subunit derived from a novel, neuron-specific, 35 kDa protein: p35. The characterization and regulation of nclk will be summarized in this minireview. The activity of nclk appears to be governed by highly complex regulatory mechanisms including protein-protein interaction, protein phosphorylation and isoforms. The histone H1 kinase activity of nclk is absolutely dependent of the interaction between the 25 kDa subunit and the catalytic subunit, cdk5. In addition, nclk interacts with other cellular proteins to form macromolecular complexes. The kinase activity of nclk is inhibited in vitro by the phosphorylation reactions of a weel-like protein tyrosine kinase and a protein serine/threonine kinase from bovine thymus. Northern blot analysis has revealed the existence of two populations of p35 mRNA of 2 and 4 kb. A novel cDNA encoding a p35 homologous protein has been obtained from a human hippocampus library.

Amino Acid Sequence↗
PubMed1996

Mitogen-activated protein kinase activity and microtubule organisation are altered by protein synthesis inhibition in maturing porcine oocytes.

Previously we have shown that mitogen-activated protein (MAP) kinase activity abruptly increases at the first metaphase (M1) and remains significantly higher than that at the germinal vesicle (GV) stage until the second metaphase (M2) in porcine oocytes cultured in vitro. The present paper describes how the mechanism of the blockage of meiotic maturation by protein synthesis inhibition involves MAP kinase regulation. Cycloheximide arrested both germinal vesicle breakdown (GVBD) and the normal transition from M1 to M2. MAP kinase activation was also reduced in these maturation-inhibited oocytes. By using immunofluorescence microscopy with the monoclonal antibody raised against rat alpha-tubulin, we showed that cycloheximide caused morphological abnormality in a spindle at M1, but not at M2. All these results indicate that in porcine oocytes: (1) GV blockage by protein synthesis inhibition involves the suppression of both histone H1 kinase and MAP kinase activation, (2) during the transition from M1 to M2, maintenance of a normal metaphasic spindle and high MAP kinase activity require protein synthesis, and (3) once the M2 cytoskeletal structures have been completed, and/or after the 'critical period', cytostatic factor activity is independent of protein synthesis.

Animals↗
PubMed2001

p38-Dependent marking of inflammatory genes for increased NF-kappa B recruitment.

We found that inflammatory stimuli induce p38 mitogen-activated protein kinase-dependent phosphorylation and phosphoacetylation of histone H3; this selectively occurred on the promoters of a subset of stimulus-induced cytokine and chemokine genes. p38 activity was required to enhance the accessibility of the cryptic NF-kappa B binding sites contained in H3 phosphorylated promoters, which indicated that p38-dependent H3 phosphorylation may mark promoters for increased NF-kappa B recruitment. These results show that p38 plays an additional role in the induction of the inflammatory and immune response: the regulation of NF-kappa B recruitment to selected chromatin targets.

Acetylation↗
PubMed1996

The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.

Animals↗
PubMed1996

Growth factor dependence of progression through G1 and S phases of adult rat hepatocytes in vitro. Evidence of a mitogen restriction point in mid-late G1.

Several hepatocyte mitogens have been identified, but the signals triggering the G0/G1 transition and cell cycle progression of hepatocytes remain unknown. Using hepatocyte primary cultures, we investigated the role of epidermal growth factor/pyruvate during the entry into and progression through the G1 phase and analyzed the expression of cell cycle markers. We show that the G0/G1 transition occurs during hepatocyte isolation as evidenced by the expression of early genes such as c-fos, c-jun, and c-myc. In culture, hepatocytes progress through G1 regardless of growth factor stimulation until a restriction point (R point) in mid-late G1 beyond which they cannot complete the cell cycle without mitogenic stimulation. Changes in cell cycle gene expression were associated with progression in G1; the cyclin E mRNA level is low early in G1 but increases at the G1/S boundary, while the protein is constantly detected during cell cycle but undergoes a change of electrophoretic mobility in mid-late G1 after the R point. In addition, a drastic induction of cyclin D1 mRNA and protein, and to a lesser extent of cyclin D2 mRNA, takes place in mitogen-stimulated cells after the R point. In contrast, cyclin D3 mRNA appears early in G1, remains constant in stimulated cells, but accumulates in unstimulated arrested cells, paralleling the cyclin-dependent kinase 4 mRNA expression. These results characterize the different steps of G1 phase in hepatocytes.

Animals↗
PubMed1993

Mitotic repression of transcription in vitro.

