Insulin-like growth factor-1 receptor and its ligand regulate the reentry of adult ventricular myocytes into the cell cycle.

To determine whether insulin-like growth factor-1 (IGF-1) stimulation in vitro of ventricular myocytes isolated from infarcted hearts is characterized by the reentry of cells into the cell cycle, the expression and kinase activity of cyclins E, A, and B and DNA synthesis were evaluated 5 days after coronary artery occlusion and 24 and 48 h following the addition of IGF-1. Myocytes surviving an acute myocardial infarction were employed because of their increase in surface insulin-like growth factor-1 receptors (IGF-1R). Western blot analysis documented that IGF-1 resulted in an upregulation of cyclins D1, E, A, and B in viable postinfarcted myocytes. Cyclin E- and A-associated histone H1 kinase activity and cyclin D1-associated retinoblastoma protein-associated kinase activity also increased, but cyclin B kinase activity was not enhanced by IGF-1. These changes in cyclins and kinase activities were characterized by a significant increase in the number of cells labeled by bromodeoxyuridine, from approximately 630/10(6) to nearly 9, 000/10(6) myocytes. This latter value was reduced by more than 50% by antisense oligodeoxynucleotide to IGF-1R mRNA. However, IGF-1 stimulation did not induce nuclear mitotic division and cytokinesis. In conclusion, the growth-promoting effect of IGF-1 on adult myocytes is regulated by the density of IGF-1R, which conditions the activation of the replicatory machinery of the cells. The failure of IGF-1 to enhance cyclin B kinase activity may be responsible for a block in the cell cycle and the inability of myocytes to progress through the M phase and subsequently divide.

Roscovitine, a novel cyclin-dependent kinase inhibitor, characterizes restriction point and G2/M transition in tobacco BY-2 cell suspension.

Although the developmental programs of plants and animals differ, key regulatory components of their cell cycle have been conserved. Particular attention has been paid to the role of the complexes between highly conserved cyclin and cyclin-dependent kinases in regulating progression through the cell cycle. The recent demonstration that roscovitine is a potent and selective inhibitor of the animal cyclin-dependent kinases cdc2 (CDK1), CDK2 and CDK5 prompted an investigation into its effects on progression through the plant cell cycle. Roscovitine induced arrests both in late G1 and late G2 phase in BY-2 tobacco cell suspensions. Both block were fully reversible when roscovitine was used at concentrations similar to those used in the animal system. Stationary-phase cells subcultured in the presence of roscovitine were arrested at a 2C DNA content. This arrest was more efficient without exogenous addition of plant growth regulator. Roscovitine induced a block in G1 earlier than that induced by aphidicolin. S-phase synchronized cells treated with roscovitine were arrested at a 4C DNA content at the G2/ M transition. The expression analysis of a mitotic cyclin (NTCYC1) indicated that the roscovitine-induced G2 block probably occurs in late G2. Finally, cells in metaphase were insensitive to roscovitine. The purified CDK/cyclin kinase activities of late G1 and early M arrested cells were inhibited in vitro by roscovitine. The implications of these experimental observations for the requirement for CDK activity during progression through the plant cell cycle are discussed.

Adhesion-dependent control of cyclin E/cdk2 activity and cell cycle progression in normal cells but not in Ha-ras transformed NRK cells.

Loss of adhesion of NRK fibroblasts to an appropriate surface leads to cell cycle arrest in late G1 and failure to produce cyclin A. Previously, we showed that adhesion-dependent expression of cyclin A is transcriptionally regulated. In an effort to identify elements of the adhesion-mediated signal transduction cascade upstream of cyclin A activation, we investigated the expression of cyclin E and its associated kinase activity in adherent and suspended NRK cells. Expression of cyclin E was found to be unaffected by suspension. However, cyclin E complexes immunoprecipitated from extracts prepared from NRK cells 12 h after release from G0 arrest were found to be catalytically inactive in suspended but not in adherent cells. This suspension-induced inhibition of cyclin E-associated kinase activity was not observed in NRK cells transformed by a c-Ha-ras oncogene containing a G12V mutation. When G0-synchronized NRK cells were transfected with a cyclin A promoter:luciferase reporter construct along with expression vectors for either wild-type cdk2 or a dominant-negative cdk2 mutant, transcriptional activation of cyclin A was found to be dependent on catalytically active cdk2. Inhibition of cyclin E/cdk2 complexes has frequently been attributed to association of the cdk inhibitors p21(Cip1) and p27(Kip1). However, no differences between adherent and suspended cells could be observed for either expression or cdk2 association of p21(Cip1) or p27(Kip1), nor were any proteins specifically associated with cdk2 or cyclin E in immunoprecipitates from metabolically labeled cell extracts. These results define a pathway through which an adhesion-generated signal controls cyclin A expression by modulating cyclin E/cdk2 activity.

