Staurosporine-sensitive Protein Kinase Research Articles

Pituitary adenylate cyclase activating polypeptide induces multiple signaling pathways in rat peritoneal mast cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is a high-affinity ligand for at least two types of G-protein coupled receptors, the PACAP type 1 and type 2 receptor. In this study it is demonstrated that the C-terminal PACAP-fragment PACAP(6–27) stimulates serotonin release from rat peritoneal mast cells with higher potency (EC 50: 0.2 vs. 2.0 μM) than the PACAP receptor ligand PACAP(1–27). PACAP-induced degranulation of rat peritoneal mast cells was abolished by pertussis toxin and by benzalkonium chloride (IC 50: 9.1 μg/ml) indicating the involvement of heterotrimeric G-proteins of the G i-type. The PACAP effect was also reduced by inhibitors of the phosphatidylinositol specific phospholipase C ((U73122), IC 50: 4 μM; (ET-18-O-CH 3), IC 50: 18 μM), by D609, a specific inhibitor of the phosphatidylcholine specific phospholipase C (IC 50: 41 μM), by the protein kinase C-inhibitor staurosporine (IC 50: 0.6 μM) and by the lipoxygenase inhibitor nordihydroguaiaretic acid (NGDA) but not by indomethacin. It is concluded that PACAP peptides stimulate secretion in rat peritoneal mast cells in a PACAP receptor-independent manner, probably via direct activation of heterotrimeric G-proteins of the G i-type; these G-proteins may lead to a sequential activation of different signaling cascades (see above), which may converge at the level of one or more staurosporine-sensitive protein kinase.

Cloning of bovine preproadrenomedullin and inhibition of its basal expression in vascular endothelial cells by staurosporine

The cDNA encoding preproadrenomedullin (preproAM) was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA ends from total RNA from bovine aortic endothelial cells (BAEC). Bovine preproAM cDNA shows high sequence homology with human, porcine and rat preproAM. Bovine-specific primers derived from this sequence were used in RT-PCR to study regulation of this gene. Treatment of BAEC or a human endothelial cell line (Ea.hy 926) with the non-selective protein kinase inhibitor staurosporine resulted in significantly reduced preproAM mRNA levels. The reduction in preproAM mRNA appeared to be absolute when Ea.hy 926 cells were exposed to 100 nM staurosporine for 2 h. However, this dramatic reduction could not be reproduced by treatment with the protein kinase A (PKA) inhibitor H-89, or the protein kinase C (PKC) inhibitors chelerythrine chloride and bisindolylmaleimide I. These observations suggest that activation of a novel staurosporine-sensitive protein kinase is necessary for basal expression of the preproAM gene in these cells.

The Role of Ca2+ and Protein Phosphorylation in Glycerol Dissimilation under Hypoosmotic Stress in the Halotolerant Alga Dunaliella.

The role of Ca2+ and protein phosphorylation in glycerol dissimilation under hypoosmotic stress in the halotorelant alga Dunaliella was investigated by a pharmacological approach. A stretch-activated Ca2+ channel blocker, GdCl3, inhibited glycerol dissimilation under hypoosmotic stress. Glycerol dissimilation under hypoosmotic stress was also inhibited in the presence of protein kinase inhibitors, K-252a and staurosporine. K-252a and staurosporine potently inhibits several types of protein kinase including serine/ threonine- and tyrosine-type protein kinases. Erbstatin analog (metyl 2, 5-dihydroxy-cinnamate), a specific inhibitor of tyrosine-type protein kinase, however, had no effect on glycerol dissimilation under hypoosmotic stress. The results of the present study suggest that Ca2+ influx from the extracellular space and protein phosphorylation by K-252a- and staurosporine-sensitive protein kinase (s) appear to be participate in the process of glycerol dissimilation under hypoosmotic stress in Dunaliella.

Reversible protein phosphorylation regulates jasmonic acid-dependent and -independent wound signal transduction pathways in Arabidopsis thaliana.

