stimulated ~itogen-Activated Protein Kinase Activator in PC12 Cells*. (Received for publication .... specific kinase that becomes tyrosine phosphorylated upon.
Vol. 268, No. 10, Issue of April 5,pp. 7055-7ffi3, 1993 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY @ 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterizationof a Nerve Growth Factorstimulated ~itogen-ActivatedProtein Kinase Activator in PC12 Cells* (Received for publication, May 6, 1992)
Rama K. Jaiswal, Michael B. Murphy, and GaryE. LandrethS From the Department of Neurology and Neurosciences and the Abheimer Center, School of Medicine, Case Western Reserve University, Cleveland,Ohio 441 06
Nerve growth factor treatmentof PC12 cells results sympathetic neuron (2-4). These changes include cessation of a variety in the rapid activation of MAP kinases. These enzymes of cell division,neurite outgrowth, and acquisition are activated through interaction with a protein &ac- of physiological and biochemical characteristics of neurons. tivator.” Themitogen-activated protein (MAP) k’ lnase The mechanisms by which NGF supports the survival of these activator has been partially purified by ion exchange cells and directs their differentiation have been extensively and gel filtration chromatography. The activator has studied but remain its action poorly understood. NGF initiates an apparentmolecular mass of 50-60 kDa. The MAP through bindingto the trkproto-oncogene product, activating kinase activator is rapidly generated in response to its intrinsic tyrosine kinase activity ( 5 , 6). Subsequently, a nerve growth factor(NGF) and canbe detected within number of serine/threonine kinases are activated, driving a 30 s of exposure, reaching maximal levels within 2 cascade of protein phospho~lation that mediates thespecific min and then d e c ~ n i n gto near basal levels by 15-20 biochemical events characteristic of hormone action. Among min. The activation of MAP kinase is dependent upon the time of incubation with the activator and on acti- the most prominent members of this cascade are the MAP kinases, also termed extracellular regulated kinases or ERKs is vator concentration. The MAP kinase activator itself a protein kinasethat phosphorylates MAP kinases and (7). In PC12 cells, two related MAP kinases, ERKl ( ~ 4 4 ~ ~ ~ ) mediates their activation. The NGF-stimulated MAP and ERK2 (p4Zmapk),are rapidly activated upon NGF treatkinase activatorphosphorylates MAP kinase on serine, ment of the cells (7-9). In PC12 cells, MAP kinases become residues threonine, and tyrosine residues, establishing this en- phosphorylated on tyrosine, serine, and threonine zyme as dual specific kinase. The MAP kinase activa- following NGF treatment (7, 10, 11).Initially, itwas thought were substrates for the hormone receptor is itself a phosphoprotein whose phosphorylation that the MAP kinases on tyrosine residues is stimulated upon NGFtreatment tor kinases andwere the most proximal members of a signal of the cells. The enzyme activity of MAP kinase acti- transduction cascade (10). Recent findings suggest that this vator isabolished by treatment withboth the tyrosine- is not thecase, and thesequence of the events mediating the specific phosphatase PTP-1 and theserinelthreonine- activation of these enzymes is considerablymore complex specific phosphatase PP2A. The activator isproduced than originally envisioned. MAP kinase activation has been in response to NGF, epidermal growth factor, and fi- shown to require functionally active p21””, although it rebroblast growth factor. The protein kinase inhibitor mains unclear as tohow ras action is coupled to subsequent K252a selectively inhibits the ability of NGF to gen- kinase activation (12, 13). erate MAP kinase activator activity. These data sugEarlierstudies byAhn et al. (14) andsubsequently by gest that the upstream events governing MAP kinase Gomez andCohen (9) havedemonstratedthattheMAP activation involve the regulated phosphorylation of are activated through interaction with another prokinases dual specificity MAP kinase activatoras an immediate or “activator.” This interaction resulted