Dissociation of p34cdc2 complex formation from phosphorylation and histone H1 kinase activity.

The cell cycle inhibitor mimosine was used to examine the activation of the p34cdc2 protein kinase in S phase of the cell cycle. Addition of mimosine to cycling epithelial cells halted cell cycle traverse in S phase, coincident with an inhibition of p34cdc2 histone H1 kinase activity. Mimosine treatment did not alter p34cdc2 synthesis or turnover; however, overall phosphorylation of p34cdc2 was decreased to near undetectable levels. Although activity of p34cdc2 was inhibited, the ability of the protein to form high molecular weight complexes, a phenomenon associated with kinase activation in vivo, was not affected. These results indicate that p34cdc2 complex formation can occur in the absence of phosphorylation and that phosphorylation of p34cdc2 is then required to activate these preformed complexes.

Cdk1 is a marker of proliferation in human lymphoid cells.

To better understand the relationship between the proliferation of human lymphoid cells and the expression of cdk1, a catalytic subunit of the histone H1 kinase (H1K), we examined its mRNA and protein content in 3 B-cell lines: Ramos, Reh-6 and IARC 963. Cells were elutriated according to their position in the cell cycle. Cell fractions were analyzed for cdk1 mRNA and protein cellular content by Northern blot and immunoblot, respectively, as well as for H1K activity. Both mRNA and protein amounts and H1K activity varied according to cell cycle phase, the lowest values being observed in G1-enriched fractions. For comparison, elutriated fractions were also tested for the expression of cdk2 and cdk4 proteins. Both showed some variations among fractions, but they were less clear than those of cdk1. We also tested 29 samples of lymphoid neoplastic and non-neoplastic tissues for proliferative activity (percentage of S and G2/M cells estimated by flow cytometry) and expression of cdk1, cdk2 and cdk4 proteins. We found a significant correlation between the percentage of cells in S or S + G2/M phases and cdk1 protein content but not cdk2 or cdk4 content. We conclude that cdk1 expression in human lymphoid cells varies during the cell cycle at both mRNA and protein levels.

Gibberellin promotes histone H1 kinase activity and the expression of cdc2 and cyclin genes during the induction of rapid growth in deepwater rice internodes.

Partial submergence or treatment with either ethylene or gibberellin (GA) promotes rapid internodal growth in deepwater rice (Oryza sativa L.). Earlier work has shown that GA is the immediate hormonal signal for this growth response, which involves induction of the cell cycle at the G2/M phase transition and subsequent enhancement in the rate of DNA synthesis. In all eukaryotes, onset of mitosis is regulated by the p34cdc2/CDC28 protein kinase, whose activity is assayed by in vitro phosphorylation of histone H1. It was found that GA enhanced the activity of p34cdc2/CDC28-like histone H1 kinase in the intercalary meristem of rice internodes. The enzyme activity showed a sharp peak that correlated with a decrease in the population of cells in the G2 phase during the first 4 h of GA treatment but not with changes in DNA synthesis. The level of histone H1 kinase activity increased again when cell division activity in the intercalary meristem is known to be high. The expression of two cdc2 homologs was examined. The mRNA level of one of these, cdc2Os-2, was increased after 1 h of GA treatment, whereas the mRNA level of the other, cdc2Os-1, was not affected. Two cDNAs, cycOs1 and cycOs2, which show high homology to cyclin cDNAs, were cloned from rice. They share 75.1% sequence identity at the amino acid level, and both of them are encoded by mRNAs of 1.6 kb. Expression of the two corresponding cyclin genes was enhanced by GA, and the time course of the induction was compatible with a role for both cyclins in regulating the G2/M phase transition. The cyclins were expressed in the intercalary meristem and the elongation zone of the internode, but the GA-induced increase in transcript levels was restricted to the meristem only. The results support the hypothesis that induction of mitosis by GA is brought about by increased p34cdc2/CDC28 protein kinase activity, which may be the result of transcriptional activation of the cdc2Os-2, cycOs1 and cycOs2 genes.

Molecular cloning and identification of two types of hamster cyclin-dependent kinases: cdk2 and cdk2L.

