Jul 15, 1993 - Mitogen-activated protein (MAP) kinases are acti- vated in response to a large variety of extracellular sig- nals, including growth factors, ...
Vol. 269, No. 8,Issue of February 25, pp. 6207-6214,
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
1994
Printed in U.S.A.
Cyclic AMP Activates the Mitogen-activated Protein Kinase Cascade in PC12 Cells* (Received forpublication, July 15, 1993)
Morten FrodinS, Pascal Peraldi, and Emmanuel Van Obbergheng From the Znstitut National de la Sank! et de la Recherche Mkdicale U 145, Faculte de Medecine, 06107 Nice Cedex 2, France
many extracellular stimuli including growth factors, hormones, and neurotransmitters (1, 2). The various stimuli which can activate theMAP kinase cascade employ distinct initial signaling pathways. Some stimuli activate receptor tyrosine kinases ( 3 4 , or non-receptor tyrosine kinases (6-8). Other stimuli activate Gprotein-coupledreceptors generatingthe second messengers diacylglycerol or calcium, or activating ion-channels (9-12). Stimuli generating the prominentsecond messenMAP ger CAMP,however, have not beenreported to activate the kinase cascade. In the case of the receptor tyrosine kinases, a sequence of MAP kinase activationis emerging. Activated events leading to receptor tyrosine kinasesincrease GTP-binding to c-Ras, through guanine nucleotide exchange factors and adaptorproteins, leading to its activation (13, 14). Activated c-Ras may activate c-Raf kinase by direct binding(15-17). c-Raf can phosphorylate and activate MAP kinase kinase (18-211, which in turn activatesMAP kinase (22,231. How other signaling pathways couple to the cascade is less understood. In some cell types, certain G protein-stimulated pathways and protein kinase C-dependent pathways may alsoactivatethe cascade through c-Ras/c-Raf (12, 24, 25), while in other cell types, G protein-stimulated pathways maybe predominantly Ras independent and employ MAP kinase kinase kinases distinctfrom c-Raf (26-29). The MAP kinase cascade is likely to serve specific functions in different cell types. In rat pheochromocytoma PC12 cells, nerve growth factor (NGF) induces a very robust and sustained activation of the MAP kinase cascade (30-33). Since NGF induces neuronal differentiation in PC12 cells, the cascade has been proposed to mediate neurotrophic signals leading to a neuronal phenotype or other neurotrophic responses in this cell line (30-33). In agreement withthis model, the transfectionof PC12 cells with constitutivelyactive, oncogenic Ras or Raf In mammalian cells the ability to activate themitogen-actimimicks NGF action by inducing activationof the MAP kinase vated protein (MAP)’kinase cascade is a feature common to cascade and neuronal differentiationof PC12 cells, respectively * This work was supported by funds from the Institut National de la (24, 25, 34, 35). Elevation of intracellular CAMP,alone or in combination with Sant6 et de la Recherche Mkdicale, Universit6 de Nice-Sophia-Antipolis, Association Pour la Recherche Contre le Cancer, ARC Grant 6760, NGF, has also been reported topromote neuronal differentiaLigue Nationale Franeaise Contre le Cancer, FBd6ration des Comit6s tion of PC12 cells (36-39). A recently identified neuropeptide, DBpartementaux, and the ComitB DBpartemental du Var, France. The pituitary 38 adenylate cyclase-activating polypeptide costs of publication of this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “aduer- (PACAP381, which generates CAMPand stimulates phosphatidyl inositol breakdown, has been shown to promote neurite tisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. outgrowth in PC12 cells (40). These observations prompted us j: Supported by the Danish Cancer SocietyGrant 92-070 and 93-007. to investigate the possibility that CAMP might stimulate the B To whomcorrespondence should be addressed: INSERM U 145, Facult6 de M6decine, Avenue de Valombrose, 06107 Nice C6dex 2, MAP kinase cascade in thesecells. We demonstrate that elevation of the intracellular cAMP France. Tel.: 93-81-54-47. Fax: 93-81-54-32. The abbreviations used are: MAP, mitogen-activated protein; BSA, stimulates theMAP kinase isozyme, extracellular signal-regubovine serum albumin; CPT-CAMP,8-(4-chlorophenylthio-cyclic-AM~ lated kinase 1 (ERKl), aswell as MAP kinase kinase inPC12 EGF,epidermalgrowth factor; ERK1, extracellular signal-regulated kinase 1; IBMX, isobutylmethylxanthine;IGF-I,insulin-like growth cells. cAMP acts synergistically with phorbol ester, and thus kinase; MAPKK, mitofactor-I; MAP kinase, mitogen-activated protein gen-activated protein kinase kinase; MBP, myelin basic protein; NGF, polypeptide, 38-amino acid form: PMA, phorbol 12-myristate 13-acnerve growth factor; PACAP38, pituitary adenylate cyclase-activating etate; PAGE, polyacrylamide gel electrophoresis.