Plants responses to mechanical injury are complex and include the induced expression of defence-related genes. The phytohormone JA has been reported to mediate some of these responses. To elucidate further the signal transduction processes involved, the action of specific agonists and antagonists of known signalling effectors on the response of Arabidopsis thaliana plantlets to JA and wounding was investigated. The identification and characterization of a reversible protein phosphorylation step in a transduction pathway leading to JA-induced gene transcription is reported. This phosphorylation event involved the opposing activities of a staurosporine-sensitive protein kinase, negatively regulating the pathway, and a protein phosphatase, most probably of type 2 A, which activated JA-responsive gene expression. JA activation via this pathway was blocked in the A. thaliana JA-insensitive mutants jin1, jin4 and coi1, and by exogenous application of cycloheximide or auxins. Wound-induced activation of JA-responsive genes was also regulated by this protein phosphorylation step. An alternative wound signalling pathway, independent of JA, was also identified, leading to the transcriptional activation of a different set of genes. This JA-independent pathway was also regulated by a protein phosphorylation switch, in which the protein kinase positively regulated the pathway while the protein phosphatase negatively regulated it. Moreover, a labile protein apparently repressed the expression of these genes. One of the genes analysed, JR3, had a complex pattern of expression, possibly because it was regulated via both of the wound signalling pathways identified. According to the function of an homologous gene, JR3 may be involved in feedback inhibition of the JA response.

Overexpression of insulin-like growth factor (IGF)-I receptor enhances inhibition of DNA replication in mouse cells exposed to x-rays.

Previous studies from our laboratory provided evidence for the operation of signal transduction pathways involving ras, myc, and staurosporine-sensitive protein kinases in the regulation of DNA replication in irradiated cells. Because ras and myc are also involved in the signal transduction elicited in response to ligand activation of growth factor receptors, we wondered whether growth factor receptors are upstream elements in the regulation of DNA replication in irradiated cells. Here, we report on the role of insulin-like growth factor I receptor (IGF-IR) in the regulation of DNA replication in irradiated cells. We compare radiation-induced inhibition of DNA replication in BALB/c 3T3 cells with that in P6 cells. P6 cells are derived from BALB/c 3T3 cells by transfection with a vector expressing IGF-IR, leading to 30-fold overexpression. We observe a significantly stronger inhibition of DNA replication after irradiation in P6 as compared with BALB/c 3T3 cells at all doses examined. Sedimentation in alkaline sucrose gradients shows that the increased inhibition in P6 cells is due to an increased inhibition of replicon initiation, the main controlling event in DNA replication. Staurosprine at 20 nM reduces radiation-induced inhibition of DNA replication in BALB/c 3T3 cells, but has only a small effect in P6 cells. Caffeine at a concentration of 1 mM, on the other hand, removes over 60% of the inhibition in both cell lines. The results implicate IGF-IR in the regulation of DNA replication in irradiated cells, but also suggest differences between cells of different origins in the proteins involved in the regulating signal transduction pathway.

Intracellular signalling by binding sites for the antipsoriatic agent monomethylfumarate on human granulocytes

Monomethylfumarate (MMF), the most active metabolite of the new antipsoriasis drug Fumaderm, stimulates an anti-inflammatory mediator profile in human leucocytes and inhibits the proliferation of keratinocytes. These effects of MMF on cells in vitro may in part explain the beneficial action of Fumaderm in patients. In addition, we have reported that MMF stimulates an increase in the intracellular free Ca2+ concentration ([Ca2+]i) and the cyclic adenosine monophosphate (cAMP) concentration in granulocytes and keratinocytes. Because Ca2+ and cAMP control many physiological cellular responses, including cell proliferation and inflammatory mediator production, the present study focused on the intracellular signal transduction pathway which links interaction between MMF and granulocytes with increases in [Ca2+]i and the cAMP concentration. The increase in [Ca2+]i in granulocytes after MMF depended both on extracellular Ca2+ and Ca2+ from intracellular stores. Ca2+ is essential for the increase in the cAMP concentration after stimulation with MMF. The results found for pharmacological inhibitors of various protein kinases suggest that a staurosporine-sensitive protein kinase different from protein kinase C (PKC) and protein kinase A is involved in the MMF-induced increase in [Ca2+]i in granulocytes. As MMF activated protein tyrosine kinase (PTK), and inhibition of this protein kinase partially reduced the increase in [Ca2+]i in granulocytes, PTK activity most likely is involved. In addition, activation of protein kinase histone 4 (PKH4) probably plays a part in the MMF-stimulated increase in [Ca2+]i in granulocytes as well. As MMF stimulated an increase in the GTP-ase activity of membranes and pertussis toxin (PTX) inhibited the increase in the [Ca2+]i and PKH4 activity of granulocytes stimulated by this compound, it is concluded that MMF activates PTX-sensitive G proteins. Competition binding studies with radiolabelled dimethylfumarate (DMF) and unlabelled DMF and MMF revealed the presence of specific binding sites for methylated fumarates on granulocytes. In summary, MMF binds to specific sites on the plasma membrane of cells. This interaction activates pertussis toxin-sensitive G proteins which then stimulate an increase in PTK and PKH4 activity. These protein kinases may regulate the rise in [Ca2+]i and the intracellular cAMP concentration. Elevated [Ca2+]i and intracellular cAMP concentration stimulate protein kinases that regulate transcription factors. Activation of these factors results in induction of downstream gene expression and thus controls cell functions, e.g. cell proliferation and production of inflammatory mediators, as has been found for cells incubated with MMF.

