ABSTRACT
BACKGROUND: Two-component systems have emerged as compelling targets for antibacterial drug design for a number of reasons including the distinct histidine phosphorylation property of their constituent sensor kinases. The DevR-DevS/DosT two component system of Mycobacterium tuberculosis (M. tb) is essential for survival under hypoxia, a stress associated with dormancy development in vivo. In the present study a combinatorial peptide phage display library was screened for DevS histidine kinase interacting peptides with the aim of isolating inhibitors of DevR-DevS signaling. RESULTS: DevS binding peptides were identified from a phage display library after three rounds of panning using DevS as bait. The peptides showed sequence similarity with conserved residues in the N-terminal domain of DevR and suggested that they may represent interacting surfaces between DevS and DevR. Two DevR mimetic peptides were found to specifically inhibit DevR-dependent transcriptional activity and restrict the hypoxic survival of M. tb. The mechanism of peptide action is majorly attributed to an inhibition of DevS autokinase activity. CONCLUSIONS: These findings demonstrate that DevR mimetic peptides impede DevS activation and that intercepting DevS activation at an early step in the signaling cascade impairs M. tb survival in a hypoxia persistence model.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Biomimetic Materials/metabolism , Gene Expression Regulation, Bacterial/drug effects , Microbial Viability/drug effects , Mycobacterium tuberculosis/drug effects , Protamine Kinase/antagonists & inhibitors , Protein Kinases/metabolism , DNA-Binding Proteins , Enzyme Inhibitors/metabolism , Mycobacterium tuberculosis/enzymology , Oxygen/metabolism , Peptide Library , Peptides/metabolism , Stress, PhysiologicalABSTRACT
Antibodies are widely utilized in cell and molecule biology for immunoblots, immunostaining, immunoprecipitation, immunoaffinity purification, and immunoassay. Some antibodies can be used for in vivo inhibition experiments. These antibodies bind to their target molecules and neutralize their functions, providing functional information in the study of their biological role. Here, we describe our methods for obtaining inhibitory antibodies against desired proteins. We then describe in the starfish oocyte system how to inhibit a target protein, even in the nucleus, by injection of antibody into the cytoplasm, and how to evaluate antibody inhibition of cell cycle regulators in small numbers of oocytes.
Subject(s)
Antibodies/pharmacology , Oocytes/drug effects , Starfish/drug effects , Animals , Antibodies/isolation & purification , Cell Culture Techniques , Cell Separation , Cell-Free System , Cells, Cultured , Enzyme Assays , Microinjections , Oocytes/enzymology , Protamine Kinase/antagonists & inhibitors , Protamine Kinase/immunology , Protamine Kinase/metabolism , Rabbits , Starfish/cytologyABSTRACT
Antitubercular treatment is directed against actively replicating organisms. There is an urgent need to develop drugs targeting persistent subpopulations of Mycobacterium tuberculosis. The DevR response regulator is believed to play a key role in bacterial dormancy adaptation during hypoxia. We developed a homology-based model of DevR and used it for the rational design of inhibitors. A phenylcoumarin derivative (compound 10) identified by in silico pharmacophore-based screening of 2.5 million compounds employing protocols with some novel features including a water-based pharmacophore query, was characterized further. Compound 10 inhibited DevR binding to target DNA, down-regulated dormancy genes transcription, and drastically reduced survival of hypoxic but not nutrient-starved dormant bacteria or actively growing organisms. Our findings suggest that compound 10 "locks" DevR in an inactive conformation that is unable to bind cognate DNA and induce the dormancy regulon. These results provide proof-of-concept for DevR as a novel target to develop molecules with sterilizing activity against tubercle bacilli.