Mitogen-activated protein ( M A P ) kinases are activated in response to a large variety of extracellular signals, including growth factors, hormones, and neurotransmitters, which activate distinct intracellular signaling pathways. Their activation by the CAMP-dependent pathway, however,has not been reported. In rat pheochromocytoma PC12 cells, we demonstrate here a stimulation of the MAP kinase isozyme extracellular signal-regulated kinase 1 (ERK1) followingelevation of intracellular CAMPafter exposure of the cells to isobutylmethylxanthine, cholera toxin,forskolin, or CAMPanalogues. CAMP acted synergistically with phorbol ester, an activator of protein kinase C, in thestimulation of ERK1. In accordance with this observation, the peptide neurotransmitter pituitary adenylate cyclase-activating polypeptide 38(PACAP38),which stimulates CAMPproduction as well as phosphatidylinositolbreakdown in PC12 cells, wasan efficient activator of ERK1. In combination with various growth factors, CAMP acted in a more than additive manner on ERKl activity. Elevation of intracellular CAMP increased in vivo szPlabeling of ERK1, suggesting that CAMP stimulated ERKl by activating MAP kinase kinase, an immediate upstream activator of ERKl in theMAP kinase cascade. Supporting this view, forskolin and a CAMP analogue were foundto increase the activity of MAP kinase kinase in PC12 cells,alone as well as incombination with phorbo1 ester. PACAP38 also stimulated in uiuo 32P-labeling of ERKl and MAP kinase kinase activity. Finally,cAMP or PACAP38 increased by %fold nerve growth factorstimulated neurite formation in PC12 cells, which may be correlated with the potentiating effect of these agents on nerve growth factor-stimulatedERKl activity.
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CAMPActivates the MAP Kinase Cascade in PC12 Cells
protein kinase C. This interaction may serve as a model for signal integrationat thelevel of the MAP kinase cascade. Such an interaction may be used by different ligands that stimulate CAMPgeneration or phosphatidylinositol breakdown, respectively, or ligands which stimulate both pathways, like PACAP38. Finally, CAMPincreased ERKlactivation as well as neurite outgrowth in response to NGF in PC12 cells. Most importantly, our observations describe a novel regulation of the MAP kinase cascade, ie. stimulationby intracellular CAMP.In addition, these data may provide new insight into the neurotrophic effects of CAMP (and physiological ligands raising intracellular CAMP) previously described in PC12 cells, but also in sympathetic and sensory neurons and neuroblastoma cells where neurotrophic effects of CAMPhave been reported (41, 42).
ERKl activity was then measured using MBPas a substrate. Briefly, pellets with immunoprecipitatedMAPKK were washedtwo times in 50 m~ HEPES, pH 7.4, dried, and resuspended in 50 ofpl50 mM HEPES, pH 7.4, containing bacterially expressed, recombinant ERKl and 0.2 m~ NaaV04, 100 unitdm1 aprotinin, 20 VM leupeptin, and 0.2 mg/ml phenylmethylsulfonyl fluoride. The phosphorylation cascade was started by the addition of [ Y - ~ ~ P I A(50 T P p ~ 50, Ci/mmol), 150 pglml MBP, 15 mM magnesium chloride, 1 mM EGTA added as a 6-fold concentrated mixture in a volume of 10 pl. The phosphorylation reaction was allowed to proceed for 10 min a t room temperature and was stopped by spotting Whatman P-81 filter papers which were then dropped into 0.1% (v/v) orthophosphoric acid. The papers were washed overnight with several shifts in this solution, rinsed once in ethanol, air dried, and radioactivitywasdetermined by Cerenkov-counting.Thereaction blank, whichreceived identical treatment, was a mixture containing all of the reagents except thecell lysate during immunoprecipitation, and was subtracted from all values. Control experiments showed that, when MBP or recombinant ERKl were omitted from the phosphorylation reaction with immunopurified MAPKK from unstimulated as well as EXPERIMENTAL PROCEDURES stimulated cells, radioactivity associated with the papers dropped by Materials-The keyhole limpet hemocyanin-coupled 12-amino-acid approximately 90%. The remaining 10% may derive from (auto-) phosphorylation of the recombinant ERKlor [-p3"?PIATPadsorbed to MBP, carboxyl-terminal of ERKl(356-367) (TAFCFQPGAPEAP),synthesized by Neosystem (Strasbourg, France) was used to generate rabbit poly- respectively. The data were not corrected for these minor contributions. clonal antisera (43). For the generation of polyclonal rabbit antisera to 32P-Labelingof PC12 Cells-Cells were washed twice in serum- and MAP kinase kinase, the 17-amino-acid amino-terminal (PKKKPTPIQLphosphate-free culture medium with 0.2% BSA and incubated for3.5 h NPAPDGS (44) was used as an antigen. Sodium orthovanadate, BSA, in thismedium containing [32Plorthophosphate (0.5 mCi/ml). At