We isolated two types of hamster cyclin-dependent kinase 2 (cdk2) cDNAs from BHK21 cells derived from Golden hamsters. One type of cdk2 (cdk2hm) encodes the 32 kDa protein consisting of 298 predicted amino acids and shows strong homology to the cdk2 cDNAs of humans and Xenopus. The other cdk2 (cdk2Lhm) encodes the 38 kDa protein containing the insertion of 48 amino acids in the cdk2hm protein. Immunoblotting analysis suggested that these two types of cdk2 protein exist in mammalian cells. The cdk2hm has the activity of protein kinase, while the cdk2Lhm does not, however, both bind with cyclin E.

Okadaic acid accelerates germinal vesicle breakdown and overcomes cycloheximide- and 6-dimethylaminopurine block in cattle and pig oocytes.

Pig and cattle oocytes, when released from the follicle, spontaneously resume first meiotic division within 20 or 8 hr, respectively. In oocytes of both species, the activity of histone H1 kinase increases during maturation, exhibiting a maximum in metaphase I. Treatment of these oocytes with okadaic acid results in acceleration of germinal vesicle breakdown (GVBD) and of histone H1 kinase activation. This effect is more important in pig oocytes, in which the acceleration rises for 6 hr, as compared to 2 hr in cattle. Moreover, under these conditions, H1 kinase activity measured after 12 hr of culture appears higher than that observed in control metaphase I oocytes. When added to prophase oocytes, both cycloheximide and 6-DMAP (6-dimethylaminopurine) block GVBD and histone H1 kinase activation. Okadaic acid, at a concentration of 2.5 microM, is able to release the inhibitory effect exerted by cycloheximide on histone H1 kinase activity; however, GVBD occurred only in two-thirds of pig and one-quarter of cattle oocytes after 20 hr of culture. In addition, okadaic acid fully reverses the effect of 6-DMAP on H1 kinase activity and on GVBD in both species. The opposite effects of 6-DMAP and okadaic acid on MPF activation are discussed, as well as the nature of the protein, which has to be synthesized during the first meiotic division and may be involved in the MPF activation cascade.

Induction of cyclin mRNA and cyclin-associated histone H1 kinase during liver regeneration.

Cyclins and cyclin-associated cdc kinases are key regulators of oocyte maturation (Maller, J. L. (1990) in The Biology and Medicine of Signal Transduction (Nishizuka, Y., Endo, M., and Tanaka, C., eds) pp. 323-328, Raven Press, New York), yeast cell cycles (Nurse, P. (1990) Nature 344, 503-508), DNA replication in cell-free systems (D'Urso, F., Marraccino, R. L., Marshak, R. R., and Roberts, J. M. (1990) Science 250, 786-791), and amphibian cell proliferative transitions (Hunt, T. (1991) Nature 350, 462-463). The extent to which these regulatory molecules participate in the growth control of differentiated epithelial cells like hepatocytes is unknown. Therefore, we investigated the expression of "G1" (E, C, and D) and "G2/M" (A, B1, and B2) cyclin mRNAs, the relative levels of cyclin A- and B1-associated histone H1-kinase activity, and the appearance of cyclin-associated kinases (p32/p33cdk2 and p33/p34cdc2) in regenerating rat liver and in control tissues from sham hepatectomized rats. To do this, we exploited a battery of human cyclin cDNAs and cyclin antisera that recognize rat molecules. The results suggest an apparent sequence of regeneration-specific changes: 1) elevated and induced expression of cyclins E (2.1 kilobases (kb)) and C (4 kb), and D mRNAs (4 kb), within 12 h, respectively; 2) induction of cyclins A (3.4 and 1.8 kb), B1 (2.5 and 1.8 kb), and B2 (1.9 kb) mRNAs at 24 h; 3) induction of cyclin A- and B1-associated nuclear histone H1 kinase at 24 h; and 4) enhanced levels of PSTAIRE-containing proteins of Mr approximately 32-33 and 33-34 kDa in nuclear extracts from 24-h regenerating liver that co-immunoprecipitate with cyclin A and B1 antisera, respectively. These observations provide an intellectual framework that unifies the biology of hepatocyte mitogenesis, proto-oncogene expression, and the machinery of the cell cycle.

M-phase-specific protein kinase from mitotic sea urchin eggs: cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis.

