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PubMed1984

S-100 and other acidic proteins promote Ca2+-independent phosphorylation of protamine catalyzed by a new protein kinase from brain.

A new protein kinase modulated by S-100 (tentatively referred to as protein kinase X) was partially purified from pig brain extracts. The activity of protein kinase X, which was independent of Ca2+, was demonstrated when protamine (free base), but not protamine sulfate and other proteins (including histone), was used as substrate. The enzyme activity, found to distribute in both soluble and particulate fractions and to occur at the highest level in brain compared with other tissues (heart, kidney, liver, skeletal muscle, spleen, and testis) of rats, was also modulated by other acidic proteins (calmodulin, troponin C, and stimulatory modulator) in a Ca2+ -independent manner. S-100 and other acidic proteins appeared to function as "substrate modifiers" by interacting with protamine (a highly basic protein), but not with the enzyme, thus rendering protamine in the complex a superior phosphate acceptor. The two isoforms of S-100 (i.e., a and b) were equally effective. Although the enzyme was not inhibited by many agents (trifluoperazine, melittin, cytotoxin I, polymyxin B, and spermine) shown to inhibit markedly phospholipid/Ca2+- or calmodulin/Ca2+ -stimulated protein kinase, gossypol was found to inhibit specifically protein kinase X. The present findings suggest that S-100, a major acidic protein specific to nervous system, may promote phosphorylation by protein kinase X of certain neural proteins resembling protamine or containing protamine-like domains, in addition to its presumed role of a low-affinity Ca2+ -binding protein.

Animals↗
PubMed2000

Detection of a mammalian histone H4 kinase that has yeast histidine kinase-like enzymic activity.

A well characterized histidine kinase purified from yeast has been shown to phosphorylate histone H4 on a histidine residue. This enzyme is unlike the two-component histidine kinases predominantly found in prokaryotes. Until now, a histidine kinase similar to this yeast enzyme has not been purified from a mammalian source. By using a purification scheme similar to that used to purify the yeast histidine kinase, a protein fraction with histone H4 kinase activity has been isolated from porcine thymus. The yeast histidine kinase was shown to be detectable using an in-gel kinase assay system and using this system, four major bands of histone H4 kinase activity were apparent in the porcine thymus preparation. Through the use of immunoprecipitation, alkaline hydrolysis and subsequent phosphoamino acid analysis it has been demonstrated that this partially purified kinase fraction is capable of phosphorylating histone H4 on histidine. In conclusion, an preparation has been made from porcine thymus that contains histone H4 kinase activity and at least one of the kinases present in this preparation is a histidine kinase.

Amino Acids↗
PubMed1996

Insulin activates a p21-activated kinase in muscle cells via phosphatidylinositol 3-kinase.

Insulin activates rapidly a complex cascade of lipid and protein kinases leading to stimulation of mitogenic and metabolic events. Here we describe a renaturable kinase of 65 kDa (PK65) that becomes rapidly activated by insulin in differentiated L6 muscle cells (myotubes) and can phosphorylate histones immobilized in polyacrylamide gels. Insulin activation of PK65 was abolished by the tyrosine kinase inhibitor erbstatin and by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, but was unaffected by inhibitors of protein kinase C or of the activation of p70(S6K). Recently, a number of protein kinases have been described which become activated through interaction with the small GTP-binding proteins Rac and Cdc42 (21-ctivated inases, or PAKs) and lead to activation of the stress-induced mitogen-activated protein kinase (MAPK) p38 MAPK. Two different polyclonal antibodies recognizing the carboxyl-terminal or the Rac-binding domain of a 65-kDa PAK (PAK65) immunoprecipitated the myotube PK65. The insulin-induced activation of PK65 in myotubes was detectable following immunoprecipitation of the kinase. Furthermore, PK65 associated with and became activated by glutathione S-transferase-Cdc42Hs in the presence of GTPgammaS (guanosine 5'-3-O-(thio)triphosphate). In myotubes insulin also induced tyrosine phosphorylation of p38 MAPK. However, this phosphorylation was insensitive to wortmannin, indicating that p38 MAPK is not activated by PK65 in insulin-stimulated cells. The results suggest that insulin activates in muscle cells a renaturable kinase (PK65) closely related to PAK65. Tyrosine kinases and PI 3-kinase act upstream of PK65 in the insulin signaling cascade. Insulin activates p38 MAPK in myotubes, but this occurs by a pathway independent of PI 3-kinase and PK65.