in the entein(s) consequence of receptor activation. hanced tyrosine and serinelthreonine phosphorylation of the enzyme and subsequent enzymatic activation of the MAP kinases. Phosphorylation at both tyrosine and threonineresNerve growth factor (NGF)’ plays an important physiolog- idues was required for expression of kinase activity, as reical role in the maintenance, survival, and differentiation of moval of tyrosine or threonine phosphate with CD45 and neurons (for review, see Ref. 1).Treatment of the rat pheo- PPBA, respectively, inactivated the MAP kinases (15, 16). It chromocytoma PC12 cell line with NGF promotes the con- has recentlybeen reported that MAP kinases are phosphorylversion of cells from a chromaffin-like phenotype to that of a ated and activatedby a member of the src family of tyrosine kinases, p56Ick (17), as well as other yet unidentified protein * This work was supported by National Institutes of Health Grant kinases (18).However, the discovery that the MAP kinases GM 34908 and NationaI Science Foundation Grant BNS 16094. The can undergo autophosphorylation on both tyrosine and threcosts of publication of this article were defrayed in part by the onine residues (19-21) raised the possibility that MAP kinases payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 may achieve their active phosphorylated state without the interaction with another protein kinase. A number of reports solely to indicate this fact. of the present study have To whom correspondence should be addressed: Alzheimer Re- published since the completion search Lab, €3504, School of Medicine, Case Western Reserve Uni- conclusively demonstrated that the MAP kinase activatoris versity, 2119 Abington, Cleveland, OH 44106. Tel.: 216-368-6101; a kinase that phosphorylates and activates MAP kinases (22”
Fax: 216-368-3079. ‘The abbreviations used are: NGF, nerve growth factor; MAP, mitogen-activated protein; EGF, epidermal growth factor; DTT, dithiothreitol; BSA, bovine serum albumin; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis.
25). Wereportherethecharacterization of a MAPkinase activator that is rapidly generated upon treatment of PC12 cells with NGF. Of particular significance is our finding that
7056
M A P Kinase Activatori n PC12 Cells
upon NGF treatment of PC12 cells, a single species of MAP tions containing peak I MAP kinase (Fig. 1)were dialyzed overnight kinase activator isvery rapidly detected. The activator is dualat 4 "cagainst 20 mM Tris-HC1, pH 7.4, containing 1 mM EGTA, 1 mM EDTA, 10 mMMgC12, and 1mM DTT. Thedialyzed enzyme (0.4 specific kinase that becomes tyrosine phosphorylated upon was treated with alkaline phosphatase-Sepharose beads (20 units) NGF treatment. The MAP kinaseactivator is inactivated ml) for 30 min a t 30 "C to inactivate the enzyme. The reaction mixture upon treatment with either PP2A or the tyrosine-specific was centrifuged in a microcentrifuge, and the supernatant was rephosphatase PTP-1. moved. The alkaline phosphatase treatment reduced the activity of the MAP kinase by 80-90%. Assay of MAP Kinase Activator-Inactive MAP kinase obtained from the untreated PC12 cells or alkaline phosphatase treatment of Materials-NGF was prepared by the method of Smith et al. (26). the NGF-stimulated MAP kinase (10 p l ) and/or recombinant MAP Epidermal growth factor (EGF) was purchased from Upstate Bio- kinase (ERK2) (1pg) were mixed with MAP kinase activator (10 pl). technology (Stoughton, MA), and basic fibroblast growth factor was Activation of the MAP kinase was then initiated by addition of 15 pl from Amgen(Thousand Oaks, CA). Okadaic acid was purchased from of buffer containing 25 mM Tris-HC1, pH 7.4, 2 mM MnCl,, 10 mM Kamiya Biochemical Co. (Thousand Oaks, CA), and K252a wasa gift MgC12,l mM DTT, 1 mg/ml BSA, 0.1 mM Na3V04,1 mM EGTA, 10 of Dr. Y. Matsuda (Tokyo Research Lab, Tokyo, Japan). Recombi- mM p-nitrophenyl phosphate, and 50 p~ ATP (all concentrations are nant rat brain protein tyrosine phosphatase-1 (rrbPTP-1) was a gift final). In control incubations, MAP kinase activator was replaced by of Dr. J. Dixon (University of Michigan), and protein