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PubMed1993

Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins.

In the budding yeast Saccharomyces cerevisiae, the G1 cyclins Cln1, Cln2 and Cln3 regulate entry into the cell cycle (Start) by activating the Cdc28 protein kinase. We find that Cln3 is a much rarer protein than Cln1 or Cln2 and has a much weaker associated histone H1 kinase activity. Unlike Cln1 and Cln2, Cln3 is not significantly cell cycle regulated, nor is it down-regulated by mating pheromone-induced G1 arrest. An artificial burst of CLN3 expression early in G1 phase accelerates Start and rapidly induces at least five other cyclin genes (CLN1, CLN2, HCS26, ORFD and CLB5) and the cell cycle-specific transcription factor SWI4. In similar experiments, CLN1 is less efficient than CLN3 at activating Start. Strikingly, expression of HCS26, ORFD and CLB5 is dependent on CLN3 in a cln1 cln2 strain, possibly explaining why CLN3 is essential in the absence of CLN1 and CLN2. To explain the potent ability of Cln3 to activate Start, despite its apparently weak biochemical activity, we propose that Cln3 may be an upstream activator of the G1 cyclins which directly catalyze Start. Given the large number of known cyclins, such cyclin cascades may be a common theme in cell cycle control.

Base Sequence↗
PubMed2005

Transcription and autoregulation of the Rv3134c-devR-devS operon of Mycobacterium tuberculosis.

DevR is a transcriptional regulator that mediates the genetic response of Mycobacterium tuberculosis to oxygen limitation and nitric oxide exposure. devR is co-transcribed along with devS, which encodes its cognate sensor kinase, and an upstream gene, Rv3134c. The transcriptional activity of this operon was characterized by primer extension, transcriptional fusion and electrophoretic mobility shift assays (EMSAs) under aerobic conditions. Transcription start points (Tsps) were detected upstream of both Rv3134c and devR, and the major transcript was derived from upstream of Rv3134c. Sequences with similarity to sigma factor consensus elements and to DevR-binding motifs were detected in the vicinity of the Tsps by in silico analysis. EMSAs with promoter regions and DevR protein showed that DevR binds to its own promoters in a sequence-specific manner with differing affinities. Consistent with the primer extension and EMSA data, Rv3134c promoters, and not devR promoters, were determined to be the principal promoters of this operon using reporter assays performed in Mycobacterium smegmatis and Escherichia coli. Furthermore, DevR modulated the activity of both devR and Rv3134c promoters. From these findings it is inferred that the Rv3134c-devR-devS operon is transcribed from multiple promoters and is autoregulated.

Bacterial Proteins↗
PubMed1997

Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae.

Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis.

Cell Cycle Proteins↗
PubMed1996

Differential effect of alkyl chain-modified ether lipids on protein kinase C autophosphorylation and histone phosphorylation.

Analogues of 1-O-octadecyl-2-O-methyl-rac-glycerol-3-phosphocholine (ET-18-OMe), containing a carbonyl group at different positions in the alkyl chain and/or a pentylammonium group in sn-3 of glycerol, were evaluated as inhibitors of protein kinase C (PKC; EC 2.7.1.37). The presence of a carbonyl group in the alkyl chain of Et-18-OMe had a dual role in decreasing the inhibitory effect on histone phosphorylation and activating this reaction at low concentrations of compound. The optimal stimulatory effect was observed with the compound having the carbonyl function in C-7 of the alkyl chain. In contrast, all of these compounds were only inhibitors of PKC autophosphorylation, its potency decreasing progressively with the distance between the carbonyl group and the sn-1 position of glycerol. Replacement of the phosphocholine group of ET-18-OMe by a pentamethylene trimethylammonium group maintained the inhibitory effect on histone phosphorylation and autophosphorylation of PKC, and the simultaneous introduction of a ketone group in C-7 of the alkyl chain did not decrease any of these effects. The effects of all these analogues on PKC autophosphorylation, but not on histone phosphorylation, correlated quite well with their known antiproliferative activity on human tumor cell lines and membranolytic activity.