Subject(s)
Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Drug Design , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/physiology , Aerobiosis , Antitubercular Agents/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Computational Biology , DNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial/drug effects , Hypoxia/metabolism , Models, Molecular , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Protamine Kinase/antagonists & inhibitors , Protamine Kinase/chemistry , Protamine Kinase/metabolism , Protein Conformation , Sequence Homology , Transcription, Genetic/drug effectsABSTRACT
NF-kappaB plays an important role in oncogenesis. Recently, we have demonstrated that loss of p53 function enhances DNA binding and transcriptional activities of NF-kappaB via IKKalpha and IKKbeta, and that glycolysis, activated by NF-kappaB, has an integral role in oncogene-induced cell transformation. Here, we show that ectopically expressed p53 induces acetylation and phosphorylation at Ser 536 of p65, an NF-kappaB component, and enhances DNA-binding activity of NF-kappaB. However, activated p53 suppresses transcriptional activity of NF-kappaB. Under non-stimulating conditions, p65 formed a complex with IKKalpha and IKKbeta. Activated p53 bound to p65 on DNA and disrupted binding of p65 to IKKbeta. Moreover, histone H3 kinase activity, which requires transcriptional activation of NF-kappaB, was diminished by p53. Thus, activated p53 may suppress transcriptional activity of NF-kappaB through inhibition of IKK and histone H3 kinase on DNA, suggesting a novel p53-mediated suppression system for tumorigenesis.
Subject(s)
DNA/metabolism , Transcription Factor RelA/antagonists & inhibitors , Transcription Factor RelA/metabolism , Transcriptional Activation , Tumor Suppressor Protein p53/metabolism , Acetylation , Animals , Humans , I-kappa B Kinase/antagonists & inhibitors , I-kappa B Kinase/metabolism , Mice , Phosphorylation , Protamine Kinase/antagonists & inhibitors , Protamine Kinase/metabolism , Protein Binding , Serine/metabolismABSTRACT
The spatial and temporal control of histone modifications is crucial for precise regulation of chromatin structure and function. Here we report that phosphorylation of H2A at threonine 119 (T119) is enriched at centromere regions in Drosophila mitosis. We found that the Aurora B kinase complex is essential for this phosphorylation at centromeres, while Polo kinase is required to down-regulate H2A phosphorylation on chromosome arms in mitosis. Cyclin B degradation triggers loss of centromeric H2A phosphorylation at anaphase onset. Epistasis analysis indicated that Polo functions upstream of the H2A kinase NHK-1 but parallel to Aurora B. Therefore, multiple mitotic kinases work together to specify the spatial and temporal pattern of H2A T119 phosphorylation.
Subject(s)
Centromere/metabolism , Drosophila Proteins/metabolism , Drosophila/physiology , Histones/metabolism , Protamine Kinase/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Aurora Kinases , Centromere/chemistry , Drosophila/cytology , Drosophila/genetics , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/genetics , Histones/analysis , Mitosis , Phosphorylation , Protamine Kinase/antagonists & inhibitors , Protamine Kinase/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/geneticsABSTRACT
This study was carried out to compare the effects of the combination of ionomycin with a H1-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on the development of reconstituted bovine eggs. For this study, the enucleated bovine oocytes were injected with a presumptive primordial germ cell pre-treated with 1% sodium citrate, and randomly allocated into three activation groups: Group 1 (ionomycin 5 microm, 5 min), Group 2 (ionomycin + DMAP 1.9 mm, 3 h), and Group 3 (ionomycin + SPP 2 mm, 3 h). The reconstituted eggs were compared on the rates of cleavage and development with the blastocyst stage and the ploidy of embryos at 96 h post-activation. Cleavage rates and blastocyst development in Groups 1, 2 and 3 were 7 and 0%, 63 and 17%, and 53 and 14%, respectively. The chromosomal composition differed significantly (p < 0.05) among treatments. Although the embryos in Group 1 had significantly lower developments, 60% of embryos evaluated had diploid chromosomal sets. In contrast, approximately 60% of embryos in Group 2 had abnormal ploidy (21% polyploid and 38% mixoploid). In Group 3, the appearance of abnormal chromosome sets was reduced with the proportion of diploid embryos being increased to 86% (19 of 22), significantly higher (p < 0.05) than in Group 2. It can be concluded that the use of SPP with ionomycin reduces greatly the incidence of chromosomal abnormalities, and may be applicable for the activation of nuclear transplant bovine embryos.