phosphatase buffer. After 15 min, the protein substrate, myelin basic protein 2A (PP2A) was from Dr. M. Mumby (University of Texas). Recom- (MBP, 1 pg), and [y3'-P]ATP (5 pCi) were added to the reaction binant ERK2 and K52R mutant expressed in Escherichia coli were mixture and incubated further for 20 min at 25 "C. After incubation, kindly provided by Dr. M. H. Cobb (University of Texas). Radiola- aliquots from the reaction were removed and pipetted onto a Whatbeled ATP was synthesized using GammaPrep A (Promega Biotec, man P-81 paper (2 X 2 cm). The P-81 paper was washed 3 X 5 min Madison, WI) and 32Pifrom ICN (Irvine, CA). All other reagents in 75 mM H3P04and 3 X 5 min with water, and radioactivity was were purchased from Sigma. measured by Cerenkov counting. In some experiments, the phosCell Culture and Preparation of Eztracts"PC12 cells were grown phorylation reaction was stopped by addition of Laemmli sample in Dulbecco's modified Eagle's medium containing 10% horse serum buffer, and the samples were then boiled for 5 min. The reaction and 5%fetal calf serum in an atmosphere of 10% CO,. The cells were mixtures were then separated by SDS-PAGE, and the bands were collected by trituration in phosphate-buffered saline containing 1mg/ excised from the gels, and incorporated radioactivity was measured. ml bovine serum albumin and 1 mg/ml glucose, washed once, and MAP Kinase Phosphorylation-MAP kinase activator (20 pl) was then resuspended at a final concentration of 2-3 X lo6 cells/ml. The mixed with inactive MAP kinase (20 pl) obtained from untreated cells were treated with growth factors for 5 min at 37 "C a t the cells, recombinant ERK2 (1.5 pg), or a kinase-defective ERK2 muindicated concentrations. The cells were then collected by centrifu- tant-K52R (1.5 pg) in a buffer containing 25 mM Tris-HC1, pH 7.4, gation and resuspended in lysis buffer containing 20mM Tris, pH 10 mM MgCI, 1 mM DTT, 1 mM EGTA, 0.1 mM Na3V04,and 15p M 7.4,l mM EGTA, 1 mM EDTA, 1 mM DTT, 0.1 mM Na3V04,0.5 mM ATP (20 FCi of [r3'-P]ATP). After 30 min at 30 "C, the reaction was phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 10 mM p- terminated by boiling in Laemmli sample buffer. The proteins were nitrophenyl phosphate. The cells were sonicated briefly (2 X 15 s a t separated by 10% SDS-polyacrylamide gel electrophoresis, dried, and 4 "C) with a Kontes Cell Disrupter at a power setting of 5 and then exposed to x-ray film (Kodak, XAR 5). The phosphorylated MAP centrifuged at 12,000 X g in a microcentrifuge a t 4 "C for 5 min. The kinases were visualized as 42 kDa bands on the autoradiogram. supernatant was collected and centrifuged for 35 min at 100,000X g. Phosphoamino Acid Analysis-Phosphoamino acid analysis was In each experiment, control and NGF-treated cells were processed in performed essentially as described by Kamps and Sefton (27). 32Pparallel. Labeled proteins were separated by 10% SDS-polyacrylamide gel Chromatography-Extracts were prepared from approximately 1- electrophoresis and transferred to Immobilon P membranes. The phosphorylated bands at 42 kDa were cut from the membrane and 1.5 X 10' cells, which had been treated for 5 min with NGF (50 ng/ ml). Anion exchange chromatography was performed on a Pharmacia digested in 50 pl of 6.7 M HC1 at 106 'C for 1 h. Water (0.5 ml) was fast protein liquid chromatography Mono Q HR5/5 column equili- then added, and the solution was transferred to a microcentrifuge brated in Buffer A (20 mM Tris, pH 7.4, 1 mM EGTA, 1 mM EDTA, tube. The tube was dried under vacuum ina SpeedVac, and the 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzami- residue was redissolved in 3 pl of a solution containing phosphoserine, dine, and 0.1 mM Na3V04). Samples were applied to thecolumn at a phosphothreonine, and phosphotyrosine (10 mg/ml). The samples flow rate of 1 ml/min, and thecolumn was then washed with 5 ml of were then applied to a thin layer cellulose plate and subjected to Buffer A and eluted with a 0-0.5 M NaCl gradient in Buffer A. electrophoresis (pyridine:acetic acidH'0; 1:10189, pH 3.5) at 1 kV Fractions were immediately assayed for MAP