Calcium- and meiotic-spindle-independent activation of pig oocytes by the inhibition of staurosporine-sensitive protein kinases.

The dependence of pig oocyte activation (both nuclear activation and cortical granule exocytosis) induced by staurosporine on intracellular Ca2+ rise and spindle assembly was studied. Nuclear activation was evaluated by pronuclear (PN) formation, cleavage and their developmental ability, and cortical granule (CG) exocytosis was assessed by electron microscopy and laser confocal microscopy of oocytes labelled with fluorescein isothiocyanate-peanut agglutinin. Exposure of pig oocytes of 0.3 and 3 microM protein kinase inhibitor staurosporine for 30 min resulted in the nuclear activation in 71.8% and 85.7% of the oocytes, respectively. The pronuclei in activated oocytes contained several compact nucleoli. When the cleaved 2-cell oocytes were further cultured in vitro, 93.5% developed beyond the 4-cell stage, and 12.9% developed to the morula stage after 4 days of culture. Of the oocytes treated with 3 microM staurosporine, 62.5% and 9.4% released their CGs partially and completely, respectively. The nuclear activation induced by staurosporine was overcome by the prior treatment of oocytes with okadaic acid, resulting in only 33.3% of the oocytes undergoing nuclear activation. However, when oocytes were exposed first to 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethanal ester), a cell permeate calcium chelator, or Colcemid, a meiotic spindle disrupter, and then to staurosporine, nuclear activation was observed in 74.2% and 82.3% of the oocytes, respectively. These data were the same as those in oocytes treated only with staurosporine (85.7%). The present study indicates that pig oocytes can be activated by the inhibition of staurosporine-sensitive protein kinase(s), and that this activation is dependent upon mitogen-activated protein kinase but independent of the intracellular Ca2+ rise and spindle integrity.

Calcium-Independent, Egg Age-Dependent Parthenogenic Activation of Mouse Eggs by Staurosporine.

The effect of protein kinase inhibitors on the activation events of mouse eggs at different ages and the dependence on Ca2+ were studied. Inhibition of protein kinase(s) by staurosporine with concentration over 2 μM first induced the destruction of meiotic spindle and then the nuclear formation in metaphase II eggs, but did not stimulate the cortical granule exocytosis; staurosporine with concentration below 0.2 μM and protein kinase C inhibitor sphingosine at concentrations of 10-100 μM did not activate mouse eggs. The activation of mouse eggs by staurosporine was egg age-dependent, but calcium-independent. The number of nucleoli in the formed nucleus or naked nucleoli in the ooplasm was dose- and egg age-dependent. To further evaluate the nuclear and cytoplasmic activation by staurosporine, interphase eggs were processed for electron microscopy and it was found that most nucleoli were highly condenced like those in the pronucleus, but vacuolated or ring-shaped ones also appeared, which implied the activation of egg genome. In addition, staurosporine also induced the formation of Golgi complexes around the nucleus. The nuclear formation induced by staurosporine was completely overcome by okadaic acid. These results suggest that inhibition of staurosporine-sensitive protein kinase(s) other than protein kinase A and C is responsible for the transition from metaphase to interphase in an egg age-dependent but calcium-independent manner in mouse eggs.