Animals↗
PubMed1976

[Structure of the active center of the catalytic subunit of histone kinase].

A number of unknown ATP analogues is isolated when studying the structure of the active site of catalytic histonekinase subunit. Adenosine-5'-chloromethanepyrophosphonate adenosine-5'-(beta-bromoethanepyrophosphonate) and adenosine-5'-(p-fluorosulphonylphenylphosphate) were isolated under the reaction of chloromethanephosphonic acid, beta-bromoethanephosphonic acid and n-phenolsulphofluoride respectively with AMP imidazolide. Adenosine-5'-(beta-chloroethylphosphate) was obtained from AMP morpholide and ethylenechorohydrine. Adenosine-5'-chloracetylaminomethanephosphonate and adenosine-5'-(p-fluorosulphonylbenzoylaminomethanephosphonate) were obtained in the reaction of chloroacetyc anhydride and n-fluorosulphonylbenzoylchloride. Adenosine-5'-(p-aminophenylphosphate) is synthesized under the reduction of AMP mononitrophenyl ester. The treatment of the former with chloroacetyc anhydride produced adenosine-5'-(p-chloroacetylaminophenylphosphate. Interaction of ATP analogues obtained and also of early synthesized adenosine-5'-chloromethanephosphonate and adenosine-5'-(beta-bromoethanephosphonate) with homogenous catalytic histonekinase subunit is studied. The decrease in the reaction rate of Hi histone phosphorylation is found to take place. pH optimum of the enzyme inactivation with adenosine-5'-chloromethanepyrophosphonate and adenosine-5'-(beta-chloroethylphosphate) and the protective effect of the substrate (ATP) indicate covalent blocking imidazole ring in the active site. The date obtained suggest that the functional group of the active site of catalytic histonekinase subunit is histidine imidazole ring located close to terminal ATP phosphate.

Adenosine Triphosphate↗
PubMed1995

Inhibition of protein synthesis affects histone H1 kinase, but not chromosome condensation activity, during the first meiotic division of pig oocytes.

The influence of protein synthesis on the regulation of the first meiotic division was studied in pig oocytes. We show that histone H1 kinase activity gradually increases during in vitro culture of pig oocytes, reaching maximum in metaphase I stage after 24 hr of culture. However, in the presence of the protein synthesis inhibitor cycloheximide, histone H1 kinase is not activated during the whole culture period, and after 24 hr it is approximately at the same level as in prophase-stage oocytes. The gradual increase in phosphorylation of six proteins of molecular weights 39, 48, 53, 66, 96, and 120 kDa, observed during the first 24 hr of culture, was not detected when cycloheximide was added to the culture medium. Similarly, the decrease in phosphorylation of a 90-kDa protein was not seen in cycloheximide-treated oocytes. On the other hand, the levels of both MPF components, p34cdc2 and cyclin B, which were found to be nearly constant during the first meiotic division, were not influenced by cycloheximide treatment as revealed by Western blotting. The process of germinal vesicle breakdown (GVBD) was totally blocked by cycloheximide. The condensation of chromatin, however, was not influenced, suggesting that GVBD and chromosome condensation could be regulated independently. The different degrees of MPF activation involved in these processes, as well as the nature of the protein(s) which must be synthesized for triggering GVBD, are discussed.

Animals↗
PubMed1993

The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF.