Subject(s)
Adenine/analogs & derivatives , Adenine/pharmacology , Blastocyst/drug effects , CDC2 Protein Kinase/antagonists & inhibitors , Clone Cells/drug effects , Diphosphates/pharmacology , Ionomycin/pharmacology , Protamine Kinase/antagonists & inhibitors , Adenine/administration & dosage , Animals , Blastocyst/physiology , Cattle , Clone Cells/physiology , Diphosphates/administration & dosage , Female , Ionomycin/administration & dosage , Pregnancy , Treatment OutcomeABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Cytoskeleton/drug effects , G2 Phase/drug effects , Ganciclovir/pharmacology , Mitosis/drug effects , Animals , Cyclin B/antagonists & inhibitors , DNA Damage , Female , Lovastatin/pharmacology , Mice , Mice, Inbred C57BL , Protamine Kinase/antagonists & inhibitors , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Cell Cycle/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Nicotiana/cytology , Nicotiana/physiology , Plants, Toxic , Purines/pharmacology , 2,4-Dichlorophenoxyacetic Acid/pharmacology , Aphidicolin/pharmacology , Cells, Cultured , Cyclin-Dependent Kinases/biosynthesis , G2 Phase , Mitosis , Protamine Kinase/antagonists & inhibitors , Protamine Kinase/biosynthesis , Roscovitine , Nicotiana/drug effectsABSTRACT
Neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe (fMLP) are known to exhibit a rapid and transient activation of a histone H4 kinase that may function in a stimulatory pathway downstream of phosphatidylinositol 3-kinase. The activity of this histone kinase in unstimulated neutrophils and cells treated with 1.0 microM fMLP for 10 sec was 8.8 +/- 5 and 43 +/- 2 pmol P/min per 10(7) cells, respectively. In this paper, we report that unstimulated neutrophils contain a latent H4 kinase in the 100,000 x g soluble fraction that can be markedly activated by treatment with trypsin. The values for the untreated and trypsin treated enzyme were 5.5 +/- 1.0 and 63.6 +/- 18 pmol P/min per 10(7) cell-equivalents, respectively. This kinase was insensitive to a selective antagonist of protein kinase C (i.e., 50 microM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) but completely blocked by 100 nM staurosporine. Only a single peak of activity was observed for this enzyme when the 100,000 x g supernatant fraction was fractionated on either an exclusion (KW-803) or an anion exchange column (DEAE), or during isoelectric focusing. The molecular weight of the latent kinase was 64 +/- 6 kDa and the isoelectric point was 7.6 +/- 0.1. During all fractionation procedures, the H4 kinase co-chromatographed with a trypsin-activated kinase that catalyzed the phosphorylation of a peptide which corresponds to residues 297-331 of the 47 kDa subunit of the NADPH-oxidase complex (p47-phox). The properties of the trypsin-activated H4 kinase from unstimulated neutrophils are very similar to those reported for this enzyme from fMLP-stimulated cells.
Subject(s)
Neutrophils/enzymology , Protamine Kinase/metabolism , Adenosine Triphosphate/pharmacology , Alkaloids/pharmacology , Amino Acid Sequence , Animals , Cell Fractionation , Enzyme Activation , Enzyme Inhibitors/pharmacology , Enzyme Stability , Guinea Pigs , Histones/metabolism , Isoelectric Point , Magnesium Chloride/pharmacology , Molecular Sequence Data , Molecular Weight , NADPH Oxidases , Phosphoproteins/metabolism , Phosphorylation , Protamine Kinase/antagonists & inhibitors , Protamine Kinase/chemistry , Protamine Kinase/isolation & purification , Staurosporine , Trypsin/metabolismABSTRACT
Calcium regulates progression through several checkpoints in the cell cycle, including the G1/S-phase transition, G2/M-phase transition, and exit from mitosis. In the GH4C1 rat pituitary cell line, calcium mobilizing polypeptides and calcium channel activation inhibit cell proliferation. This report examines the effects of maitotoxin (MTX), an activator of type L voltage-dependent calcium channels (L-VDCC), on calcium influx and cell cycle progression in GH4C1 cells. MTX causes both a block from G1 to S-phase and a concentration-dependent accumulation of cells in G2+M. MTX does not increase the mitotic index; thus, sustained calcium channel activation by MTX results in an accumulation of cells in G2. In order to temporally localize the MTX-induced G2 block relative to cell cycle regulatory events at the G2/M transition, we assessed the relative activity of two cell cycle regulatory protein kinases, CDC2 and CDK2, in MTX-treated cells. CDC2-specific histone kinase activity in MTX-treated cells is lower than either in cells blocked in mitosis with the microtubule destabilizing agent demecolcine or in randomly cycling cells. In contrast, the activity of CDK2 is highest in MTX-treated cells, consistent with a G2 block prior to CDC2 activation. Together, these results implicate with a G2 block prior to CDC2 activation. Together, these results implicate calcium as an intracellular signal required for progression through G2 phase of the cell cycle prior to CDC2 kinase activation.