Ca 2+-independent contraction induced by hyperosmolar K +-rich solutions in rat uterus

The present experiments were designed to investigate the mechanisms involved in the contractile responses evoked by KCl, added either isoosmotically or hyperosmotically, in the rat uterus. Exposure of uterine strips to a Ca 2+-free, 3 mM EGTA-containing solution abolished the responses induced by isoosmotic KCl solutions. Conversely, addition of hyperosmolar KCl induced concentration-dependent tonic responses in a Ca 2+-free, 3 mM EGTA-containing solution. The maximum increase in tension was reached with 210 mM K +. The response to hyperosmotic K + was unaffected by previous depletion of intracellular Ca 2+ stores with oxytocin (1 μM), by inhibition of refilling of the intracellular Ca 2+ stores using cyclopiazonic acid (10 μM) or by increasing the concentration of EGTA in the medium to 10 mM. Sucrose and mannitol (60–420 mM) induced concentration-dependent sustained contractions which were not reproducible and were significantly smaller in size than those evoked by the maximally effective concentration of hyperosmotic K + (210 mM). The contraction induced by hyperosmotic K + in Ca 2+-free solution was not altered by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide hydrochloride (W-7, 100 μM), the Ca 2+ calmodulin protein kinase II inhibitor 1-[ N,O-bis(1,5-isoquinoline-sulphonyl)- N-methyl- l-tyrosyl]-4-phenylpiperazine (KN-62, 10 μM) or the tyrosine kinase inhibitor genistein (10 μM). The protein kinase C inhibitor calphostin C (1–3 μM) failed to modify the K +-effect curve, which was however partially inhibited in the presence of the non-selective protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride (H-7, 3–100 μM). The protein kinase inhibitor staurosporine (30–300 nM) depressed the contraction induced by hyperosmolar K + in a concentration-dependent manner. The contraction induced by sucrose in Ca 2+-free solution was unaffected by W-7 (100 μM) and KN-62 (10 μM) but was partially reduced by calphostin C (1 μM), H-7 (30 μM), staurosporine (100 nM) and genistein (10 μM). These results suggest that different mechanisms are involved in the responses evoked by isoosmotic and hyperosmotic KCl in the rat uterus. A component of the contraction induced by hypertonic KCl seems mainly independent of both external and internal Ca 2+ and of hyperosmolar stress. This contraction is not mediated by protein kinase C, Ca 2+ calmodulin-dependent kinases or protein tyrosine kinases but involves activation of other, at the present unknown, staurosporine-sensitive protein kinase(s).

Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor alpha 6 subunit protein over the 2-12 day period. 2. GABA treatment for 2 days down-regulates the alpha 1 subunit protein in a dose-dependent (10 microM-1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 microM). At 10 microM and 1 mM GABA the reduction in alpha 1 subunit expression compared to controls was 31% and 66%, respectively. 3. The flunitrazepam-induced decrease in alpha 1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABAA receptor channel activity. 4. Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha 1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5. The flunitrazepam-induced down-regulation of alpha 1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 microM). 6. This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor alpha 1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect is independent of GABA, and that the benzodiazepine and GABA sites may not be equally coupled to the down-regulation process, with the benzodiazepine site being the more dominant. The biochemical mechanism underlying the benzodiazepine-mediated down-regulation of the alpha 1 subunit protein seems to involve the activity of staurosporine-sensitive protein kinases.

Losartan-sensitive AII Receptors Linked to Depolarization- dependent Cortisol Secretion through a Novel Signaling Pathway