kinase activity. For for 45 min, air-dried, and autoradiographed after ninhydrin staining. Inactivation of MAP Kinase Activator by Phosphatases-MAP kication exchange chromatography, peak fractions from the Mono Q column were dialyzed for 2 h against a buffer containing 20 mM nase activator obtained from Mono Q fractions from NGF-treated HEPES, pH 7.4,2 mM EGTA, 1 mM EDTA, and 1mM DTT at4 "C. PC12 cells were pooled and dialyzed overnight at 4 "C against a buffer The dialyzed samples were applied to a Mono S HR5/5 column containing 25 mM Tris-HC1, pH 7.4, 1 mM EDTA, 1 mM EGTA, and preequilibrated in the same buffer. The column was eluted with a 1 mM DTT specifically to reduce the orthovanadate concentration to linear gradient of 0-0.4 M NaCl (40 ml) in the same buffer at a flow levels that would not inhibit the phosphotyrosine phosphatase. Dialyzed MAP kinase activator (0.4 ml, 0.1 mg of protein) was treated rate of 1ml/min, and 1-ml fractions were collected. Gel filtration chromatography was performed by first concentrat- with either alkaline phosphatase-Sepharose (20 units) or with ing column fractions using Centricon ultrafiltration units (Amicon). rrbPTP-1 (300 units/ml). The protein tyrosine phosphatase 1 is a The samples were then applied to a Superose 12 HR10/30 column truncated form of rat brain PTP-1 expressed in E. coli (28). The equilibrated with Buffer A containing 0.2 M NaCI. The flow rate was MAP kinase activator was incubated at 30 "C with the various phos0.25 ml/min, and 0.25-ml fractions were collected. The column was phatases. At the indicated times, aliquots (10 pl) were removed from calibrated using IgG (97 kDa), bovine serum albumin (68 kDa), the reaction, and thePTP-1was inactivated by the addition of 2 mM ovalbumin (45 kDa), and cytochrome c (12.4 kDa) as molecular mass Na3V04or by simply removing the alkaline phosphatase-Sepharose resin by centrifugation. PP2A treatment was performed by incubating standards. For time course and ligand activation experiments, the MAP kinase this phosphatase (10 milliunits/ml) with Mono Q fractions (0.4 ml) activator was isolated using a stepwise elution method. PC12 cells containing MAP kinase activator, which had been dialyzed against (1-2 X lo6 cells) were treated with growth factors for the indicated 25 mM HEPES, pH 7 . 4 , l mM EDTA, 1 mM EGTA, 1 mM DTT, 2 times. The cells were centrifuged briefly in a microcentrifuge and mMMgC12, and 0.5 mg/ml BSA. At the indicated times, aliquots (10 then lysed in 1.0 ml of lysis buffer as described above. The supernatant pl) were removed, okadaic acid was added to a final concentration of , the activator activity was then assayed. The reactivation was mixed with 0.2 ml of DE-52 ion exchange resin (Whatman) in a 1 p ~ and microcentrifuge tube and incubated for 10 min on ice with occasional of MAP kinase was measured using MAP kinase that had been shaking. The resin was collected by centrifugation and then washed inactivated by alkaline phosphatase as described above. The phos3-4 times with 1 ml of Buffer A. The resin was washed with 1.0 ml phatase-treated activator was combined with inactive MAP kinase of Buffer A containing 0.01 M NaCl and activator eluted with 0.7 ml and incubated for 15 min at 22 'C. Radiolabeled ATP (6.6 cpm/fmol) and MBP (1pg) were added, and the incubation was carried out for of Buffer A containing 0.04 M NaCl. Alkaline Phosphatase Inactivation of MAP Kinase-Mono Q frac- an additional 20 min at 22 "C. The 32Pincorporation into MBP was EXPERIMENTALPROCEDURES
7057
