Raf-1 and ERK2 kinases are required for phorbol 12,13-dibutyrate-stimulated proliferation of rat lymphoblasts. ERK2 activation precedes Raf-1 hyperphosphorylation

Rat lymphoblasts are arrested in the G₁ phase of the cell cycle and can be promoted to proceed up to the S phase, when they are stimulated by phorbol ester. In this work, we have studied some details of the phorbol 12,13-dibutyrate (PBu₂)-stimulated proliferation. We show that in response to PBu₂ at least four different protein kinase C (PKC) isoforms translocate to the membrane. A specific PKC ζ antibody recognizes two bands of 75 and 82 kDa. These two activities are separated using a Mono Q chromatography and we show that p75 is the classical PKC ζ isoform, while p82 might be a related isoform which is PBu₂ sensitive. Our data show that there is a correlation between the ability of PBu₂ to promote mitogenesis and to activate ERK2 kinase, suggesting that ERK2 kinase might be the limiting step of the process. We also show that ERK kinase activation precedes Raf-1 kinase hyperphosphorylation, suggesting that Raf-1 kinase activation is not required for ERK kinase activation. This idea was checked using a Raf-1 kinase antisense (AS) oligonucleotide. The results obtained with the Raf-1 AS oligonucleotide indicate that this serine/threonine kinase is dispensable for ERK kinase activation, but needed for the PBu₂ mitogenic signaling even as late as 7 h after the delivery of the signal.     

Adenosine reduces the potassium conductance of guinea pig submucosal plexus neurons by activating protein kinase A

Intracellular recordings were made from S neurons of the submucosal plexus isolated from the guinea pig ileum. Adenosine or its analog 2-chloroadenosine (CADO) depolarized about 80% of neurons; previous work has shown that this results from activation of an A₂ receptor. The depolarization was associated with an increase in membrane input resistance, became smaller with membrane hyperpolarization, reversed polarity at the potassium equilibrium potential and was mimicked and occluded by calcium-free solutions or by cadmium, suggesting that it is due to a reduction in a calcium dependent potassium conductance. Both forskolin (though not 1,9-dideoxyforskolin) and phorbol 12,13-dibutyrate (PDBu) mimicked and occluded the action of CADO. Staurosporine (a nonspecific inhibitor of protein kinases) blocked the depolarization induced by the phorbol ester within 5 min, and blocked the effects of forskolin and CADO in 15–35 min. The depolarization caused by CADO was inhibited by the specific inhibitor of protein kinase A KT5720 (8R^*,9S^*,11S^*)-(–)-9-hydroxy-9-n-hexylester-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H, 8H,11H-2,7b-11a-triazadibenzo [a,g]cycloocta[cde]trin-den-1-one, whereas this inhibitor did not affect the depolarization induced by PDBu. The results are consistent with the control of this potassium conductance by protein kinase C, protein kinase A and intracellular calcium, and they indicate that adenosine reduces the conductance by activating protein kinase A.     

Comparison of the effects of phorbol dibutyrate and low-frequency stimulation of synaptic inputs on the excitability of myenteric AH neurons

Low-frequency stimulation of synaptic inputs to after-hyperpolarising (AH) neurons in the guinea-pig small intestine causes sustained increases in excitability that far outlast the stimulus period. This excitation has been called sustained, slow, post-synaptic excitation (SSPE). Intracellular microelectrodes were used to record the effects of the protein kinase C (PKC) stimulant, phorbol dibutyrate (PDBu), and compare these with changes seen during the SSPE, in AH neurons of the small intestine of the guinea-pig. PDBu (1 nM–1 µM) increased excitability, depolarised the membrane and increased input resistance concentration dependently, mimicking the effects of low-frequency stimulation of pre-synaptic inputs. These changes developed slowly after the start of infusion and were only slowly reversible after wash out. PDBu suppressed a late after-hyperpolarising potential (AHP) that depends on Ca²⁺ entry via voltage-gated Ca²⁺ channels during the action potential. The effects of PDBu (10 nM) on the late AHP were indistinguishable from those observed during the SSPE. PDBu, at a concentration that inhibited the AHP, had no effect on the action potential half-width or the slope of its first repolarisation phase (the early phase of repolarisation is slowed by the Ca²⁺ influx of the action potential). Thus PDBu inhibited K⁺ channel opening underlying the late AHP, but did not suppress Ca²⁺ entry during the action potential. The hyperpolarisation-activated cation current (I _h) in intrinsic primary afferent neurons (IPANs) was not affected by PDBu. We conclude that PDBu mimics the sustained excitation caused by low-frequency stimulation of synaptic inputs to IPANs by closing IK channels responsible for the AHP or restricting their opening by Ca²⁺ and by reducing the current carried by K⁺ channels that are active at rest. IK channels, the opening of which results in the AHP, have consensus sites for PKC and are likely targets for phosphorylation during the SSPE.     