Histone H1 kinase (H1K) undergoes a transient activation at each early M phase of both meiotic and mitotic cell cycles. The mechanisms underlying the transient activation of this protein kinase were investigated in mitotic sea urchin eggs. Translocation of active H1K from particulate to soluble fraction does not seem to be responsible for this activation. H1K activation cannot be accounted for by the transient disappearance of a putative H1K inhibitor present in soluble fractions of homogenates. Aphidicolin, an inhibitor of DNA synthesis, and actinomycin D, an inhibitor of RNA synthesis, do not impede the transient appearance of H1K activity. H1K activation therefore does not require DNA or RNA synthesis. Fertilization triggers a rise in intracellular pH responsible for the increase of protein synthesis. H1K activation is highly dependent on the intracellular pH. Ammonia triggers an increase of intracellular pH and stimulates protein synthesis and H1K activation. Acetate lowers the intracellular pH, decreases protein synthesis, and blocks H1K activation. Protein synthesis is an absolute requirement for H1K activation as demonstrated by their identical sensitivities to emetine concentration and to time of emetine addition. About 60 min after fertilization, H1K activation and cleavage become independent of protein synthesis. The concentration of p34, a homolog of the yeast cdc2 gene product which has been recently shown to be a subunit of H1K, does not vary during the cell cycle and remains constant in emetine-treated cells. H1K activation thus requires the synthesis of either a p34 postranslational modifying enzyme or another subunit. Finally, phosphatase inhibitors and ATP slow down in the in vitro inactivation rate of H1K. These results suggest that a subunit or an activator of H1K is stored as an mRNA in the egg before mitosis and that full activation of H1K requires a phosphorylation.

M phase-promoting factor: its identification as the M phase-specific H1 histone kinase and its activation by dephosphorylation.

A major protein kinase independent of Ca2+, cyclic nucleotide or diacylglycerol, the activity of which becomes maximal when cells enter M phase, decreases at ana-telophase, and is low during interphase, has been purified to near homogeneity from starfish oocytes and its catalytic subunit identified as p34cdc2. M phase-promoting factor (MPF) was found to co-purify with the M phase-specific kinase throughout its purification. p34cdc2 does not have to be associated with any specific protein for expression of H1 histone kinase or MPF activities. When p34cdc2 is phosphorylated its protein kinase activity is inhibited, preventing entry into M phase, but once p34cdc2 becomes dephosphorylated, its protein kinase activity increases and M phase is initiated. A second peak of MPF activity was separated from p34cdc2 in the ammonium sulfate fraction treated with ATP-gamma-S. It induced p34cdc2 dephosphorylation and the concomitant stimulation of its kinase activity when injected in Xenopus or starfish oocytes.

Increase in type I cyclic adenosine 3':5'-monophosphate-dependent protein kinase activity and specific accumulation of type I regulatory subunits in adenovirus type 12-transformed cells.

Types I and II cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases were compared in extracts from untransformed rat 3Y1 cells and 3Y1 cells transformed by the highly oncogenic human adenovirus type 12. Analysis of the kinases through diethylaminoethyl Sepharose column chromatography showed a significantly increased activity of type I, but not of type II, cAMP-dependent protein kinase in two transformed cell clones tested. 8-Azidoadenosine 3':5'-monophosphate, a sensitive photoaffinity-labeling analogue of cAMP, was used to quantitate the regulatory subunits of the type I kinase. A 3- to 6-fold increase in the amount of incorporation of the photoaffinity label into the Mr 48,000 regulatory subunit of type I kinase was obtained in three transformants tested. The increased incorporation was attributed to an increase in the actual amount of the subunit rather than to an increase in the affinity of type I regulatory subunit for the photoaffinity label. These results suggest that type I cAMP-dependent protein kinase activity may be a biochemical marker for adenovirus transformation.

The hormonal induction of maturation in amphibian oocytes.

The maturation of the amphibian oocyte has been analyzed. Progesterone as well as organomercurials, lanthanum chloride and propranolol rapidly induce maturation. These chemicals are active only is applied on the cell surface. The mechanism seems to be an induction of the migration of Ca2+ from the cell membrane to the cytoplasm. K + may also play a role. Progesterone induced maturation involves synthesis of histone and histone kinase as well as several biologically active but chemically unidentified factors. cAMP does not seem to be directly involved, whereas protein phosphorylation is so.

Protein kinase of bacteriophage T7. 2. Properties, enzyme synthesis in vitro and regulation of enzyme synthesis and activity in vivo.

Protein kinase, which was isolated from cells infected with T7, is indeed a viral gene product. This is shown by DNA-dependent synthesis in vitro. The protein kinase transfers phosphate from ATP to seryl or threonyl residues in protein. The enzyme has only a relative requirement for magnesium ions, but is only active at low ionic strength. The best substrate is lysozyme. T7 protein kinase activity is not stimulated by cyclic 3':5'-AMP and/or cyclic 3':5'-GMP. The T7 protein kinase carries -- SH groups essential for activity. There is indication that the enzyme phosphorylates itself and causes self inactivation, which may explain the fast disappearance of enzyme activity in vivo. Bacteriophage T3 also induces a protein kinase which is similar to the T7-induced enzyme in all respects tested.