the end leupeptin, phenylmethylsulfonyl fluoride, MBPfrom bovine brain, pro- of the labeling period, cells were stimulated as indicated. Following X-lOO,8-(4-chlorophenylthio~-cAMP, forskolin, stimulation the monolayers were washed once in ice-cold phosphatetein A-Sepharose, Triton cholera toxin, N6,2'-O-dibutyryl CAMP,iP,2'-O-dibutyryl-cGMP, 3-iso- buffered saline and solubilized for 15 min in the solubilization buffer. butyl-1-methyl-xanthine (IBMX), and phorbol 12-myristate 13-acetate The cell extracts were clarified by centrifugation at 18,000 x g for 15 (PMA) were purchased from Sigma. The final experimental concentra- min and incubated for 2 h with antibodies to ERKl preadsorbed to tion of dimethyl sulfoxide, used as carrier for PMA and forskolin, was protein A-Sepharose beads.Following the incubation period, the beads 0.04% which had no effect on ERKl activity in itself. OvinePACAP38 were washed six times in solubilization buffer. Pellets were dried, re(identical to rat PACAP38) from Peninsula Laboratories, Inc, was in suspended in Laemmli buffer (3% SDS), andboiled for 5 min. Dissolved part a generous gift from Prof. J. Fahrenkrug, Bispebjerg Hospital, proteins were submitted toSDS-PAGE under reducing conditionson a Copenhagen, Denmark. Mouse 7 s NGF was purified and generously 10% acrylamide resolving gel followed by autoradiography of the dried provided tous by Dr P. Kitabgi (Nice-Sophia Antipolis,France). gel. [T-~~PIATP, [32Plorthophosphate, and RPMI without phosphate were Neurite Assay-A single cell suspension of PC12 cells wasproduced purchased from ICN (Costa Mesa, CAI. by trituration of cells through a syringe needlefollowing trypsination. Cell Culture-Rat pheochromocytoma PC12 cells (45) were cultured Cells were seeded in 12-well dishes at lo4 cells/cm2 and cultured for 1 in RPMI supplemented with 10% ( d v ) horse serum 5% (v/v) fetal calf day before they were shifted to medium containing 0.5% serum and serum, 50 unitdm1 penicillin, and 50 pg/ml streptomycin. Cells were 0.2% BSA and stimulatedas indicated. After 24,48, and 72 h of treatplated a t 1.5 x lo5 cells/cm2 in 24-well dishes or in 6-well dishes for ment, all cells in randomly selected fields were analyzed for neurite kinase assay (ERK1) or 32P-labeling, andMAPKK assay, respectively, outgrowth using phase contrast microscopy. Neurites were defined as cultured for 3-4 days and incubated overnight 0.25% in (v/v) serum and processes exceeding the length of one cell soma diameter and were 0.2% (w/v) BSA prior t o stimulation. generally tipped witha growth cone. 150-250 cells were scoredper well Immunopurification of ERKl or MAPKKActivated in IntactCellsand all treatments were performed in duplicates. Following the serum starvation period, PC12 cells were incubated for various times with peptides or agonists. Medium was aspirated and the RESULTS cell monolayerswere solubilized for 15 min in solubilization buffer containing 1% (v/v) Triton X-100,50 mM HEPES, pH7.5, 150 mM NaCl, CAMP Stimulates ERKl in PC12 Cells-We first measured 10 mM Na,P,O,, 2 mM Na3V04, 100mM NaF, 100 unitdml aprotinin, 20 ERKl activity in PC12 cells treated with a variety of agents p~ leupeptin, and 0.2 mg/ml phenylmethylsulfonyl fluoride. The solu- which share theability t o raise the intracellularlevel of CAMP bilized cell extracts were clarifiedby centrifugation at 18,000 x g for 15 min and then incubated for 2 h with antibodies to ERKl or MAPKK or being themselves a CAMP analogue. After stimulation of preadsorbed on protein A-Sepharose beads.Following the immunopre- ERKlin PC12 cells, the cell monolayerswere solubilized. ERKl wasimmunopurified from the cell lysates, and itsactivcipitation period pellets were washed three times with solubilization buffer. ity was measured in vitro using myelin basic protein as a subERKl Assay-Pellets with immunoprecipitated ERKl were washed strate. two times with HNTG buffer (50 mM HEPES, 150 mM NaC1, 10% (v/v) The various agents, shown in Fig. 1,were found to increase glycerol, 0.1% (v/v) Triton X-100 with 0.2 mM Na,VOJ, dried, and rethe activity of ERKl from 2- to 12-fold above the level seen in suspended in 50 pl of HNTG buffer supplemented with 0.2 mM Na3V04, 100 unitdml aprotinin, 20 p~ leupeptin, and 0.2mg/ml phenylmethyl- unstimulated cells (Fig. 1).Maximal activation occurred rapsulfonyl fluoride. Phosphorylation of MBP was started by addition of idly, within 5-25 min depending on the agent,except for chol150 pg/ml MBP, 10 m~ magnesium acetate, 1 mM dithiothreitol, and era toxin, which may be due to thefact that it raises intracel[y-32P]ATP(5 p ~33, Ci/mmol) added as a 6-fold concentrated mixture in lular CAMP only slowly. The agents increase CAMPin widely a volume of 10 pl. The phosphorylation reaction was allowed to proceed differing ways. IBMX increases CAMPlevels by