Subject(s)
CDC2 Protein Kinase/antagonists & inhibitors , Calcium Channel Agonists/pharmacology , Cell Cycle/drug effects , Marine Toxins/pharmacology , Oxocins , Animals , CDC2 Protein Kinase/metabolism , Calcium/physiology , Calcium Channels/metabolism , Cell Division/physiology , Cell Line/cytology , Cell Line/enzymology , DNA/biosynthesis , Electric Conductivity , G1 Phase/drug effects , G2 Phase/drug effects , Mitosis/drug effects , Pituitary Gland/cytology , Protamine Kinase/antagonists & inhibitors , Protamine Kinase/metabolism , Rats , S Phase/drug effects , Signal Transduction/physiology , Thymidine/metabolism , Tritium/metabolismABSTRACT
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.
Subject(s)
CDC2 Protein Kinase/metabolism , Maturation-Promoting Factor/metabolism , Protamine Kinase/metabolism , Animals , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/biosynthesis , Cell Cycle/drug effects , Cell Cycle/physiology , Cells, Cultured , Enzyme Activation , Epithelial Cells , Epithelium/drug effects , Epithelium/enzymology , Lung/cytology , Lung/drug effects , Lung/enzymology , Mimosine/pharmacology , Mink , Molecular Weight , Phosphorylation , Protamine Kinase/antagonists & inhibitors , Protamine Kinase/biosynthesis , S Phase/drug effects , S Phase/physiologyABSTRACT
The present study investigated the role of intracellular Ca2+ (Ca2+i) elevation on the inactivation of maturation promoting factor (MPF) in rabbit oocytes. The effects of the number of Ca2+ stimulations and of the amplitude of Ca2+i elevation on the profile of histone H1 kinase activity were determined. A Ca2+ stimulation consisted of transferring mature oocytes from culture medium to 0.3 M mannitol containing 0.1-1.0 mM CaCl2, and pulsing them at 1.25 kV/cm for 10 microseconds, or microinjecting 2-8 mM CaCl2 into the oocyte cytoplasm. The number of electrically-induced Ca2+ stimulations was varied, and amplitude of the Ca2+i rise was controlled by altering Ca2+ concentration in the pulsing medium or the injection pipette. Ca2+i concentration was determined with fura-2 dextran; oocytes were snap-frozen at indicated time points and assayed for H1 kinase activity. The activity was quantified by densitometry and expressed as a fraction of activity in nonstimulated oocytes. Electrically-mediated Ca2+i rises inactivated H1 kinase in a manner dependent on the number of Ca2+ stimulations. A single Ca2+ stimulation inactivated H1 kinase to 30-40% of its initial activity. However, H1 kinase inactivation was only transient, regardless of the amplitude of the electrically- or injection-mediated Ca2+i elevation. Increasing the number of Ca2+ stimulations helped to maintain H1 kinase activity at basal (pronuclear) levels. The results show the necessity of a threshold of Ca2+i concentration to trigger MPF inactivation, and suggest a role for the extended period of time over which Ca2+i oscillates at fertilization.
Subject(s)
Calcium/metabolism , Calcium/pharmacology , Oocytes/enzymology , Protamine Kinase/antagonists & inhibitors , Animals , Cells, Cultured , Electric Stimulation , Kinetics , Oocytes/physiology , Rabbits , Time FactorsABSTRACT
The activities of nuclear histone-H1 kinase and C-kinase as well as the amount of phosphate bound to histone-H1 following partial hepatectomy were studied in rat. It was found that the nuclear histone-H1 kinase activity increased twice within 80 h, first 20 to 30 h, and second at 50 to 70 h after partial hepatectomy. The timing of increase of the enzyme activity correlated with increased amount of bound phosphate. On the other hand, the increase of the C-kinase activities occurred between 5 and 15 h after partial hepatectomy. Antibodies raised against human cdk2, human cyclin-A and mouse cdc2 kinase showed no detectable effect on the nuclear histone H1 kinase activity. These results suggest that phosphorylation of histone-H1 in liver regeneration may be catalysed by a putative kinase(s).