MAP Kinase Activator in PC12 Cells determined by applying aliquots of the reaction mixture onto P-81 paperandwashingasdescribedabove.Controlincubations were performed by inactivation of the phosphatases with 2 mM Na3V04or 1PM okadaic acid for20 min prior to the addition of the MAP kinase activator. Immunoblotting with Anti-phosphotyrosine Antibody-Protein present in Mono Q column fractions obtainedfrom NGF-treated and untreated control PC12 cell lysates were trichloroacetic acid-precipitated and separated on 10% SDS-PAGE gel. The proteins were then transferred to an Immobilon P membranefor 4 h at 20 mA in a solution containing 15 mM Tris, 112 mM glycine, and 9% methanol. After blotting, the membrane was blocked with TBS (20 mM TrisHC1, pH 7.6, 137 mM NaCl) containing 1%BSA, 0.1% Tween 20 and 0.01% sodium azide overnight at 4 "C. The membrane was washed once with TBS containing 0.1% Tween 20 (TBST) and then twice with TBST for 5 min. Membrane was incubated with monoclonal anti-phosphotyrosineantibody (Upstate Biotechnology Inc.) at a dilution of 1:lOOO in TBST containing 1%BSA and 0.01% sodium azide for 45 min at room temperature. The membrane was washed three times with TBST for 5 min. After washing,the membrane was incubated for 45 min with peroxidase-conjugated goatantimouse IgG at a dilution of 1:10,000 inTBST containing 1%BSA. The membrane waswashed threetimeswith TBST. Phosphotyrosine-containing proteins were detected by ECL Western blotting detection reagents (Amersham Corp.) exactly followingthe manufacturer protocol.
2000-( A
/P 5
I
/I
1
E
.25~ m
z
0
10
20
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"
Fraction Number
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2000 B
/
b.5
RESULTS
Chromatographic Characterization of MAP Kinase Actiuator-To characterize the NGF-stimulated MAP kinase activFraction Number ity in PC12 cells, cytosolic extract prepared from PC12 cells FIG. 1. Identification of MAP kinase activator activity by treated with 50 ng/ml NGF for 5 min was chromatographed Mono Q chromatography of extracts of NGF-stimulated PC12 on a Mono Q column. The Mono Q chromatography resolved cells lysate. PC12 cells (1-2 X 10') were treated with 50 ng/ml NGF two major peaks of NGF-stimulatedkinaseactivities. As for 5 min at 37 "C. Cell lysate (14 mg of protein) were prepared and chromatographed on a Mono Q columnusing a 40-mllinear salt shownin Fig. L 4 , NGF-stimulatedMAPkinaseactivity gradient (solid line) from 0-0.5 M NaCl as described under "Experi( ~ 4 2 designated ~ ~ ~ ~peak ; I) which eluted from the Mono Q mental Procedures." One-ml fractionswere collected, and aliquotsof column at 0.09-0.1 M NaCl, and the MAP2 kinase ( ~ 4 4 ~ "each ~ ~fraction ; were assayed for phosphorylation of MBP. A, MBP phosphorylation by fractions from NGF-treated ( 0 )and control (0) designated peak 11) eluted at 0.18-0.2 M NaCl. Both peaks exhibited almost equallevels of kinase activity when assayed PC12 cells chromatographed on a Mono Q column. B , MBP phoswith MBP and consistently showed a >16-fold increase, as phorylation by NGF-treated (0)and control (0)Mono Q fractions in the presence of inactive MAP kinase (peak I) or fractions from compared with control cells. The chromatographic behavior untreated cellscorresponding to peak I (fraction 21). The peak and MBP phosphorylation activityof these two peaks of MAP fraction containing MAP kinase activator activity isshownby an kinase activity are identical with thatpreviously reported in arrow. No MAP kinase activator activity was detected in any other these cells and many other cell types in response to NGF or fractions; similar resultswere obtained in more than 10 experiments. other growth factors (14,15, 29). was eluted a t 0.1 M NaCl In order to ascertain whether NGF treatment of PC12 cells A single MAP kinase activator peak generated a species that is able to activate MAP kinase (a (Fig. 2). MAP kinase activator),we screened the MonoQ fractions for Theapparent molecularweight of theNGF-stimulated the abilityto reactivate the peakI MAP kinase that has been MAP kinase activator was determined by gel filtration chromatography. Fractions from Mono Q columns corresponding previously inactivated by alkalinephosphatasetreatment. Although we have routinely used alkaline phosphatase-inac- to MAP kinase-activating activitywere concentrated 10-fold tivated MAP kinase, identical results were obtained in these and then applied to a Superose 12 gel filtration column. MAP assays whether we employed peak I MAP kinase obtained kinase-activating activity was detected in a peak that eluted from untreated control cells or following alkaline phosphatase with an apparent M, = 50-60,000 (Fig. 3). A 26-fold increase inactivation