inhibiting its for 15 min a t room temperature(linearassaycondition)andwas degradation by CAMP phosphodiesterase (46). Cholera toxin stopped by spottingWhatman P-81 filterpaperswhichwerethen dropped into 0.1% (v/v) orthophosphoric acid. The papers were washed activates the G protein Gs through ADP-ribosylation of its a overnight with several changes inthis solution, rinsed once in ethanol, subunit, which subsequently stimulates adenylyl cyclase (47). air dried, and radioactivity was determined by Cerenkov-counting. The Forskolin increases intracellular CAMP by direct binding and reaction blank which received identical treatment was a mixture con- activation of adenylyl cyclase (48). Dibutyryl-CAMP (dbt-cAMP) taining all of the reagents but with the omission of cell lysate during permeates the cell membrane and is metabolized in thecell to immunoprecipitation was subtractedfrom a11 values. MAP Kinase Kinase Assay-MAPKK activity was measured in a generate the active CAMP analogue (491, while chlorophenylreconstitution assay as the abilityof immunopurified MAPKK, stimu- thio-CAMP (CPT-CAMP)is a membrane permeant CAMPanalated in intact cells, t o activate added recombinant rat ERKl (19, 20). logue (50). The second messenger cGMP is structurally related
6209
CAMP Activatesthe MAP Kilqase Cascade in PC12 Cells
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FIG.1. Stimulating effectof agents raising intracellular cAMP on the activity of ERKl in PC12 cells. Serum-starved PC12 cells were incubated with the various agents at the following concentrations and for the following periods of time: IBMX: 1mM, 10 min; cholera toxin: 500 ng/ml, 90 min; forskolin(FORj: 10 PM,15 min; dbt-CAMP: 1mM, 20 min; CPT-CAMP:1 mM, 20 min. At the end of the incubation period, cells were solubilized.ERKl was immunoprecipitated from cell extracts, and its kinase activity was measured using exogenous MBP as substrate as described under “Experimental Procedures.” ERKl activity is expressed as -fold stimulation of the basallevel of unstimulated PC12 cells (buffer condition). Data are means k S.D. of three to six experiments performed in triplicate.
to CAMP and elicits some cellular responses equipotently to CAMP (51). However, cGMP, when added as dibutyryl-cGMP, had no effect on ERKl activity, indicating that the CAMPeffect is not due to cross-reaction with a cGMP-dependent signaling pathway (data notshown). The stimulatory effect of CAMPon ERKl is not limited to the PC12 cell subclone used in this study, since we observed similar effects of CAMPin two other PC12 subclones (data not shown). Importantly, the five agents used in Fig. 1increase intracellular CAMPeach in their own distinct way. Their sole common denominator is the ability to elevate intracellular CAMP.We therefore conclude that CAMPcan activate ERKl PC12 in cells. CAMP Acts Synergistically with Phorbol Ester to Stimulate ERKl in PC12 Cells-Although CAMP stimulates ERK1, it has a rather modest effect compared with certain other activators in PC12 cells, e.g. phorbol ester (PMA), an activatorof protein kinase C (52). CAMP,however, was found to greatlyamplify the stimulatory effect of PMAon ERKl inPC12 cells. Fig. 2.4 shows the time course of ERKl activation by CPT-CAMPand PMA, both used at maximally stimulating concentrations. At each time point tested, theeffect of PMA was potentiatedby a factor of 3 to 4 by the presence of CPT-CAMP. IBMX, cholera toxin, forskolin,and dibutyryl-CAMP (but not dibutyryl-cGMP, not shown) were also found to potentiate the effect of PMA on ERK1, indicating that the potentiation was indeed due to an increase in intracellularCAMP(Fig. 2 B ) . ERK2, another M A P kinase isozyme expressed in PC12 cells ( 5 ) ,appeared tobe regulated by cAMPin much the sameway as ERK1. In experiments performed like the ones shown in Fig. 2A, but instead using antibodies directed against the COOH terminus of ERK2, we found that CAMP by itself stimulated ERK2 activity only slightly but potentiated severalfold the effect of PMA (data not shown). Finally, we tested a neuropeptide, PACAP38, which stimulates CAMP synthesis as well as phosphatidyl inositol breakdown in PC12 cells (40, 53). PACAP38 was found to be an efficient activator of ERK1, showing a rapid, but transient, activity peak, reached within 5 min (lower curves in Fig. 8). Half-maximal and maximal stimulationof ERKl by PACAP38 were observed a t approximately 25 and 200 nM, respectively