In bovine adrenal zona fasciculata (AZF) cells, angiotensin II (AII) may stimulate depolarization-dependent Ca2+ entry and cortisol secretion through inhibition of a novel potassium channel (IAC), which appears to set the resting potential of these cells. Aspects of the signaling pathway, which couples AII receptors to membrane depolarization and secretion, were characterized in patch clamp and membrane potential recordings and in secretion studies. AII-mediated inhibition of IAC, membrane depolarization, and cortisol secretion were all blocked by the AII type I (AT1) receptor antagonist losartan. These responses were unaffected by the AT2 antagonist PD123319. Inhibition of IAC by AII was prevented by intracellular application of guanosine 5'-O-2-(thio)-diphosphate but was not affected by pre-incubation of cells with pertussis toxin. Although mediated through an AT1 receptor, several lines of evidence indicated that AII inhibition of IAC occurred through an unusual phospholipase C (PLC)-independent pathway. Acetylcholine, which activates PLC in AZF cells, did not inhibit IAC. Neither the PLC antagonist neomycin nor PLC-generated second messengers prevented IAC expression or mimicked the inhibition of this current by AII. IAC expression and inhibition by AII were insensitive to variations in intracellular or extracellular Ca2+ concentration. AII-mediated inhibition of IAC was markedly reduced by the non-hydrolyzable ATP analog adenosine 5'-(beta, gamma-imino)triphosphate and by the non-selective protein kinase inhibitor staurosporine. The protein phosphatase antagonist okadaic acid reversibly inhibited IAC in whole cell recordings. These findings indicate that AII-stimulated effects on IAC current, membrane voltage, and cortisol secretion are linked through a common AT1 receptor. Inhibition of IAC in AZF cells appears to occur through a novel signaling pathway, which may include a losartan-sensitive AT1 receptor coupled through a pertussis-insensitive G protein to a staurosporine-sensitive protein kinase. Apparently, the mechanism linking AT1 receptors to IAC inhibition and Ca2+ influx in adrenocortical cells is separate from that involving inositol trisphosphate-stimulated Ca2+ release from intracellular stores. AII-stimulated cortisol secretion may occur through distinct parallel signaling pathways.

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.

Establishment of Lipopolysaccharide-dependent Nuclear Factor κB Activation in a Cell-free System

Nuclear factor kappa B (NF-kappa B), consisting of p50 and p65, is bound to a cytoplasmic retention protein, I kappa B, in a resting state, and the stimulation of cells with a variety of inflammatory stimuli induces the dissociation of NF-kappa B from I kappa B and the nuclear translocation of NF-kappa B, thereby activating several genes involved in inflammatory responses, such as interleukin (IL)-6, IL-8, and tumor necrosis factor alpha. In order to elucidate the precise mechanism of NF-kappa B activation, we have established lipopolysaccharide (LPS)-dependent NF-kappa B activation in a cell-free system using plasma membrane-enriched, cytosol, and nuclear fractions extracted from a human monocytic cell line, THP-1, by disruption with sonication followed by a differential centrifugation. The combination of plasma membrane-enriched fraction and cytosol was sufficient to activate NF-kappa B in a LPS/CD14-dependent manner only in the presence of ATP as judged by the binding of NF-kappa B to the IL-8 gene kappa B site on an electrophoretic mobility shift assay. LPS-dependent NF-kappa B activation was inhibited by protein kinase inhibitors, such as staurosporine, herbimycin A, tyrphostin, and genistein, but not mitogen-activated protein kinase substrate, cGMP-dependent protein kinase, cAMP-dependent protein kinase, protein kinase C, and calmodulin-dependent protein kinase II inhibitory peptides, suggesting that staurosporine-sensitive kinase(s) as well as tyrosine kinase(s) are involved in LPS-mediated NF-kappa B activation. In addition, LPS induced the phosphorylation of I kappa B-alpha, starting at 5 min after the stimulation in a cell-free system. Moreover, the phosphorylation was inhibited by herbimycin A and tyrphostin, but not staurosporine, suggesting that these protein kinase inhibitors act at distinct steps of signal transmission. Establishment of ligand-dependent activation of NF-kappa B in a cell-free system will facilitate identification of protein kinase(s) and its substrate(s) involved in LPS-mediated NF-kappa B activation.

Requirement of a colchicine-sensitive component of the cytoskeleton for acetylcholine receptor recovery.