A normal consequence of mitosis in eukaryotes is the repression of transcription. Using Xenopus egg extracts shifted to a mitotic state by the addition of purified cyclin, we have for the first time been able to reproduce a mitotic repression of transcription in vitro. Active RNA polymerase III transcription is observed in interphase extracts, but strongly repressed in extracts converted to mitosis. With the topoisomerase II inhibitor VM-26, we demonstrate that this mitotic repression of RNA polymerase III transcription does not require normal chromatin condensation. Similarly; in vitro mitotic repression of transcription does not require the presence of nucleosome structure or involve a general repressive chromatin-binding protein, as inhibition of chromatin formation with saturating amounts of non-specific DNA has no effect on repression. Instead, the mitotic repression of transcription appears to be due to phosphorylation of a component of the transcription machinery by a mitotic protein kinase, either cdc2 kinase and/or a kinase activated by it. Mitotic repression of RNA polymerase III transcription is observed both in complete mitotic cytosol and when a kinase-enriched mitotic fraction is added to a highly simplified 5S RNA transcription reaction. We present evidence that, upon depletion of cdc2 kinase, a secondary protein kinase activity remains and can mediate this in vitro mitotic repression of transcription.

Animals↗
PubMed1994

Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells.

The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.

2-Aminopurine↗
PubMed1992

Transforming growth factor beta 1 (TGF beta 1) reduces cellular levels of p34cdc2, and this effect is abrogated by adenovirus independently of the E1A-associated pRB binding activity.

We have used E1A probes to study the roles of the p34cdc2 kinase and the retinoblastoma tumor susceptibility gene product (pRB) in transforming growth factor beta 1 (TGF beta 1)-mediated growth suppression in mink lung epithelial (Mv1Lu) cells. In agreement with previous reports, we see a decline in p34cdc2 kinase activity and a loss of pRB phosphorylation after TGF beta 1 treatment. We report here that TGF beta 1 induces not only a change in p34cdc2 kinase activity but a strong repression of p34cdc2 synthesis. Loss of p34cdc2 kinase activity is not seen until the steady-state level of p34cdc2 declines, suggesting that the intra-cellular signals induced by TGF beta 1 affect p34cdc2 at the level of expression, rather than by altering the posttranslational modifications of p34cdc2 that regulate its kinase activity. Infection with adenovirus expressing either wild-type E1A or a mutant E1A (pm928) defective for pRB binding alleviated TGF beta 1-mediated suppression of DNA synthesis, indicating that E1A does not need to bind pRB physically to keep cell growth-suppressing functions from being activated by TGF beta 1. The E1A.928 mutant virus is able to maintain p34cdc2 expression and kinase activity, as well as pRB phosphorylation in the presence of TGF beta 1, which may account for its ability to maintain cell cycle activity without directly sequestering pRB. Overall our results suggest that TGF beta 1 acts by signaling changes at the level of control of G1 gene expression, not at the level of posttranslational modification of p34cdc2 or its substrates.

Adenoviridae↗
PubMed1994

Direct inhibition of the yeast cyclin-dependent kinase Cdc28-Cln by Far1.

Cell cycle arrest of Saccharomyces cerevisiae in G1 by the antimitogen alpha-factor is mediated by activation of a signal transduction pathway that results in inhibition of the cyclin-dependent kinase Cdc28-Cln. The Far1 protein is required for cell cycle arrest and associates with the Cdc28-Cln complex. The kinase activity of Cdc28-Cln was directly inhibited by Far1 both in vivo and in vitro, thus demonstrating that Far1 acts at the final step in the alpha-factor response pathway by inhibiting a G1 cyclin-dependent kinase.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1992

A cdc2-related kinase oscillates in the cell cycle independently of cyclins G2/M and cdc2.

The Eg1 gene in Xenopus laevis is related in sequence to the cdc2+ gene. We show here that the Eg1 gene product (cdk2) possesses histone H1 protein kinase activity and binds to PSTAIR antibodies as well as to Sepharose beads linked to the 13-kDa product of the suc 1 gene (p13suc1). Eg1 protein kinase is active only in an Mr approximately 200,000 complex with other proteins but is not associated with any of the three known Xenopus mitotic cyclins or with any newly synthesized protein in egg extracts that exhibit cell cycle oscillations in vitro. The protein kinase activity of Eg1 oscillates in the mitotic cell cycle, being high in M-phase and low in interphase. Hyperactivation of cdc2 kinase by the addition of cyclin A has no effect on the activity or oscillatory behavior of Eg1. Inhibition of cdc2 kinase activation by emetine or RNase treatment of oscillating extracts does not inhibit the activation of Eg1 but does block deactivation normally seen during exit from mitosis. These results indicate that Eg1 is regulated by a cell cycle clock independently of cyclin and cdc2 kinase.

Amino Acid Sequence↗