M-current suppression by agonist and phorbol ester in bullfrog sympathetic neurons

Activation of protein kinase C (PKC) by phorbol esters is known to suppress M-current. 4--Phorbol 12,13-dibutyrate (PDBu) irreversibly suppressed M-current in a concentration-dependent manner (K _i 38 nM). Inhibitors of PKC, the pseudo-substrate peptide PKCI (19–31), staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) antagonized PDBu-mediated suppression of M-current. Suppression of M-current by muscarine and luteinizing hormone-releasing hormone (LHRH) was unaffected by PKCI (19–31) and H7, but was antagonized by staurosporine. The balance of data suggests that suppression of M-current by agonists is probably not mediated by activation of PKC. Addition and subsequent removal of PDBu to M-current suppressed by muscarine prevented the action of PDBu, while closing M-channels by voltage or blocking by barium did not. This suggests that M-channel closure by muscarine protects those channels from the effects of PDBu. Partial suppression of M-current by low concentrations of muscarine antagonized the response to PDBu, with the magnitude of suppression equivalent to that seen with PDBu alone. It is suggested that two interconvertable populations of M-channels exist, one that is sensitive to both agonist and PDBu and another that can only be suppressed by agonist.     

Alterations in AMPA receptor phosphorylation in the rat striatum following acute and repeated cocaine administration

Protein phosphorylation is an important mechanism for the posttranslational modulation of ionotropic glutamate receptors and is subject to regulation by changing synaptic inputs. In this study, we investigated the regulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit phosphorylation by cocaine exposure in the rat dorsal striatum in vivo. We found that acute cocaine challenge followed by 6 days of repeated systemic injections of cocaine (20 mg/kg once daily) enhanced the sensitivity of the GluR1 subunit in its phosphorylation at serine 831 (Ser831) in the dorsal striatum. This enhancement of the sensitivity of Ser831 phosphorylation was reduced, at the receptor and ion channel level, by blocking (1) group I metabotropic glutamate receptors (mGluRs), (2) N-methyl-d-aspartate receptors, and (3) L-type voltage-operated Ca²⁺ channels. Similar reduction of the enhancement was also induced, at the protein kinase level, by inhibiting (1) protein kinase C, (2) calcium/calmodulin-dependent protein kinases, and (3) c-Jun N-terminal kinases. In addition, inhibition of protein phosphatase 1/2A or calcineurin increased GluR1-Ser831 phosphorylation in the dorsal striatum of normal rats, whereas inhibition of these phosphatases did not further enhance the Ser831 phosphorylation in rats pretreated with 7 daily injections of cocaine. These data suggest that the phosphorylation of AMPA receptor GluR1 subunits at Ser831 is subject to upregulation by acute and repeated cocaine administration. Complex signaling integrations among glutamate receptors, Ca²⁺ channels, protein kinases, and protein phosphatases participate in this upregulation.     

Recombinant TB10.4 of Mycobacterium bovis induces cytokine production in RAW264.7 macrophages through activation of the MAPK and NF-κB pathways via TLR2