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FIG.2. Synergistic effectof CAMP and phorbolester on ERR1 activity in PC12 cells. A, time course of ERKl activation by CFTCAMP and PMA. Serum-starved PC12 were exposed to CPT-CAMP (1 m ~ or)PMA (2 PM) added separately or together for the indicated periods of time. At the end of the incubation period cells were solubilized. MBP phosphotransferase activity of immunoprecipitated ERKl was measured and expressedas -fold stimulation of basal. Data are means S.D. of three experiments performed in triplicates. B , synergistic stimulation of ERKl activity by PMA and agents raising intracellular CAMP. Serum-starvedPC12cellswereincubatedwiththevarious agents at the following concentrations andfor the following periods of time: IBMX, 1 mM, 10 min; cholera toxin,500 ng/ml, 90 min; forskolin (FORj, 10 PM, 15 min; dbt-cAMP, 1m ~ 20, min. PMA was addedat 2 p~ for the same lengthof time as the agent with which its interaction was investigated, except in the experiment with cholera toxin, where PMA was addedfor the final 10 min. At the of end the incubation period cells were solubilized. MBP phosphotransferase activity of immunoprecipitated ERKl was measured and expressed in percent of ERKl activity stimulated by PMA alone. Data are meansof triplicate wells k S.D. of one representative experiment performed three times or more with similar results.
(data notshown). These values are consistent with thebinding affinity of the PACAP type 1receptor and thebiological potency of PACAP38 in PC12 cells and chromaffin cells (40, 53-55) suggesting receptor specificity and physiological relevance of ERKlstimulation byPACAP38.PACAP38 stimulation of ERKl was inhibited by 40% following protein kinase C downregulation, through overnight preincubation with PMA (data not shown), suggesting protein kinase C involvement in the PACAP38 response. Forskolin acted non-additively with PACAP38 on ERKl activation (data not shown), indicating CAMP involvement in the PACAP38 response. Taken together, the observations with PACAP38 suggest that the synergistic stimulation of ERKl by CAMPand activatorsof protein kinase C, shown in Fig. 2, may occur in response to a physiological stimulus in PC12 cells. CAMP Acts Synergistically with Receptor Orosine Kinases on
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CAMPActivates the MAP Kil %useCascade in PC12 Cells
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FIG.3. c A ” acts synergistically with IGF-I on ERKl activation in PC12 cells. Serum-starved PC12 cells were incubated for 10 min with IGF-I (10nM), CF’T-CAMP(1 mM), or forskolin (FOR)(10 VM) in combination as indicated. MBP phosphotransferase activity of immunoprecipitated ERKl was measured and expressed as -fold stimulation of basal. Data are means f S.D. of three experiments performed in triplicates.
ERKl Activation in PC12 Cells-In PC12 cells, peptide growth factors like NGF or epidermal growth factor (EGF) areamong the strongest activatorsof ERKl (30-33) capable of increasing ERKl activity more than 100-fold in our assaysystem. In contrast, we found that insulin-like growth factor I (IGF-I) by itself has a weak effect in PC12 cells. However, a strong synergistic activation of ERKl was observed by CPT-CAMPor forskolin in the presence of a maximal concentration of IGF-I (Fig. 3). In combination with the strongly stimulating growth factors,CPTcAMP or forskolin were also found to potentiate ERKlactivation in a more than additive manner, as shown with NGF, used at a maximally stimulating concentration (see Fig. 9). In combination withEGF, however, a potentiation by CAMPwas most clearly observed, when EGF was used at a half-maximal concentration (data not shown). These observations indicate that in PC12 cells CAMP also potentiates activation of ERKl by growth factors, which initiate their signaling through tyrosine kinase receptors. CAMP Increases ERKl Phosphorylation in IntactPC12 Cells-The activity of ERKl is known to be stimulated upon phosphorylation. We therefore investigated whether elevation of intracellular CAMP increased the level of ERKl phosphorylation. Cells were metabolically labeled with [32Plorthophosphate and exposed to various agonists. Thereafter, cells were solubilized, and cell extracts were subjected to immunoprecipitation with antibodies to ERK1. The immunoprecipitateswere analyzed by SDS-PAGE followed by autoradiography. We found that both CPT-CAMP and forskolin increased [32P]phosphate incorporation in a 44-kDa protein (Fig. 4, lefc and right panels, respectively). This protein has previously been identified as ERKl(33,43,56).PMA also increasedERKl phosphorylation. However, when PMA was added in combination with CPT-CAMPor forskolin, the level of ERKl phosphorylation was markedly higher than with either agent alone. Phosphorylation of ERKl in response to NGF was also increased byCPT-CAMP. Finally, PACAP38 increasedERKl phosphorylation to a high level after 5 min of stimulation, which had returned to near-basallevels within 45 min. The labeling of an additionalphosphoprotein, having a M, of approximately 90,000, was increased by stimulation with CPTCAMPand PMA, NGF, or PACAP38 at 5 min. We have previously shown that this protein corresponds to the pp90rsk protein kinase, which coimmunoprecipitates with ERKl (33, 56) and which is a presumed physiological substrate of ERKl (4, 57). In summary, the increase in thephosphorylation of ERKl in response to CAMP(alone or in combination with other stimuli) paralleled the stimulation of its kinase activity observed in