In unfertilized eggs from vertebrates, the cell cycle is arrested in metaphase of the second meiotic division (metaphase II) until fertilization or activation. Maintenance of the long-term meiotic metaphase arrest requires mechanisms preventing the destruction of the maturation promoting factor (MPF) and the migration of the chromosomes. In frog oocytes, arrest in metaphase II (M II) is achieved by cytostatic factor (CSF) that stabilizes MPF, a heterodimer formed of cdc2 kinase and cyclin. At the metaphase/anaphase transition, a rapid proteolysis of cyclin is associated with MPF inactivation. In Drosophila, oocytes are arrested in metaphase I (M I); however, only mechanical forces generated by the chiasmata seem to prevent chromosome separation. Thus, entirely different mechanisms may be involved in the meiotic arrests in various species. We report here that in mouse oocytes a CSF-like activity is involved in the M II arrest (as observed in hybrids composed of fragments of metaphase II-arrested oocytes and activated mitotic mouse oocytes) and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation. In addition, the presence of an intact metaphase spindle is required for cyclin B degradation. Finally, MPF activity is preferentially associated with the spindle after bisection of the oocyte. Taken together, these observations suggest that the mechanism maintaining the metaphase arrest in mouse oocytes involves an equilibrium between cyclin synthesis and degradation, probably controlled by CSF, and which is also dependent upon the three-dimensional organization of the spindle.

Animals↗
PubMed1998

Irreversible G2-M arrest and cytoskeletal reorganization induced by cytotoxic nucleoside analogues.

The mechanisms by which cytotoxic agents perturb the normal cell biology and cell cycle progression of cancer cells were explored using B16F10 cells genetically modified to express the Herpes Simplex Virus-thymidine kinase gene. Culture in the presence of the nucleoside analogue ganciclovir induced a profound morphological change that required entry of treated cells into S phase and was dependent on prenylated proteins such as those of the rho gene family. Cell cycle arrest occurred in late S phase or G2 phase due to the activation of the G2-M DNA damage checkpoint. This checkpoint control operated at the level of inhibition of the activity of Cdc2/cyclin B and occurred by two mechanisms: (a) p53-mediated up-regulation of p21CIP/WAF1 expression and its association with Cdc2/cyclin B; and (b) prevention of the dephosphorylation of tyrosine 15 of Cdc2. These events occurred in vitro and in vivo, and were shown to mediate bystander killing in this model. The mechanism of cell death seemed to be due to the irreversible cell cycle arrest at the G2-M checkpoint, rather than induction of apoptosis. These data link DNA damage checkpoints with cytoskeletal signaling pathways and the core cell cycle machinery and may represent a general mechanism of cytotoxicity of this class of nucleoside analogues.

Animals↗
PubMed1982

Phosphorylation of histones 1 and 3 and nonhistone high mobility group 14 by an endogenous kinase in HeLa metaphase chromosomes.

We have developed a simple in vitro system for studying phosphorylation in isolated HeLa metaphase chromosomes which utilizes the endogenous protein kinase and phosphoprotein phosphatase activities in the chromosomes. Because the isolated chromosomes retain the specificity of phosphorylation seen in vivo, this system offers unique possibilities for studying the properties and regulation of the kinase and phosphatase by adding exogenous substances and observing their effects. It should also be useful for studying the sites of phosphorylation, since proteins can be more easily labeled to high specific activity with 32P in this system than in vivo. The pattern of proteins phosphorylated in isolated metaphase chromosomes appears to be nearly identical with the pattern found in vivo. Among the histones (H) only H1 and H3 are phosphorylated, but several nonhistone proteins, including high mobility group (HMG) 14, are also phosphorylated. Since HMG 14 has been implicated as a structural protein of actively transcribing chromatin, our results suggest that phosphorylation of chromatin proteins may be involved in the shutoff of transcription during mitosis. Tryptic peptide maps and analysis of the phosphorylated amino acids indicate that H1A, H1B, HMG 14, and H3 are phosphorylated at the same sites in vitro in metaphase chromosomes as in mitotic cells in vivo. The major site of phosphorylation of histone H3, both in vivo and in vitro, has been identified as serine 10. HMG 14 is phosphorylated both at serine and threonine residues.

Adenosine Triphosphate↗
PubMed1995

The role of protein phosphorylation in the assembly of a replication competent nucleus: investigations in Xenopus egg extracts using the cyanobacterial toxin microcystin-LR.