The TB10.4 antigen of Mycobacterium bovis/Mycobacterium tuberculosis induces a strong Th1 CD4+ T-cell response. Thus, it is currently under intensive study as a possible vaccine candidate. However, how TB10.4 activates innate immune cells is unclear. How TB10.4 interacts with toll-like receptors (TLRs) and signaling pathways responsible for active inflammation have also not been fully elucidated. Here, as stimulated RAW264.7 cells with recombinant TB10.4 (rTB10.4), derived from M. bovis, increased TNF-α, IL-6 and IL-12 p40 secretin in a dose-dependent manner. Blocking assays showed that TLR2-, but not TLR4-neutralizing antibody reduced expression of TNF-α, IL-6 and IL-12 p40 in RAW264.7 cells. rTB10.4 stimulation activated p38 kinase (p38) and extracellular-regulated kinase (ERK) was TLR2-dependent, whereas inhibition of p38 and ERK activity significantly reduced TNF-α, IL-6 and IL-12 p40 production. Furthermore, rTB10.4 stimulation of RAW264.7 cells resulted in TLR2-mediated activation of NF-κB and induced translocation of NF-κB p65 from the cytoplasm to the nucleus via IκBα degradation. rTB10.4-induced TNF-α, IL-6 and IL-12 p40 release was attenuated by the specific IκB phosphorylation inhibitor, BAY 11-7082. These findings indicate that the M. bovis-derived rTB10.4 induced production of TNF-α, IL-6 and IL-12 p40 involves p38, ERK and NF-κB via the TLR2 pathway.     

Effects of phorbol esters on excitation-contraction coupling and protein kinase C activity of frog twitch muscle fibers

 By recording the calcium transients evoked by voltage-clamp depolarizing pulse with arsenazo III as a calcium indicator, it has been shown that 1 μmol/l phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) agonist, causes a transient potentiation and then a depression of the calcium transients of twitch muscle fibers in frogs. PDBu also produces an initial translocation and activation of PKC, which is followed by a down-regulation. To find out whether the effect of PDBu on the calcium transients depends on PKC, a correlated study of the effect of phorbol esters on calcium transients and PKC activity was performed. The calcium transients and PKC activity were similarly affected by PDBu in ordinary and cold-accommodated frogs, but the effects occurred more quickly in the latter. However, they still changed in parallel as in ordinary frogs. 1 or 10 μmol/l, 4-α-phorbol, a PKC-inactive analogue of phorbol ester, caused a partial depression of the calcium transients in cold-accommodated frogs, while PKC activity was not affected. Moreover, the transient potentiation of the calcium transients induced by 1 μmol/l PDBu could be antagonized by the PKC inhibitors 10 μmol/l chelerythrine chloride or 10 μmol/l polymyxin B (PMB). All these results suggest that: (1) the transient potentiation of calcium transients induced by PDBu is caused by activation of PKC; (2) phorbol ester can depress the calcium transients by a mechanism that is independent of PKC. Received: 25 September 1998 / Received after revision: 14 December 1998 / Accepted: 19 January 1999     

NMDA-mediated activation of the tyrosine phosphatase STEP regulates the duration of ERK signaling

The intracellular mechanism(s) by which a cell determines the duration of extracellular signal-regulated kinase (ERK) activation is not well understood. We have investigated the role of STEP, a striatal-enriched tyrosine phosphatase, in the regulation of ERK activity in rat neurons. Glutamate-mediated activation of NMDA receptors leads to the rapid but transient phosphorylation of ERK in cultured neurons. Here we show that activation of NMDA receptors led to activation of STEP, which limited the duration of ERK activity as well as its translocation to the nucleus and its subsequent downstream nuclear signaling. In neurons, STEP is phosphorylated and inactive under basal conditions. NMDA-mediated influx of Ca(2+), but not increased intracellular Ca(2+) from other sources, leads to activation of the Ca(2+)-dependent phosphatase calcineurin and the dephosphorylation and activation of STEP. We have identified an important mechanism involved in the regulation of ERK activity in neurons that highlights the complex interplay between serine/threonine and tyrosine kinases and phosphatases.     