of peak I activity obtained from NGF-treated in MAP kinase activity was observed when peak fractions cells. Upon combination of Mono Q fractions from NGFfrom Superose 12 column were incubated with inactive MAP treated PC12 cells with inactive MAP kinase, we detected kinase (Fig. 3). reactivation of the enzyme confinedto a peakincluding The capacity of the MAP kinase activator to stimulate the fractions 14-15, which eluted between 0.03-0.05 M NaC1. A MAP kinase activity was time- and dose-dependent. MAP 5-6-fold stimulationintherate of MBP phosphorylation kinase activity increased as a function of incubation times above control levels was observed. No MAP kinase-activating and was evident within 5 min, reachinga maximum enhanceactivity was found in any other fractions, nor in corresponding ment after 25 min. The rate of MAP kinase activation was fractionsobtainedfromuntreatedcontrol cells(Fig. 1B), linear for up to 20 min and was directly proportional to the indicating that this activator was specifically generated upon amount of MAP kinase activator added to the reaction mixNGF treatmentof the cells. ture (data not shown). The peak activator fractions from the Mono Q column were Time Course of Activation of MAP Kinase Activator-We furthercharacterized by cation exchange chromatography investigated the time course of stimulation of MAP kinaseusing a Mono S column. Fractions were examined for MAP activating activity by NGF (Fig. 4). Exposure of PC12 cells kinase-activatingactivity by preincubationwithinactive to 50 ng/ml NGF resulted ina rapid but transient rise in the to addition of MBP as previously described. activator levels that was first detected after 30 s, reaching a MAP kinase prior
7058
PC12M Ain Activator P Kinase
Cells
0.4
0
7
0
E. 0.2 0
z 0
.-
”
’..
30
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10
A
40
Fraction Number FIG. 2. Cation exchange chromatography of MAP kinase activator. The peakfractionscontainingMAPkinase-activating activity froma Mono Q column were dialyzed and loaded onto a Mono S column as described under “Experimental Procedures.” The column was developed with a linear gradient (dotted line) from 0-0.4 M NaC1.One-ml fractions were collected, and alternate fractionswere assayed for MBP phosphorylation by preincubating with peak1MAP kinaseobtained from untreatedPC12 cells (0) or buffer (0)as described in the legend to Fig. 1. Similar results were obtained in a t least three different experiments.
.-0
iij-
3$
20
30
TIME (rnin)
FIG. 4. Time course of induction of MAP kinase-activating activity. PC12 cells (1-2 X lo6) were incubated with NGF (50 ng/ ml) for the indicated time periods. The cells were collected by brief centrifugation (15 s), and the cells were lysed. The clarified lysate was incubated with DE-52, and the MAP kinase activator was eluted from the resinby buffer A containing 0.04 M NaCl asdescribed under “Experimental Procedures.” Aliquots (10 pl) of each fraction were preincubatedwithequal volumes of inactiveMAPkinaseinthe presence of Mg-ATP for 15 min, followed by 20-min incubation with MBP (1 pg) and [-y3’P]ATP ( 5 pCi). Phosphorylation of MBP was measured asdescribed under “Experimental Procedures.” Values were (kf3.D.) of normalizedfor proteinconcentration.Dataaremean triplicate determinations.
0
c
10
0
6 0
25000
8oool
-
20000
22
ts
:
15000
Cn.
m 2
10000
5000
1 60
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20
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80
Fraction Number FIG. 3. Gel filtration chromatography of MAP kinase activator. Mono Q fractions corresponding to MAP kinase activator were pooled and concentrated (10-fold). The sample (200 pl) was loaded on Superose12 as described under “ExperimentalProcedures.” Aliquots (10 ~ l of) eluting fractions were preincubated with equal volumes of inactivepeak I MAP kinase (0)or buffer (0) in the presence of Mg-ATP for 15 min priorto addition of MBP and [y”P] ATP. MBP phosphorylation was then measured after a further 20min incubation. The position of void volume ( Vo) and elution of the proteinstandardsareindicated by arrows. OA, ovalbumin; CyC, cytochrome c.