1. The effect of colchicine treatment on acetylcholine receptor function was examined in potassium depolarized, voltage-clamped snake twitch fibre endplates. Receptor function was assessed by analysis of miniature endplate currents (m.e.p.c.) as well as acetylcholine (ACh)-induced single channel currents. 2. Pretreatment of snake muscle fibres with colchicine (10 microM to 100 microM) for 16-18 h had no effect on m.e.p.c. amplitude or decay rates. At higher concentrations (1 mM), there was a slight decrease in the average m.e.p.c. amplitude. 3. Colchicine produced a concentration-dependent decrease in the extent of m.e.p.c. amplitude recovery following a 10 min exposure to 540 microM carbachol. Exposure of 100 microM colchicine-treated preparations to 0.5 microM staurosporine further reduced the extent of m.e.p.c. amplitude recovery following carbachol exposure. 4. The decrease in m.e.p.c. amplitude following carbachol exposure was not due to a shift in the m.e.p.c. reversal potential. In addition, the distribution of m.e.p.c. amplitudes remained unimodal in both control and colchicine (100 microM)-treated preparations following carbachol exposure. 5. In addition to the normal, large conductance (approximately 48 pS) ACh-activated channels, a population of small conductance (approximately 29 pS) channels was observed in colchicine-treated preparations following exposure to carbachol. In preparations treated with both colchicine and staurosporine and then exposed to carbachol, the conductance of these small channels was identical to that of colchicine or staurosporine alone. 6. We suggest that prolonged exposure of snake twitch fibre endplates to agonist results in the activation and desensitization of ACh receptors. Furthermore, we propose that for a subpopulation of the inactivated receptors, restoration of function requires both the integrity of a subsynaptic cytoskeletal component and phosphorylation by a staurosporine-sensitive protein kinase. One plausible mechanism is that some receptors become destabilized in the membrane and phosphorylation of a cytoskeletal component, whose distribution may depend on an intact microtubular system, is required to re-anchor these receptors. If this anchoring process is inhibited either by disruption of the cytoskeleton with colchicine, or inhibition of the kinase by staurosporine, these receptors remain activatable, but have a reduced conductance.

Redox reagents and staurosporine inhibit stimulation of the transcription regulator NF-κB following tumour necrosis factor treatment of chronic B-leukaemia cells

B-chronic lymphocytic leukaemia (B-CLL) and hairy cell leukaemia cells (HCL) are refractory to stimulation by several cytokines which activate normal B-cells. However, tumour necrosis factor (TNF) promotes the proliferation of these cells. TNF regulates some of its cellular responses via the transcription factor NF-κB. Using an electrophoretic mobility shift assay, we demonstrate that TNF treatment of B-CLL and HCL cells in vitro resulted in the augmentation of NF-κB levels. In haemopoietic cell lines. TNF induction of NF-ϰB is mediated via the generation of reactive oxygen intermediates and by the activation of protein kinase C (PKC). We have used activators and inhibitors of these pathways to unravel TNF signalling in the cells of ten patients with B-CLL and two with HCL, using the increase in NF-ϰB levels following TNF treatment as an end point. Raising glutathione levels with N-acetyl cysteine substantially reduced NF-ϰB induction by TNF in two of four samples, as did treatment of cells with the antioxidant butylated-hydroxytoluene in all three samples tested. These data suggest that redox mechanisms are involved in TNF signalling in these cells. Treatment with the P KC activator phorbol myristate acetate failed to activate NF-ϰB suggesting that this enzyme does not mediate the induction of NF-ϰB in these cells. However, the protein kinase inhibitor staurosporine inhibited TNF induction of NF-ϰB in four of five samples, suggesting that staurosporine-sensitive protein kinases (other than P KC) are involved in the signalling pathway. Our results suggest that P KC-independent pathways, including pathways sensitive to redox reagents, mediate the induction of NF- KB by TNF in chronic B-leukaemia cells. Additionally, these data suggest that defects in P KC-mediated pathways may contribute to the general reluctance of B-CLL and HCL cells to respond to mitogenic signals.

Enhancement by staurosporine of platelet-activating factor formation in N-formyl peptide-challenged human neutrophils is mediated by intracellular platelet-activating factor binding sites

Staurosporine potentiates the formation of platelet-activating factor (PAF) and causes a sustained elevation of intracellular Ca 2+ ([Ca 2+] i). WEB 2086, a specific PAF-receptor antagonist, inhibits both potentiation of PAF formation and elevation of [Ca 2+] i by 78% and 65%, respectively. Moreover, the PAF produced by FMLP and/or Staurosporine was completely retained in the cell. This suggests that the effect of staurosporine in FMLP-stimulated neutrophils may be mediated by the action of endogenously produced PAF, which in turn leads to an increase in [Ca 2+] i and PAF formation. We conclude that PAF is the major product of human neutrophils which reacts via specific intracellular PAF binding sites to stimulate the phospholipase A 2, and its synthesis is under control of a staurosporine-sensitive protein kinase.