Phorbol 12,13-dibutyrate-mediated contraction of the stimulated bronchial smooth muscles of mice

Increasing evidence suggests that protein kinase C (PKC) is involved in the Ca²⁺ sensitization of various smooth muscle contractions. However, the exact role of PKC on bronchial smooth muscle (BSM) contraction is still unclear. In the present study, to determine the role of PKC activation in the BSM contraction, the effects of phorbol 12,13-dibutyrate (PDBu) on BSM tone were examined in the absence and presence of contractile stimulation. Although PDBu had no effect on the basal tone, the contraction induced by acetylcholine, high K⁺ depolarization or Ca²⁺ ionophore A23187 was significantly augmented by PDBu. Western blot analyses also revealed that the increase in the level of phosphorylated myosin light chain (MLC) induced by high K⁺ depolarization was significantly augmented by PDBu treatment. Interestingly, neither high K⁺ depolarization alone nor PDBu alone caused CPI-17 phosphorylation, but a significant phosphorylation of CPI-17 was observed when BSMs were co-stimulated by high K⁺ and PDBu. Thus, a certain level of intracellular Ca²⁺ might be needed both for an activation of CPI-17 and an induction of contraction induced by PDBu in mouse BSMs.     

Phosphatases regulate histamine synthesis in rat brain

Cyclic AMP-dependent protein kinase (PKA) and Ca(2+)-calmodulin dependent protein kinase II (CaMKII)-mediated phosphorylation activate histamine synthesis in nerve endings, but the phosphatases deactivating it had not been studied. In this work we show that the protein phosphatase 2A (PP2A)/protein phosphatase 1 (PP1) inhibitor okadaic acid increases histamine synthesis up to twofold in rat cortical miniprisms containing histaminergic nerve endings. This effect was mimicked by the PP2A/PP1 inhibitor calyculin, but not by the inactive analog 1-norokadaone. Other phosphatase inhibitors like endothall (PP2A), cypermethrin and cyclosporin A (protein phosphatase 2B, PP2B) had much lower effects. The effects of okadaic acid appeared to be mediated by an activation of the histamine synthesizing enzyme, histidine decarboxylase. PKA-mediated activation of histamine synthesis decreased the EC(50) and maximal effects of okadaic acid. On the other hand, CaMKII-mediated activation of histamine synthesis decreased okadaic acid maximal effects, but it increased its EC(50). In conclusion, our results indicate that brain histamine synthesis is subjected to regulation by phosphatases PP2A and PP1, and perhaps also PP2B as well as by protein kinases.     

Hydrogen sulfide protects neurons against hypoxic injury via stimulation of ATP-sensitive potassium channel/protein kinase C/extracellular signal-regulated kinase/heat shock protein90 pathway

Cerebral hypoxia is one of the main causes of cerebral injury. This study was conducted to investigate the potential protective effect of H₂S in in vitro hypoxic models by subjecting SH-SY5Y cells to either oxygen–glucose deprivation or Na₂S₂O₄ (an oxygen scavenger) treatment. We found that treatment with NaHS (an H₂S donor, 10–100 μM) 15 min prior to hypoxia increased cell viability in a concentration-dependent manner. Time-course study showed that NaHS was able to exert its protective effect even when added 8 h before or less than 4 h after hypoxia induction. Interestingly, endogenous H₂S level was markedly reduced by hypoxia induction. Over-expression of cystathionine-β-synthase prevented hypoxia induced cell apoptosis. Blockade of ATP-sensitive K⁺ (K_ATP) channels with glibenclamide and HMR-1098, protein kinase C (PKC) with its three specific inhibitors (chelerythrine, bisindolylmaleide I and calphostin C), extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059 and heat shock protein 90 (Hsp90) with geldanamycin and radicicol significantly attenuated the protective effects of NaHS. Western blots showed that NaHS significantly stimulated ERK1/2 activation and Hsp90 expression. In conclusion, H₂S exerts a protective effect against cerebral hypoxia induced neuronal cell death via K_ATP/PKC/ERK1/2/Hsp90 pathway. Our findings emphasize the important neuroprotective role of H₂S in the brain during cerebral hypoxia.     

Conserved cross-interactions in Drosophila and Xenopus between Ras/MAPK signaling and the dual-specificity phosphatase MKP3.

The extracellular signal-regulated kinase (ERK) is a key transducer of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) signaling pathways, and its function is required in multiple processes during animal development. The activity of ERK depends on the phosphorylation state of conserved threonine and tyrosine residues, and this state is regulated by different kinases and phosphatases. A family of phosphatases with specificity toward both threonine and tyrosine residues in ERK (dual-specificity phosphatases) play a conserved role in its dephosphorylation and consequent inactivation. Here, we characterize the function of the dual-specificity phosphatase MKP3 in Drosophila EGFR and Xenopus FGFR signaling. The function of MKP3 is required during Drosophila wing vein formation and Xenopus anteroposterior neural patterning. We find that the expression of the MKP3 gene is localized in places of high EGFR and FGFR signaling. Furthermore, this restricted expression depends on ERK function both in Drosophila and Xenopus, suggesting that MKP3 constitutes a conserved negative feedback loop on the activity of the Ras/ERK signaling pathway.     