Cell-free extracts of Xenopus eggs support nuclear assembly and DNA replication in vitro. Extracts supplemented with the protein phosphatase inhibitor microcystin-LR displayed various inhibitory effects at different concentrations of the toxin. In the presence of cycloheximide, additions of microcystin did not induce histone H1-kinase activity. Nevertheless, increasing concentrations of microcystin did sequentially prevent DNA replication, nuclear lamina assembly and nuclear envelope assembly. DNA replication was prevented when microcystin was added at 250 nM. Furthermore, this effect could be reversed after the addition of the catalytic sub-unit of protein phosphatase 2A to inhibited extracts. At a concentration of 250 nM microcystin, nuclear membrane assembly, nuclear lamina assembly and nuclear transport all occurred in egg extracts. In addition single-stranded M13 DNA replication was also permitted. However, it appeared that replicase assembly was not completed, since nuclei assembled in microcystin-treated extracts displayed an unusual distribution of proliferating cell nuclear antigen (PCNA). Although PCNA was located at sites that resembled pre-replication foci, this nuclear protein was readily solubilised when nuclei were isolated and extracted sequentially with Triton, nucleases and salts. Despite this, nuclei containing pre-assembled replication forks could synthesise DNA when transferred into microcystin-treated extracts.

Animals↗
PubMed1994

Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs.

Results of several lines of experimentation suggest that sperm-induced egg activation has several features in common with G protein-coupled receptor signal transduction mechanisms. We report that microinjection of GDP beta S into metaphase II-arrested mouse eggs blocks sperm-induced egg activation. Since GDP beta S inactivates both heterotrimeric and monomeric classes of G proteins, the involvement of members of each of these families in sperm-induced egg activation was evaluated. Neither pertussis toxin treatment of eggs nor microinjection of eggs with inhibitory antibodies toward G alpha q blocked sperm-induced egg activation. Nevertheless, microinjection of phosducin, a protein that binds tightly to free G protein beta gamma subunits, specifically inhibited second polar body emission, the fertilization evoked decrease of H1 kinase activity and pronucleus formation. Microinjection of phosducin, however, did not inhibit the fertilization-induced modifications of the zona pellucida and microinjection of beta gamma t did not result in egg activation in the absence of sperm. Inactivation of the monomeric Rho family of G proteins with C3 transferase from Clostridium botulinum inhibited emission of the second polar body and cleavage to the 2-cell stage, but did not affect the modifications of the zona pellucida or pronucleus formation. Microinjection of Rasval12, which is a constitutively active form of Ras, did not result in egg activation in the absence of sperm. Moreover, microinjection of either an anti-Ras neutralizing antibody (Y13-259) or a dominant negative form of Ras (RasT) did not affect events of sperm-induced egg activation. In contrast, microinjection of RasT inhibited embryo cleavage to the 2-cell stage. These results suggest that both heterotrimeric and monomeric G proteins are involved in various aspects of sperm-induced egg activation.

ADP Ribose Transferases↗
PubMed1993

Okadaic acid induces spindle lengthening and disrupts the interaction of microtubules with the kinetochores in metaphase II-arrested mouse oocytes.

The cell cycle is regulated by phosphorylation events via a cascade of protein kinases and phosphatases, but many of their substrates remain unknown. To study whether proteins of the metaphase II-arrested mouse oocyte meiotic spindle are substrates for phosphorylation events, we used okadaic acid (OA), a potent phosphatase inhibitor, upon fully mature spindles. Incubation of oocytes for 3 hr with 1 microM OA led to a dramatic lengthening of the spindle and a disorganization of the metaphase plate. Electron microscope studies revealed that this was due to a disruption of the interactions between the microtubules and the kinetochores. Biochemical analysis including MPM-2 immunoblotting and [32P]phosphate labeling of whole oocytes revealed several changes in the phosphorylation pattern following the OA treatment. Moreover, meiotic spindle purification or microtubule-associated proteins (MAPs) isolation showed that some of these phosphorylations occur on proteins associated with the microtubules or with structures closely related to the spindle. These results suggest that the changes occurring in the microtubule network during the cell cycle are partly due to the phosphorylation of some proteins associated with the microtubules.