Time-dependent inhibition of stimulated adrenal catecholamine release by staurosporine.

Staurosporine, a potent inhibitor of protein kinases, is used to study the involvement of protein kinases in cellular processes. In the present studies, the effect of prolonged staurosporine treatment on catecholamine secretion in cultured bovine adrenal chromaffin cells was examined. Staurosporine inhibits catecholamine release stimulated by 10 microM nicotine, depolarizing concentrations of potassium (56 mM KCl), and 2 mM BaCl2. The effects of staurosporine on KCl-stimulated release are time dependent, with a half-time of approximately 50 min and a maximal inhibition at 2 h. Our results indicate that activation of a staurosporine-sensitive protein kinase is not directly involved in the stimulus-secretion coupling process. This does not rule out the possibility that Ca2+/phospholipid-dependent protein kinase or other protein kinases may acutely modulate release. However, these results suggest that a protein(s), which is phosphorylated by a staurosporine-sensitive protein kinase(s), is required to maintain the integrity of the stimulus-secretion coupling process.

Thrombin promotes actin polymerization in U937 human monocyte-macrophage cells. Analysis of the signalling mechanisms mediating actin polymerization.

The U937 human monocyte-macrophage cell line was used to examine the effect of thrombin, an ill-defined chemoattractant, on the polymerization of actin, a process essential for cell motility. In differentiated macrophage-like U937 cells, thrombin (0.5-50 units/ml) caused a rapid dose-dependent increase in the formation of filamentous (F-) actin, detected by the staining of F-actin with the fluorescent toxin, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In contrast with other chemoattractants such as N-formylmethionyl-leucylphenylalanine or C5a, actin polymerization in response to thrombin occurred via a pertussis-toxin-insensitive G1-(inhibitory G-protein) independent signalling pathway. Further, this response was not affected by the Ca2+ chelator EGTA or by the specific protein kinase C (PKC) inhibitor RO-31-8220. The response to thrombin was not mimicked by the Ca2+ ionophore ionomycin or by the direct PKC activator phorbol 12-myristate 13-acetate. The thrombin response was, however, inhibited by the non-specific protein kinase inhibitor staurosporine. The present results suggest that in U937 cells thrombin stimulates the formation of F-actin via a signalling pathway independent of (i) the activation of PKC, (ii) the mobilization of intracellular Ca2+ and (iii) the activation of Ca(2+)-dependent protein kinases, but dependent on the activation of an undefined staurosporine-sensitive protein kinase.

Arachidonic acid release and platelet-activating factor formation by staurosporine in human neutrophils challenged with n-formyl peptide

Staurosporine, a putative protein kinase C (PKC) inhibitor, increased the release of [ 14C]arachidonic acid dose dependently between 100 nM and 1000 nM in human neutrophils challenged with 100 nM N-formyl-methionine-leucine-phenylalanine (FMLP). Staurosporine also increased the formation of leukotriene B 4 (LTB 4) and platelet-activating factor (PAF) in a dose-dependent manner. In addition, exogenously added lyso-PAF further augmented [ 3H]PAF formation in staurosporine-pretreated human neutrophils stimulated by FMLP, thus suggesting an activation of acetyl-CoA: lyso-PAF acetyltransferase by staurosporine. The potentiation of [ 14C]arachidonic acid release and [ 3H]PAF formation by staurosporine was further enhanced in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA), which pinpoints a mechanism other than the modulation of PKC in this process, inasmuch as staurosporine antagonizes PMA-induced O 2 − production and [ 3H]PAF formation. Additional studies with other putative PKC inhibitors also revealed the potentiating effects of 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7, 20 μM) and sphingosine (2.5 μM) on FMLP-induced [ 14C]arachidonic acid release and [ 3H]PAF formation. We therefore conjecture that staurosporine-sensitive protein kinases including PKC are not involved in the activation of phospholipase A 2 and acetyl-CoA: lyso-PAF acetyltransferase.