Figs. 1and 2. These data suggest that CAMPactivates ERKlby increasing itsphosphorylation. CAMP Activates MAP Kinase Kinase in PC12Cells-MAPKK functions immediately upstream of ERKl in the MAP kinase cascade, stimulating itsactivity by phosphorylating it on threonine and tyrosine residues (23). To investigate whether cAMP stimulation of ERKl phosphorylation and activation could be traced upstream in the MAP kinase cascade, we investigated the effect of CPT-CAMP and forskolin on the activity of MAPKK. MAPKK activity was measured in a reconstitution assay as the ability of W K K , immunopurified from intact cells, to activatebacterially expressed, recombinantERK1. The activity of ERKl was measured using MBP as a substrate. In Fig. 5 the results of three experiments areshown (panels A-C, respectively). Data were not pooled in this series of experiments, since the variationbetween individual experiments would obscure the cross-talk between CAMP-and PMA-stimulatedpathways. We found that CPT-CAMP or forskolin inK K from 75 t o 250% above its level creased the activity of W inunstimulated PC12 cells (Fig. 5, A X ) . PMA increased MAPKK activity to approximately the same extentas cAMP at the time point tested. The effects of CPT-CAMPor forskolin in combination with PMA were additive at the least or significantly synergistic, increasing MAPKK activity from 400 t o 1100% above basal levels. Finally, we tested the effect of the physiological ligands on MAPKK activity. PACAP38 stimulated MAPKK with a time course paralleling thatobserved for ERKl activation and phosphorylation (Fig. 6). For comparison, NGF K K activity from 1000 t o 2000% was found to increase W over basal levels depending on theexperiment(datanot shown). In summary, the patternof MAPKK activation by the various stimuli generally reflected the pattern observed for ERKl activation (and phosphorylation) in PC12 cells. The stimulation as expressed in percent over basal wasapproximately 10 times lower in theMAPKK assay as compared with the ERKlassay. This may reflect the fact that the reconstitution assay did not mirror theexact extent of stimulation of W K K activity that may occur in theliving cell. It could also be due t o the fact that W K K is one step upstream of ERKl in a potential signal amplification cascade. In conclusion, elevation of intracellular CAMP stimulates MAPKK in PC12 cells. CAMP may therefore act, at least in part, at the level of MAPKK to increase ERKlphosphorylation and activity. CAMPcan therefore be said t o stimulate theW kinase cascade in PC12 cells. CAMP and PACAP38 Potentiate NGF-stimulated Neurite Outgrowth witha Concomitant Potentiationof NGF-stimulated ERKl Activation-Finally, we investigated ERKlactivation by differentiation of CAMPor PACAP38 in relation to the neuronal PC12 cells. NGF is known to promote the differentiation of PC12 cells into a sympathetic neuron-like phenotype which is associated with the progressive outgrowth of neuronal processes as originally described (45) and illustrated in Fig. 7. Likewise, it hasbeen reported that CAMPpromotes stable neurite outgrowth in some PC12 subclones. In other PC12 subclones, however, cAMP has only a modest and transient neurite promoting effect by itself, but it can potentiate the effect of NGF on long term, stable differentiation. The PC12 cells used in this studybelong t o the latter category as evidenced by the potentiating effect of CPT-CAMPor forskolin on NGF-stimulated differentiation(Fig. 7). Likewise, PACAP38 had only very little neurite-promoting effect by itself in our PC12 cells, but it effectively potentiated neurite formation in response to NGF. After prolonged exposure to NGF (1-2 weeks), in thepresence or absence of CPT-CAMP, forskolin or PACAP38, most of the PC12 cells appeared to have neurites, and the cultures dis-
the MAP Kinase Cascade PC12 Cells in
CAMP Activates
Time (min): Mr x l o 3
FIG. 4. In vivo [SzPlphosphorylation of ERK1. PC12 cells, metabolically labeled with I:'2Plorthophosphate, were incubated with agonists (200 nM PACAP38, 1 mM CF'T-CAMP( C P T ) , 10 PM forskolin (For),2 PM PMA, 50 ng/ml NGF) for the period of time and in combination a s indicated. Following solubilizationERKl was immunoprecipitated from the cell extracts, dissolved in Laemmli buffer, and subjected to SDS-PAGE. We show an autoradiograph of the dried gel from two representative experiments(left and right panels, respectively) outof several experiments with similar results.