Animals↗
PubMed1993

Vitamin A-deficient testis germ cells are arrested at the end of S phase of the cell cycle: a molecular study of the origin of synchronous spermatogenesis in regenerated seminiferous tubules.

Vitamin A deficiency in male rats arrests spermatogenesis and leads to the loss of advanced germ cells. Retinol treatment of these rats seems to result in a synchronous spermatogenesis initiated from the remaining type A1 spermatogonia. To determine at a molecular level the onset of the M phase of the cell cycle occurring in the retinol-treated germ cells, the H1 histone kinase activity associated with the cdc2 kinase/cyclin B complex was measured. This kinase activity is an excellent molecular indicator of mitosis (M phase) since it is known to be high only for approximately 2 h between the G2/M phase transition of the cell cycle and the metaphase of mitosis. This activity was low in vitamin A-deficient testis, increased by 4 h after retinol treatment, and reached a 15.6-fold level at 12 h. These results suggest that the germ cells of vitamin A-deficient testis begin to enter the M phase around 4 h after retinol injection, with the highest percentage of cells entering at 12 h. These results are consistent with placing the origin of synchronous spermatogenesis in regenerated seminiferous tubules at the end of the S phase of the cell cycle.

Amino Acid Sequence↗
PubMed1975

Purification of a protein kinase and two phosphate acceptor proteins from vaccinia virions.

A novel protein kinase that requires protamine as an activator to catalyze the phosphorylation of viral acceptor proteins was extracted from vaccinia virus cores with deoxycholate and purified 250-fold by DNA-cellulose and DEAE-cellulose column chromatography. The enzyme has a molecular weight of 62,000 as determined by sucrose gradient sedimentation. Two heat-stable phosphate acceptor proteins were extracted from virus particles with a nonionic detergent and purified by heat treatment, precipitation with organic solvents, and CM-cellulose chromatography. The molecular weights of the phosphate acceptor proteins, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, are 38,500 and 11,700.

Centrifugation, Density Gradient↗
PubMed1993

Cyclin E and cyclin A as candidates for the restriction point protein.

Progression of cells into S phase is proposed to be determined by accumulation of a labile protein (the restriction point protein R; A. B. Pardee, Proc. Natl. Acad. Sci. USA, 71: 1286-1290, 1974). We report here that cyclin E and cyclin A proteins as well as their dependent histone H1 kinases satisfy all of the criteria for the R protein, which includes late G1 phase increase, an excess delay of appearance after inhibition of protein synthesis in nontransformed cells, and a faster recovery in transformed cells. We suggest that the molecular basis of the R protein could be cyclin production and inactivation.

3T3 Cells↗
PubMed1983

Characterization of a cyclic nucleotide-independent protein kinase highly active in human adrenocortical carcinoma.

Study of the protein kinase activity pattern of four human adrenocortical carcinoma showed that in all the samples examined a histone kinase (HK III) activity was present at high level, whereas it was barely detectable in normal tissue. HK III was separated from other known adrenocortical protein kinases by diethylaminoethyl cellulose chromatography. Isolated HK III exhibited a histone (H2B) protamine-phosphotranferase selectivity and used adenosine triphosphate but not guanosine triphosphate as phosphate donor. Serine was identified as the only target amino acid phosphorylated in the protein substrate. HK III showed an apparent molecular weight of 65,000 upon gel filtration and an apparent sedimentation coefficient of 3.7S. HK III activity was cyclic adenosine 3':5'-monophosphate independent and was not influenced by calcium, calmodulin, polyamines, and heparin. The significance of HK III activity in adrenocortical carcinoma extracts at a high level as compared to that of normal tissue remains to be clarified with regard both to its possible relationship with tumoral cell growth and differentiation processes and to its potential interest as a marker of human tumoral tissue activity.

Adrenal Cortex Neoplasms↗
PubMed1994

Inhibition of G1 phase cyclin dependent kinases by transforming growth factor beta 1.