FIG. 5 . Effect of various growth factors and kinase activators on stimulation of MAP kinase-activating activity. PC12 cells (1-2 X lo6) were incubated with NGF(50 ng/ml), EGF (100 ng/ ml), basic fibroblastgrowthfactor (bFGF, 100 ng/ml),and 12-0tetradecanoylphorbol-13-acetate(TPA, 1 p ~ for) 5 min. In experiments where K252a was used, cells were preincubated for 20 min with 25 nM of K252a before the addition of growth factors. Cells were pelleted by brief centrifugation, and the activator was isolated by absorbtion to DEAE-cellulose as described under “Experimental Promaximum level at 2 min, and subsequentlydeclining to basal cedures.” MAP kinase activity was determined as described in the levels by 20-25 min. It is interesting to note that the appear-legend to Fig. 4.The values represent means (+S.D.) of three experance of the MAP kinase activatorpreceded that of the NGF- iments performed in duplicate andnormalized for protein concentrastimulated MAP kinase activity in PC12 cells. The activity tion.
of MAP kinase is maximal after 5 min and then returns to basal level within 30 min (8). Thedevelopment of MAP EGF (29, 30) and fibroblast growth factor (31), all of which kinase-activating activitywas correlated with the time coursehave been shown to activate MAP kinase (32). MAP kinase of tyrosine phosphorylationof MAP kinase inPC12 cells (11). activity is also stimulated by exposure of the PC12 cells to (33) The tyrosine phosphorylation of MAP kinase preceded the phorbolesters (12-0-tetradecanoylphorbol-13-acetate) activation of MAP kinase activity andwas apparent within 1 and the phosphatase inhibitor okadaic acid (34). Therefore, we investigated the ability of these agents to stimulate the min of NGF exposure, with the peak tyrosine phosphorylation occurring after 2 min and then subsequently declining signif- MAP kinase activator activity inPC12 cells. The data shown in Fig. 5 demonstratethe level of MAP kinaseactivator icantly over the next 30 min. Ligand Specificity of MAP Kinase Actiuator”PC12 cells measured following 5 min of exposure to NGF (50 ng/ml), possess receptors for a variety of growth factors, including EGF (100 ng/ml), basic fibroblast growth factor (100 ng/ml),
M AActioator P Kinase
in PC12 Cells
so59
and 12-0-tetradecanoylphorbol-13-acetate(1 p ~ ) NGF . was [AI I I 40000 the most potent stimulus, resulting in a 7-fold stimulation of the MAP kinase activator, whereas EGF and basic fibroblast growth factor stimulated the activator by 4- and 3-fold, respectively. In general, the magnitude of stimulation of the PC12 MAP kinase activator was correlated with that of MAP kinase activity and was consistent with that reported earlier by Ahn et al. (14) in EGF-stimulated 3T3cells. However, 120-tetradecanoylphorbol-13-acetatewas able to produce only a modest increase in activator activity. This correlates with the modest stimulation of MAP kinase activityby this agent in PC12 cells (35). We tested the effect of K252a on the growth factor-stimulatedMAPkinase-activatingactivity. K252a is a protein kinase inhibitor which has been shown to specifically inhibit NGF-mediated responsesin PC12 cells a t nanomolar concentrations, while having no effect on the EGF- and fibroblast growth factor-stimulated responses (36, 37), presumably by selective inhibition of the trk kinase(38).Pretreatment of the PC12 cells with 25 nM K252a specifically inhibited the production of the NGF-stimulated MAP kinase activator by more than 60%. K252a did not have any inhibitoryeffect on EGFand fibroblast growth factor-stimulated levels of MAP kinase e activator. Paradoxically, K252a was found to have a stimulatory effect on fibroblast growth factor-mediated stimulation of the MAP kinase activator. Activation and Phosphorylation of MAP Kinases by MAP Kinase Activator-We tested whether the NGF-stimulated MAP kinase activator would directlyphosphorylateMAP kinase andmediate its activation. Phosphotransferase activity towardMBP was measuredasan index of MAPkinase