Subject(s)
CDC2-CDC28 Kinases , Histones/metabolism , Liver Regeneration/physiology , Protamine Kinase/metabolism , Animals , Antibodies/pharmacology , Cell Nucleus/enzymology , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , DNA/metabolism , DNA Primase , Hepatectomy , Humans , Male , Mice , Phosphorylation , Protamine Kinase/antagonists & inhibitors , Protamine Kinase/drug effects , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA Nucleotidyltransferases/metabolism , Rats , Rats, WistarABSTRACT
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.
Subject(s)
CDC2-CDC28 Kinases , Cyclin-Dependent Kinases/antagonists & inhibitors , G1 Phase/drug effects , G1 Phase/physiology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Transforming Growth Factor beta/pharmacology , Animals , Cell Line , Cyclin-Dependent Kinase 2 , Cyclins/metabolism , Genes, ras , Mink , Protamine Kinase/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/physiology , S Phase/drug effects , S Phase/physiology , TransfectionABSTRACT
HSA at appropriate concentrations shows cytostatic and/or cytotoxic effects on murine Lewis carcinoma cell line C108. The cytostatic effect is mediated by an arrest in the cell cycle machinery, with accumulation of cells in G2-M. The combination of enzymatic assays, cell cycle kinetics studies and immunoprecipitation shows that HSA causes to a certainty an accumulation of cells in the M phase, while a similar effect in G2 has still to be demonstrated. It also inhibits histone H1 kinase activity up to 95% of that of mitotic cells, having as a direct or indirect target the cdc2 complex.
Subject(s)
CDC2 Protein Kinase/metabolism , Carcinoma, Lewis Lung/enzymology , Protamine Kinase/antagonists & inhibitors , Stearic Acids/pharmacology , Animals , Carcinoma, Lewis Lung/pathology , Cell Cycle/drug effects , DNA, Neoplasm/drug effects , DNA, Neoplasm/isolation & purification , DNA, Neoplasm/metabolism , Flow Cytometry/methods , Hydroxyurea/pharmacology , Kinetics , Mice , Nocodazole/pharmacology , Tumor Cells, CulturedABSTRACT
In this study, we have analyzed the effects of the immunosuppressive agent rapamycin on the activation of highly purified normal human B lymphocytes. When the polyclonal activators Staphylococcus aureus (SA) and soluble CD40 ligand (CD40L) were used to stimulate B cells, rapamycin inhibited both interleukin 2 (IL2)-dependent and -independent proliferation, as well as IL2-dependent differentiation into antibody-secreting cells. Cell cycle analysis indicated that rapamycin inhibited the progression of SA+IL2-stimulated B cells past the mid-G1 phase of the cell cycle. To begin to identify rapamycin-sensitive signaling events essential for B cell activation, we examined the effects of rapamycin on p34cdc2 and p33cdk2 kinase activities. SA+IL2 stimulation induced the activation of both cyclin-dependent kinases. Of interest, rapamycin abrogated the activation of both p34cdc2 and p33cdk2. Our results indicate therefore that rapamycin inhibits a number of SA- and CD40L-inducible events that may be necessary for both entry into S phase and for permitting subsequent B cell differentiation. These studies emphasize the utility of this drug as a tool to begin to dissect the activation pathways utilized by human B cells, as well as to provide implications for the therapeutic use of rapamycin in vivo.
Subject(s)
B-Lymphocytes/drug effects , CDC2-CDC28 Kinases , Cyclin-Dependent Kinases , Immunosuppressive Agents/pharmacology , Polyenes/pharmacology , Protein Serine-Threonine Kinases , Adult , Antibody-Producing Cells/cytology , Antibody-Producing Cells/drug effects , Antibody-Producing Cells/immunology , B-Lymphocytes/cytology , B-Lymphocytes/immunology , CD40 Ligand , CDC2 Protein Kinase/metabolism , Cell Cycle/drug effects , Cell Differentiation/drug effects , Cyclin-Dependent Kinase 2 , DNA/biosynthesis , Humans , In Vitro Techniques , Kinetics , Lymphocyte Activation/drug effects , Membrane Glycoproteins , Protamine Kinase/antagonists & inhibitors , Protein Kinases/metabolism , RNA/biosynthesis , Sirolimus , Staphylococcus aureus/immunology , Tacrolimus/pharmacologyABSTRACT
In mammalian cells, hyperphosphorylation of histone H1 and phosphorylation of histone H3 correlate well with the G2 phase to metaphase condensation of chromosomes, and these phosphorylations most probably have a role in initiating and controlling the entry into mitosis. The protein kinase inhibitor staurosporine has been used to examine the role of H1 and H3 phosphorylations in controlling chromosome condensation in the mouse FM3A cell line. We present evidence that (i) staurosporine inhibits the protein kinases that phosphorylate histone H1 during mitosis, (ii) staurosporine also inhibits the histone H3-specific kinase, (iii) the inhibition of these kinase activities prevent cells from entering mitosis, and (iv) addition of staurosporine to cells already arrested at metaphase by nocodazole causes a rapid dephosphorylation of histones H1 and H3 and the decondensation of the metaphase chromosomes. The results show that the hyperphosphorylation of histone H1 and phosphorylation of histone H3 are required to maintain metaphase chromosomes in their condensed state.