Inhibitory effect of phorbol 12, 13 dibutyrate on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

 The effect of protein kinase C (PKC) on carbachol (CCh)-activated nonselective cationic current (I _CCh) was investigated in guinea-pig gastric myocytes using a PKC activator, phorbol 12, 13 dibutyrate (PDBu). Pretreatment with 1 μ M PDBu suppressed I _CCh by 96.5 ± 2.9% (n = 14) in a reversible manner in nystatin-perforated mode. In the presence of 1 μM chelerythrine , a PKC inhibitor, inhibition of I _CChby PDBu was not seen. In whole-cell mode, the inhibition of I _CCh by PDBu was dependent on intracellular MgATP. In the presence of MgATP in the pipette, PDBu decreased I _CCh by 98.8 ± 1.2% (n = 5) as was observed in nystatin-perforated mode. However, PDBu had little effect on I _CCh in the absence of MgATP in the pipette; the extent of inhibition was 12.7 ± 4.3% (n = 8). PDBu also suppressed the generation of cationic current induced by intracellularly perfused GTP[γS]. In the PDBu-pretreated group (n = 9) and PDBu-untreated control group (n = 6), GTP[γS]-induced currents were 6.7 ± 2.4 pA and 236 ± 23 pA, respectively. These results suggest that PKC modulates I _CCh at postreceptor sites via protein phosphorylation. Received: 4 April 1997 / Received after revision: 27 June 1997 / Accepted: 3 June 1997     

The role of protein kinase C in the regulation of extracellular signal-regulated kinase by the T cell antigen receptor

The aim of this study was to explore the role of protein kinase C (PKC) in the activation of mitogen-activated protein kinases (MAPK) in T lymphocytes. The MAPK extracellular signal-regulated kinase-2 (ERK2) is activated in response to phorbol esters which stimulate PKC, by transient expression of a constitutively active ras mutant, by cell activation via the G protein-coupled type 1 muscarinic acetylcholine receptor (HM1R) or in response to triggering of the T cell antigen receptor (TCR). The relative contribution of PKC to TCR and HM1R regulation of ERK2 was explored by examining the effects of a PKC inhibitor (Ro 31-8425) on ERK2 activation. The data demonstrate that phorbol ester and HM1R regulation of ERK2 was prevented by the PKC inhibitor, but that the inhibitor had no effect on ERK2 activation induced by expression of a constitutively active ras mutant p21v-Ha-ras. Furthermore, the TCR stimulates both PKC and p21^ras but TCR regulation of ERK2 was only weakly suppressed by the PKC inhibitor. These data indicate that PKC has a potential but not a predominant role in TCR regulation of ERK2.     

Phorbol ester-induced hippocampal long-term potentiation is counteracted by inhibitors of protein kinase C

Summary As was shown previously (Reymann et al. 1988), the protein kinase C (PKC)-inhibitor polymyxin B prevents the maintenance of electrically induced long-term potentiation (LTP) of synaptic transmission to CA1 neurons, indicating that post-translational phosphorylation processes mediated by PKC are involved in mechanisms underlying this form of synaptic plasticity. To make sure that 1.) the polymyxin B actually acts against PKC activation and 2.) the long-lasting potentiation elicited by phorbol esters (Malenka et al. 1986) is mediated by PKC-activation, we have tested polymyxin B as well as the potent PKC-inhibitor K-252b during phorbol ester-induced LTP. 4-beta-phorbol-12,13-dibutyrate (PDBu) — a known activator of protein kinase C, induces a remarkable potentiation at concentrations as low as 0.5 M. When 20 M polymyxin B or 40 nM K-252b was administered to rat hippocampal slices prior to such a weak phorbol ester treatment, this potentiation did not develop with the exception of a small increase in the population spike in spite of polymyxin B-treatment (42% instead of 120% increase at 2 h after PDBu). In contrast, spike potentiation induced by high concentrations of PDBu (10 M) could not be counteracted by 100 M polymyxin B. It is concluded that at low concentrations the phorbol ester-induced potentiation is mainly mediated by a selective activation of protein kinase C and that the prevented maintenance of electrically induced LTP by polymyxin B is in fact due to inhibition of this kinase. The spike potentiation developed faster than that of the EPSP raising the possibility that PDBu activates two separate PKC-dependent processes.     