Transforming growth factor beta 1 (TGF beta 1) inhibits epithelial cell proliferation late in the G1 phase of the cell cycle. We examined the effect of TGF beta 1 on known late G1 cell cycle regulators in an attempt to determine the molecular mechanism of growth inhibition by this physiological inhibitor. The results demonstrate that TGF beta 1 inhibits the late G1 and S phase specific histone H1 kinase activity of p33cdk2. This inhibition is not due to TGF beta 1's effect on p33cdk2 synthesis, but rather due to its negative effects on the late G1 phosphorylation of p33cdk2. It is also shown that TGF beta inhibits both late G1 cyclin A and cyclin E associated histone H1 kinase activities. The inhibitor has no effects on the synthesis of cyclin E but is shown to inhibit the synthesis of cyclin A protein in a cell cycle dependent manner. If TGF beta 1 is added to cells which have progressed further than 8 hours into G1, then it is without inhibitory effect on cyclin A synthesis. These effects of TGF beta 1 on late G1 cell cycle regulators correlate well with its inhibitory effects on cellular growth and suggest that these G1 cyclin dependent kinases might serve as targets for TGF beta 1-mediated growth arrest.

Animals↗
PubMed1994

Cyclin/cdk2 complexes in the nucleus of HeLa cells.

Two different fractions of cdk2 and cdc2 have been found in the nucleus of HeLa cells. One, which can be extracted by nuclease treatment, possibly associated with DNA- or RNA-containing structures and another one, which is bound to the nuclear matrix. Nuclear cdk2 forms high molecular weight complexes which migrate at the same position as DNA polymerase alpha and proliferating cell nuclear antigen in sucrose gradient centrifugation experiments. These results suggest that nuclear cdk2 complexes could be associated with the replication factories. Immunoprecipitation experiments reveal that nuclear cdk2 complexes display histone H1-kinase activity and phosphorylate a protein of 18 kDa which is present in these complexes.

Blotting, Western↗
PubMed1993

TGF beta inhibition of Cdk4 synthesis is linked to cell cycle arrest.

Transforming growth factor beta 1 (TGF beta 1) causes G1 growth arrest and the accumulation of unphosphorylated retinoblastoma protein (Rb) in responsive cells. Cdk4 (cyclin-dependent kinase), a major catalytic subunit of the mammalian D-type G1 cyclins, can phosphorylate Rb in vitro, and at least one D-type cyclin, D2, directs the phosphorylation of Rb in vivo. Here we show that TGF beta 1 induces suppression of cdk4 synthesis in G1 in mink lung epithelial cells. Constitutive cdk4 synthesis in these cells led to TGF beta 1 resistance. It also resulted in growth in low serum medium when these cells were released from contact inhibition. Cdk2 activity was also suppressed by TGF beta 1 action, but its constitutive expression failed to override a TGF beta 1-induced G1 block. Hence, the TGF beta 1 block is primarily mediated by cdk4 modulation. Further evidence suggests that TGF beta 1-induced down-modulation of cdk4 leads to inhibition of cdk2 activation and that both events might contribute to TGF beta 1 growth suppression.

Animals↗
PubMed1991

Role for cyclin A in the dependence of mitosis on completion of DNA replication.

THE cyclins were first identified by their cell-cycle-dependent synthesis and destruction and have a key role in the control of mitosis in Xenopus embryonic cell cycles. All higher eukaryotes have at least two types of cyclins, the A- and B-type, which can be distinguished by sequence motifs and the timing of their destruction in the cell cycle. The degradation of both cyclins is required for exit from mitosis, but the activation and destruction of cyclin A occur earlier in the cell cycle than with the B-type cyclins. This suggests that cyclin A has a distinct role in cell-cycle progression. We have used an antisense oligodeoxy-nucleotide directed against cyclin A to investigate this role. Ablation of cyclin A messenger RNA in cytostatic factor/metaphase-arrested extracts of Xenopus eggs, followed by in vitro progression into interphase, resulted in the premature appearance of cyclin B-cdc2-associated H1 kinase activity and premature entry into mitosis. Although cyclin A-ablated extracts could initiate DNA synthesis during interphase, S phase was not completed before entry into mitosis. The effects of cyclin A ablation were reversed by the addition of cyclin A mRNA or cyclin A protein to the extracts.

Animals↗
PubMed1995

Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils.