Animals↗
PubMed1982

Characterization of highly phosphorylated subcomponents of rat thymus H1 histone.

The phosphorylation of electrophoretically homogeneous preparations of the five major subcomponents of that thymus H1 histone by growth-associated histone kinase isolated from Ehrlich ascites tumor or Novikoff hepatoma cell chromatin results in the introduction of three to six phosphates/molecule into different subcomponents. Fully phosphorylated preparations of subcomponents 1 through 4 consist of H1 molecules containing a uniform number of phosphate groups, and run as single bands in long acid-urea gels. Fully phosphorylated preparations of subcomponent 5 consist of a mixture of molecules containing five and six phosphate groups. Phosphorylation of subcomponents 2, 4, and 5 occurs in both the NH2- and carboxyl-terminal regions of the molecules. Phosphorylation of subcomponents 1 and 3 occurs only in the carboxyl-terminal region. The central globular region of the histones is not phosphorylated. The major sites of phosphorylation in rat H1 histone subcomponents are similar to, but not entirely identical with, the major sites of phosphorylation previously characterized in total calf thymus H1, as determined by comparison of phosphopeptide maps. Highly phosphorylated rat H1 molecules, similar in phosphate content to those found in mitotic cells, have distinct chromatographic properties, compared to lightly phosphorylated molecules of the type found in interphase cells. This change in chromatographic properties appears to depend on the number of phosphate groups present in the histone rather than on the presence of phosphate in any specific sites.

Animals↗
PubMed1992

Evidence for interaction between v-Mos and a p34cdc2 isoform, p35cdk.

The c-mos gene product (c-Mos) encodes a serine/threonine kinase required for activation of pre-MPF (maturation-promoting factor) to MPF in oocytes undergoing meiosis and also for stabilization of MPF leading to metaphase arrest in unfertilized eggs. In order to determine whether the v-mos gene product (v-Mos) causes neoplastic transformation via interaction with cell cycle control elements, we have searched for proteins that interact with v-Mos. Extracts of NIH3T3 cells transformed by v-Mos encoded by Moloney murine sarcoma virus (Mo-MuSV) were examined by gel filtration, by immunoprecipitation with antibodies to a conserved region of p34cdc2, and by binding to beads that contain cross-linked p13suc1, a protein known to bind p34cdc2. Gel filtration detected a 500-kDa complex that contained v-Mos and a p34cdc2 isoform, termed p35cdk. The 500-kDa macromolecular complex also exhibited histone H1 phosphorylation activity, consistent with the presence of a cdc2 isoform. The identity of p35cdk is based on its recognition by anti-cdc2 PSTAIR but not by anti-cdc2 C-terminal antibodies, which detect authentic p34cdc2. Structures containing v-Mos and p35cdk were also detected by experiments involving co-immunoprecipitation of v-Mos with anti-cdc2 PSTAIR antibodies. Furthermore, both v-Mos and the p35cdk co-precipitated with p13suc1-Sepharose beads. Our findings raise the possibility of a v-Mos-p35cdk regulatory interaction in cells transformed by Mo-MuSV.

3T3 Cells↗
PubMed1999

Regulation of protein kinase C delta by estrogen in the MCF-7 human breast cancer cell line.

We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) delta in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC delta isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC delta was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC delta expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC delta protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10(-10)-10(-8) M estrogen for 7 days down-regulated specifically PKC delta mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC delta expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor beta while the MCF-7 cells expressed predominantly estrogen receptor alpha and barely detectable levels of estrogen receptor beta. These results suggest that the differential ability of estrogen to regulate PKC beta expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.

Animals↗
PubMed1989

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.