Ceramide from sphingomyelin hydrolysis differentially mediates mitogen-activated protein kinases (MAPKs) activation following cerebral ischemia in rat hippocampal CA1 subregion

Objective

To explore the role that ceramide plays in the activation of mitogen-activated protein kinases (MAPKs) during cerebral ischemia and reperfusion.

Methods

Rats were subjected to ischemia by the four-vessel occlusion (4-VO) method. The sphingomyelinase inhibitor TPCK was administered to the CA1 subregion of the rat hippocampus before inducing ischemia. Western blot was used to examine the activity of extracellular-signal regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK) using antibodies against ERK, JNK and diphosphorylated ERK and JNK.

Results

At 1h reperfusion post-ischemia, JNK reached its peak activity while ERK was undergoing a sharp inactivation (P < 0.05). The level of diphosphorylated JNK was significantly reduced but the sharp inactivation of ERK was visibly reversed (P < 0.05) by the sphingomyelinase inhibitor.

Conclusion

The ceramide signaling pathway is up-regulated through sphingomyelin hydrolysis in brain ischemia, promoting JNK activation and suppressing ERK activation, culminating in the ischemic lesion.     

The phosphoprotein phosphatase calcineurin controls calcium-dependent apoptosis in B cell lines

Group I Burkitt's lymphoma cell lines and the B104 lymphoma cell line which expresses a phenotype of immature B cells undergo apoptosis after cross-linking of their surface immunoglobulin (Ig) receptors or after exposure to a calcium ionophore, while protein kinase C (PKC)-activating phorbol esters prevent such apoptosis. We show here that blockade of the phosphoprotein phosphatase calcineurin or phosphatase 2B by cyclosporin A (CsA) also protects these B cell lines against Ca²⁺-dependent apoptosis but not against apoptosis triggered by the PKC inhibitor chelerythrine or by serum deprivation. Okadaic acid, an inhibitor of phosphatases 1, 2A and 2C was ineffective. Among a series of human cytokines tested, only interferon-α and tumor necrosis factor-α were shown to protect against Ca²⁺-dependent apoptosis when used alone or in combination with CsA. In contrast to phorbol esters which block the progression into the S/G₂ phases of the cell cycle, CsA partially restored the proliferation of cells exposed to the calcium ionophore. Altogether these data provide indirect evidence for the control of B cell apoptosis by the serine/threonine phosphorylation status of yet undefined key cellular substrates.     

Mechanisms of H4/ICOS costimulation: effects on proximal TCR signals and MAP kinase pathways

H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals have been analyzed in mouse CD4(+) Th2 cells, expressing H4/ICOS at higher levels than Th1 clones. Anti-H4/ICOS antibodies strongly enhanced CD3-mediated tyrosine phosphorylation of ZAP-70, zeta, or Vav, as well as extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phosphoinositide 3-kinase (PI-3K) to H4/ICOS was enhanced by H4/ICOS cross-linking, and PI-3K inhibitors inhibited ERK and JNK activation and IL-4/IL-10 secretion, but not p38 MAP kinase or ZAP-70 activation. H4/ICOS-mediated activation of JNK, but not ERK or p38, is partially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulation is partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymerization, inhibited ZAP-70, MAP kinase activation, or IL-4/IL-10 secretion. Neither cyclosporin A nor inhibitors of PKC produced detectable inhibition of ZAP-70 phosphorylation or MAP kinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL-4, but not IL-10 secretion. ERK or JNKinhibitors partially inhibited IL-4 and IL-10 secretion, while PKC or p38 inhibitors had no significant effects on IL-4 or IL-10 secretion. Taken together, our data show clear similarities of costimulation mechanisms between H4/ICOS and CD28 during the early steps of TCR activation.