Subject(s)
Alkaloids/pharmacology , Chromosomes/drug effects , Histones/metabolism , Protamine Kinase/antagonists & inhibitors , Animals , Cell Cycle/drug effects , Cell Line , Chromosomes/ultrastructure , Electrophoresis, Polyacrylamide Gel , Female , G2 Phase/drug effects , Histones/isolation & purification , Mammary Neoplasms, Experimental , Metaphase/drug effects , Mice , Mitosis/drug effects , Phosphoproteins/isolation & purification , Phosphoproteins/metabolism , Phosphorylation , Staurosporine , Tumor Cells, CulturedABSTRACT
The plant amino acid mimosine has been shown to reversibly arrest mammalian cells in late G1 phase of the cell cycle. However, the underlying molecular mechanisms of this block are not as yet understood. Here we show that mimosine prevents the serum-stimulated synthesis and activation of histone H1 kinase, a crucial regulator of cell cycle progression. The same effect is observed in logarithmically growing primary cells as well as transformed cells. Concomitantly, hyperphosphorylation of the retinoblastoma tumor suppressor gene product is partially inhibited. These effects are fully reversible, because removal of mimosine restores histone H1 kinase activity and the cells resume growth. Because the activity of histone H1 kinase has been shown to be absolutely required for cell cycle progression, it is conceivable that the cytostatic effect of mimosine is due to its negative effects on synthesis and activity of this enzyme.
Subject(s)
Cell Cycle/drug effects , Mimosine/pharmacology , Protamine Kinase/antagonists & inhibitors , CDC2 Protein Kinase/metabolism , Cell Line, Transformed , Cells, Cultured , Cyclins/metabolism , Down-Regulation , Phosphorylation , Protamine Kinase/metabolism , RNA, Messenger/analysis , Retinoblastoma Protein/metabolismABSTRACT
Upon entering a cell the natural product rapamycin, like the structurally related immunosuppressant FK506, associates with members of the FKBP family of proteins. One or more of the resulting FKBP-rapamycin complexes blocks signaling pathways emanating from some growth factor receptors. Recently, the addition of rapamycin was shown to inhibit the phosphorylation and activation of a 70-kDa ribosomal S6 protein kinase, which normally occurs minutes after the activation of certain cytokine and growth factor receptors. We now report that rapamycin can be added 4 to 6 h after the addition of serum growth factors to quiescent human osteosarcoma cells and still arrest these cells in G1. This window of action correlates with the inducible appearance of a cyclin-dependent kinase (cdk) activity, and the induction of this activity is inhibited by the addition of rapamycin. Furthermore, p36cyclin D1 associates with this cdk protein complex in lysates of untreated cells, but does not associate with this cdk protein complex in lysates of rapamycin-treated cells. Together, these studies demonstrate that FKBP-rapamycin can modulate a cyclin-dependent kinase activity and a cyclin D1-cdk association during early G1 in MG-63 human osteosarcoma cells.
Subject(s)
CDC2-CDC28 Kinases , Carrier Proteins/pharmacology , Cyclin-Dependent Kinases , Cyclins/antagonists & inhibitors , Heat-Shock Proteins/pharmacology , Immunosuppressive Agents/pharmacology , Polyenes/pharmacology , Protein Kinase Inhibitors , Protein Serine-Threonine Kinases , Cyclin-Dependent Kinase 2 , G1 Phase/drug effects , Humans , Osteosarcoma , Protamine Kinase/antagonists & inhibitors , Protein Binding , S Phase/drug effects , Sirolimus , Tacrolimus Binding Proteins , Tumor Cells, CulturedABSTRACT
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.