Animals↗
PubMed1998

Natural (alpha-tocopherol) and synthetic (phenosan potassium salt) antioxidants regulate the protein kinase C activity in a broad concentration range (10(-4)-10(-20) M).

The effects of natural lipid-soluble antioxidant, alpha-tocopherol (alpha-TP), and the synthetic water-soluble antioxidant, phenosan potassium salt (Ph-K), in a broad range of concentrations down to ultralow doses (10(-4)-10(-20) M) on the activity of protein kinase C (PKC) have been studied. It was shown that alpha-TP is a potent inhibitor of the rabbit heart enzyme: the maximum extent of inhibition is 80%. The effects of alpha-TP on the main kinetic parameters of the PKC activity differ at the alpha-TP physiological (10(-4) M) and ultralow (10(-14) M) concentrations: at 10(-14) M, alpha-TP acts as an allosteric inhibitor with Hill's coefficient about 2 and doubles the PKC affinity to the substrate (histone H-1). It was concluded that alpha-TP is more efficient inhibitor at ultralow concentration. Ph-K added to normal (A7r5 rat vascular smooth muscle cells, VSMC) and tumor cells (Saos-2 human osteosarcoma) growing in a culture has been found to be a PKC superactivator. The maximum activation is 400-500%, which is more than two times as high as the effect of the best activator of this enzyme, phorbol ester (TPA). It was demonstrated that irrespective of the effector action (activation or inhibition), the dose-effect curves are of the bimodal type with two maxima at the high (or physiological) (10(-4)-10(-7) M) and ultralow (10(-14)-10(-19) M) concentrations of the antioxidants and the so-called "silence zones" between them, in which the effect of antioxidants is significantly reduced or absent (tumor cells). For the first time, these bimodal curves were observed at the enzyme level. The results obtained are discussed considering various hypotheses of the effect of ultralow doses of biologically active compounds and the PKC activity regulation in normal and tumor cells by the antioxidants.

Animals↗
PubMed1987

Arachidonate metabolites, platelet-activating factor, and the mobilization of protein kinase C in human polymorphonuclear neutrophils.

In contrast to our previous report (Biochem. Biophys. Res. Comm. 134:587, 1986), we now find that protein kinase C (PKC) is mobilized in human polymorphonuclear neutrophils (PMN) stimulated with platelet-activating factor (PAF) or leukotriene (LT)B4. Thus nanomolar concentrations of each compound caused PMN to lose cytosolic, PKC-specific protein phosphorylating activity, as well as receptors for phorbol myristate acetate (PMA). Smaller gains in membrane-associated PMA receptors accompanied these changes. Diacylglycerol and PMA had very similar effects on PKC. However, unlike these direct PKC activators, PAF and LTB4 induced only moderate decreases in cytosolic PKC; acted only on PMN pretreated with cytochalasin B; did not mobilize PKC in disrupted PMN or activate PKC in a cell-free system; and with respect to PAF, induced responses that partially reversed within 30 min. Furthermore, PAF, LTB4, and several of their structural analogues mobilized PKC at concentrations correlating closely with their respective affinities for cellular LTB4 or PAF receptors. Thus PAF and LTB4 acted by indirect and apparently receptor-mediated mechanisms. Four observations indicated that the cytochalasin B-dependent degranulating actions of PAF and LTB4 involved PKC. First, PKC mobilization and degranulation occurred at the same stimulus concentrations. Second, 5-hydroxyicosatetraenoate dramatically enhanced both PKC mobilization and degranulation when elicited by PAF; it had relatively little influence on LTB4-induced responses. Third, PAF-induced mobilization (t1/2 less than 7 sec) preceded degranulation (t1/2 approximately 20 sec). Finally, a PKC blocker, polymyxin B, was similarly effective in inhibiting degranulation responses to PAF, LTB4, and PMA. Because stimulated PMN may produce and use PAF, LTB4, and 5-hydroxyicosatetraenoate as secondary intracellular mediators, our results implicate PKC as a central and potentially critical regulator of function.

Arachidonic Acid↗