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FIG.5. CAMPactivates MAP kinase kinase in PC12 cells. Serum-stawed PC12 cells were incubatedfor 10 min with CF'T-CAMP (1 mM), forskolin (10 PM), or PMA (2 PM) alone or in combination as indicated. At the end of the incubationperiod, cells were solubilized and MAP kinase kinase was immunoprecipitated from cell extracts.Theactivity of immunopurified MAP kinasekinasewas measured as its ability to activate recombinant ERK1, the activity of which was determined using MBP as a substrate as described under "Experimental Procedures.'' Phosphorylation of MBP by ERK1, activated by MAP kinase kinase immunopurified from stimulated PC12 cells, is expressed as percent over MBP phosphorylation by ERK1, incubated with MAP kinase kinase immunopurifiedfrom unstimulated PC12 cells (basal). Data are means of triplicate wells 2 S.D. of three representative experiments ( A X , respectively)out of severalexperimentswith similar results.
PMA PMA
played a similar, dense networkof neurites. Theeffect of CAMP or PACAP38 may thus be characterized as an acceleration of the NGF-induced neuronal differentiation process. As PACAP38 has thepotential to stimulate also protein kinase C, we tested the effect of PMA in combination with forskolin or NGF (Fig. 7). PMA led to only a small potentiation of NGFinduced neurite outgrowth during thefirst 24 h, but thereafter did not potentiate the neuronal differentiation of PC12 cells. Prolonged exposure of cells to PMA, however, is known to downregulate protein kinase C, which may explain the lack of any detectable effects in this long term experiment. NGF is known to induce a robust andprolonged activation of ERKl inPC12 cells as previously described (30-33) and shown in Fig. 8. Interestingly, PACAP38, while having a transient effect itself, increased the sustained activation of ERKl by NGF in a more than additive manner. This potentiation was seen with a half-maximally as well as a maximally stimulating concentration of NGF (Fig. 8, A and B, respectively). Forskolin, CPT-CAMP, and PMA also increased NGF-stimulated ERKl activity during the sustained secondary phase of the NGFresponse as measured after1 h of stimulation (Fig. 9). At this
time point, all of these agents hadvery little stimulatoryeffect on ERKl activity in the absence of NGF. In summary, these data suggest that there may be a causal relationship between the potentiating effect of cAMP (and PACAP38) onNGF-induced ERKl activation and NGF-induced neurite outgrowth. DISCUSSION
In the present study we describe the activation and phosphorylation of ERKl in response to several agents known to increase the intracellularlevel of CAMP.Since these agents act at distinct levels in thepathway of cAMP generation or degradation we conclude that the second messenger CAMP can stimulate ERKl in PC12 cells. In mammalian cells, the vast majority of CAMPeffects can be attributed to activation of the CAMP-dependent protein kinase. This may also be the case for cAMP stimulation of ERKl in PC12 cells. However, protein kinase A-independent CAMP responses have been described. For instance, theinhibitory effect of CAMP on glucosetransport is thought tobe mediated by direct bindingof the nucleotide to the glut 4 transporter molecule (58). Further, cardiac pace-
CAMP Activatesthe MAP Kinase Cascade in PC12 Cells
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T i m (hours) 0 5 mln
45 rnin
NGF: 50 ng Iml
PACAP 38 FIG.6. PACAP38 activates MAP kinase kinase in PC12 cells. Serum-starved PC12 cellswereincubated for 5 or 45 minwith PACAP38 (200 nM). At the end of the incubation period, cells were solubilized, M A P kinase kinase was immunopurified from the cell extracts, and its activity was measured asdescribed in Fig. 5 and under “Experimental procedures.” Phosphorylation of MBP by ERK1, activated by MAP kinase kinase immunopurified from stimulated PC12 cells, is expressed as percent over MBP phosphorylation by ERKl, incubated with MAP kinase kinase immunopurified from unstimulated PC12 cells (basal). Data are means 2 S.D. of three experiments performed in triplicate.
I
0
1
2
3
4
5
’
6
Time (hours)
FIG.8. Time course of activation of ERKl in PC12 cells treated with PACAP38 or NGF. Serum-starved PC12 cells were incubated with PACAP38 (200 nM) andlor NGFat 2 ng/ml (A ) or 50 ng/ml ( B) for the indicated periodsof time. At the end of the incubation period, cells were solubilized. MBP phosphotransferase activity of immunoprecipitated ERKl was measured and expressed as-fold stimulation of basal. Data are meansof triplicate wells 2 S.D. of two representative experiments out of several.