Several novel protein kinases are known to be rapidly activated in neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP). These kinases include a histone H4 protein kinase and several renaturable kinases with molecular masses of about 69, 63, 49, and 40 kDa. The renaturable kinases can catalyze the phosphorylation of a peptide that corresponds to residues 297-331 of the 47-kDa subunit of the NADPH-oxidase system (p47-phox). Previous studies have indicated that the activation of all of these protein kinases involves an uncharacterized stimulatory pathway and/or novel second messenger. The studies reported herein were undertaken to determine if phosphatidylinositol 3-kinase (PI3-K) is a component of this pathway. We report that certain chromosome derivatives (e.g. 2-(4-morpholinyl)-8-phenylchromone (LY294002)) and wortmannin, which inhibit PI3-K by distinct mechanisms, blocked activation of all of these novel kinases. These antagonists also inhibited the phosphorylation of p47-phox (about 50%) and O2.- release (about 80%) in cells stimulated with fMLP, but not with 4 beta-phorbol 12-myristate 13-acetate. A strong correlation exists between the amounts of these antagonists required to produce 50% inhibition of PI3-K in vitro and O2.- release in vivo. In contrast, a single atom substitution of LY294002 produced a compound (LY303511) that did not inhibit PI3-K. Compound LY303511 did not appreciably inhibit the activation of the novel protein kinases or O2.- generation. These data strongly suggest that PI3-K is involved in the activation of several novel protein kinases in neutrophils, one or more of which may be involved in O2.- release.

Androstadienes↗
PubMed1998

Activation of the p42 mitogen-activated protein kinase pathway inhibits Cdc2 activation and entry into M-phase in cycling Xenopus egg extracts.

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.

Amino Acid Sequence↗
PubMed1993

Simian virus 40 large T antigen associates with cyclin A and p33cdk2.

In this paper we provide evidence that a fraction of large T antigen of simian virus 40 (SV40) interacts with cyclin A and p33cdk2 in both virus-infected and stably transformed cells. Immunoprecipitates of SV40 large T antigen from SV40-infected or SV40 large-T-antigen-transformed cells contain cyclin A, p33cdk2, and histone H1 kinase activity. Conversely, immunoprecipitates of cyclin A from these cells contain SV40 large T antigen. In this respect, SV40 large T antigen has properties similar to those of the E1A oncogene of adenoviruses and the E7 oncogene of human papillomaviruses.

Animals↗
PubMed1996

Cell cycle protein suppression and p21 induction in differentiating Caco-2 cells.

Despite intensive efforts, the exact cellular mechanisms leading to gut differentiation and development remain largely undefined. The cyclins, the cyclin-dependent kinases (Cdks), and the Cdk inhibitors (e.g., p21 and p27) are proteins that are important for cell cycle progression, subsequent growth inhibition, and differentiation of various cell types. The purpose of our study was to better define the role of these cell cycle proteins in gut differentiation using the Caco-2 human cell line, which spontaneously differentiates to a small bowel phenotype, as demonstrated by induction of sucrase-isomaltase (SI) gene expression. We found that protein levels of the cyclins (both D- and E-type) and the Cdks (both Cdk2 and Cdk4) progressively decreased in postconfluent Caco-2 cells. Moreover, cyclin E-associated histone H1 kinase activity decreased in an analogous fashion as the cyclins and Cdks. In contrast, induction of the Cdk inhibitor p21 occurred by 3 days postconfluency, which was before the increase in SI mRNA levels. These changes in the cell cycle proteins, which include a progressive decrease of the cyclins and Cdks and a concomitant induction of p21, suggest an important role for these proteins in Caco-2 cell differentiation. Identifying the cell cycle mechanisms responsible for intestinal cell differentiation will be important to our understanding of both normal gut development as well as gut neoplasia, which involves aberrant regulation of cell cycle arrest.

CDC2-CDC28 Kinases↗
PubMed1997

Cyclins in a dinoflagellate cell cycle.

The dinoflagellates are distinct eukaryotes in having and extranuclear spindle and permanently condensed chromosomes. These cytologic features implicate special adaptations to the molecular mechanisms of cell cycle control. We have demonstrated the presence of cyclin-box-containing polypeptides in dinoflagellates by immunoblotting using peptide-generated antibodies. We identified four major cyclin-box-containing polypeptides. The cell cycle dynamics of these polypeptides were also investigated in synchronized populations, using a newly developed method. Of the four major cyclin-box-containing polypeptides detected, a triplex with apparent molecular weight of 75 kDa did not change appreciably during the cell cycle. For two other cyclin-box-containing polypeptides with apparent molecular weights of 50 and 65 kDa, we observed an early expression in the cell cycle, with the level accumulating and eventually being degraded on the exit of mitosis. At least on cyclin-box-containing polypeptide (50 kDa) was also observed in a protein complex bound to p13suc1 beads. The bound complex head associated histone kinase activity. Variation of this activity corresponded well with the periodic expression of the 50-kDa cyclin-box-containing polypeptide during the cell cycle of Crypthecodinium cohnii. This demonstrates the presence of cyclins and cyclin-dependent kinases in dinoflagellates.

Animals↗