-
-
-
-
NGF:+ + + + + FIG.7. Effect of CAMP, PACAP38, or PMA on NGF-stimulated I 1 I I neurite formation in PC12 cells. PC12 cells were cultured in 0.5% PAC FOR CPT PMA serum in the presence of NGF (50ng/ml), forskolin (10 p ~ )CPT-CAMP , (1mM), PACAP38 (200 nM), or PMA (2p ~in) combinationas indicated. FIG.9. Effect of CAMP, PACAP38, or PMA on NGF-stimulated After 1, 2, and 3 days, the fraction of neurite bearing PC12 cells was ERKl activity. Serum-starved PC12 cells were incubated for 60 min determined and expressed as a percentage of total cells. Data are mean with NGF(50 ng/ml), PACAP38 (200nM), forskolin (FOR, 10 p ~ ) CPT, -c S.D. of three to six experiments performed in duplicate. CAMP(CPT, 1m),or PMA(2 p ~in) combination as indicated. Data are expressed in percent of ERKl activity stimulatedby NGF alone and are mean 2 S.D. of three experiments performed in triplicate.
maker ion channels (591, ion channels inolfactory receptor cilia (511, and potassiudcalcium-specific Drosophila eug channels (60)have been reported t o be activated directly by CAMP.PC12 cells are neuroendocrine cells in which several biological responses to extracellular stimuli are elicited via ion channel activation. A mechanism for ERKl regulation involving the activation of ion channels by cAMP may therefore be a possibility. cAMP increased the in vivo phosphorylation of ERKl aswell as the activity of MAPKK in PC12 cells. It therefore appears that CAMP stimulates ERKl through activation of the MAP kinase cascade operating at the level of MAPKK or upstream of it. The synergistic interaction observed between CAMP and PMA on ERKl was reflected in MAPKK activation, suggesting M A P kinase that this interaction also occurred upstream in the cascade, again being at least at the level of MAPKK. The MAP kinase cascade seems to utilize several activators upstreamof
MAPKK including Raf-1kinase, MAPKK kinases, andpossibly other functional equivalents, perhaps in a cell type-specific or pathway-specific manner (27,29).We found a very pronounced electrophoretic mobility shift of Raf-1 kinase in response to NGF, which is indicative of its activation, as described previously in PC12 cells (25,611. We observed no such mobility shift of Raf-1 in response to CPT-CAMPor forskolin (data notshown). While we cannot exclude that CAMPis activatingRaf-1 without causing a concomitant mobility shift, our data would indicate that CAMPactivates MAPKK through a MAPKK kinase, or a functional equivalent, which is distinct from Raf-1 kinase. We are currently investigating whether the other members of the Raf-family, A-Raf and B-Raf, could be involved. Our observations may have several biological implications. An interesting featureof the CAMPstimulation of ERKl is its
cAMP Activates the MAP Kinase Cascade in
PC12 Cells
62 13
Addendum- During the reviewing process of our manuscript, three synergistic interaction with PMA, which presumably reflects synergy with the protein kinase C-dependent pathway. This papers were published demonstrating that, in contrast to the stimulatory action of CAMP in PC12cells shown in this work, increasing intrafinding opens the possibility that theMAP kinase cascade may cellular CAMP can attenuate activation of the MAP kinase cascade in integrate and amplify signals originating from receptors em- other cell systems. Thus,cAMP antagonizes the stimulatory action of (i) ploying CAMPand receptors using phosphatidyl inositol break- platelet-derived growth factor in aortic smooth muscle cells (Graves, L. down products as second messengers, respectively. By exten- M., Bornfeldt, K. E., Raines,E. W., Potts, B. C., Macdonald, S. G., Ross, sion, receptors activating both pathways, may equally well R., andKrebs, E. G.(1993) Proc. Nutl. Acud. Sci. USA 90, 10300employ this mechanism. With PACAP38 wefound a time course 103041, (ii) insulin in rat adipocytes (Sevetson, B.R., Kong, X., and Lawrence, J . C., Jr. (1993)Proc. Nutl. Acud. Sci. USA 90,10305-10309), and efficiency of activatiodphosphorylation of ERK1, MAPKK, and (iii)EGF, platelet-derived growthfactor, and insulin in NIH3T3 and and pp90rsk, which were overall similar t o that observed with ratl-fibroblasts (Burgering, B. M.T., Pronk, G. J., van Weeren, P. C., CAMPin combination with PMA. It isreasonable to believe that Chardin, P., and Bos, L. J. (1993) EMBO J. 12, 42114220). InterestPACAP38 employs the synergistic interaction between CAMP ingly, in these cell types CAMP also inhibits the biological responses and phosphatidyl inositol-derived second messengers to acti- induced by these agents. vate theMAP kinase cascade in PC12 cells. With the exception REFERENCES of the early events,which are CAMPsynthesis, phosphatidyli1 Pelech, S. E., and Sanghera, J. S. (1992) Pends Biochem. Sci 17,233-238 2 Crews, C. 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