activity in the absence or presence of MAP kinase activator FIG.6. Activation and phosphorylation of w i l d type (Wn. (Fig. 6A). Incubation of normal wild type (WT) MAP kinase recombinant ( E R K 2 ) . and kin--defective ( K M MAP obtained from PC12 cells, recombinant ~ 4 2 ' " "(ERK2) ~~ and/ kinasesbyMAPkinaseactivator. A , the peakMAf' kinase or ERK2-K52R mutant (KM) with Mg-[y"-PIATP alone did activator fraction ( A ( ' 7 ' ) from Mono Q chromatography wns incubated with WT. ERKZ, and KMin the presence of Mg-ATP. After not result in detectable activation of MAP kinase activity, 15 min, M R P ( 1 p a ) and [y" PJATP( 5 pCi) were added, and incorindicating littleor no autoactivationof the catalytically active poration of the radioactivity in .MRI' was determined after 20 min as ERK2 occurred.However,in the presence of MAP kinase described under "Experimental Procedures." H . the WT, ESIKZ, and activator, WT and ERK2 were both activated and to equal KM MAP kinases were phosphorylated in the presence or ahsence of levels. The ERK2-K52R mutant, in which lysine 52 in the MAP kinase activator as described under "Experimental Procedures." ATP-binding site had been changed to arginine, renderingit The phosphorylated proteins were suhjected to SDS-PAGE. dried, catalytically inactive, did not exhibit any phosphotransferase and autoradiographed. The arrow indicates the position ofphosphorylated 42-kI)a protein bands representing the various MAP activity toward MBP when incubated with activator under kinases. similar conditions.We also tested the capacity of MAP kinase activator to phosphorylate WT, ERK2, and KM in parallel MAP kinase activatoris a dual specificity kinase. experiments. As shown in Fig. 6R, MAPkinaseactivator Phosphatase Inactivation of MAP Kinasp Activator-The phosphorylated WT, ERK2, and KM, as indicated by detec- MAP kinase activator was detected following activation of tion of radioactivity incorporated into the 42-kDa protein trk receptor kinase, and it was possible that activator was bands. Under similar conditions, no phosphorylation of these itself regulated by phosphorylation. T o test this possibility. proteins was observed if MAP kinase activator was omitted. we treated the MAP kinase activator with alkaline phosphaThe phosphorylation of ERK2-K52R mutant, aswell a s W T tase, the serine/threonine specific protein phosphatase 2A and ERK2, provided direct evidence that MAP kinase acti(PP2A). and tyrosine-specific recombinant rat brain tyrosine vator is itself a protein kinase. phosphatase-1 (rrbPTP-1) for different time periods and then Phosphoamino Acid Analysis of in Vitro Phosphorylated tested the ability of the activator to stimulate the activity of WT, ERK2, and K52R Mutant MAPKinase-We carried out MAP kinase (Fig. 8).The treatmentof MAP kinase activator phosphoamino acid analysis of MAP kinases after in vitro with alkaline phosphatase immobilized on agarose resulted in phosphorylation by MAPkinaseactivator.Theautoradithe inactivation of MAP kinase activator. More than 80% ograms reveal the presence of phosphoserine, phosphothreo- inactivation was observed within 10 min, and a complete loss nine, and phosphotyrosine residues. The relative amount of in activator activity was achieved within 30 min. This obserthe phosphoamino acids observed was identical in WT, ERK2,vation indicated that the activator was itself a phosphoproand KM (Fig. 7). After the in vitro phosphorylation by MAP tein, and its capacity to stimulate MAP kinasewas exhibited kinase activator, all three 42-kDa phosphoproteins contained only by the phosphorylated MAP kinase activator species. predominantly phosphotyrosine withlesser amounts of phos- Alkaline phosphatase acts primarily as serine/threonine phosphothreonine and phosphoserine. The finding that the cata- phatase but also exhibits tyrosine phosphatase activity (39). lytically inactive ERK2-K.52R mutant was phosphorylateda t The specific involvement of the serine/threoninephosphorylall residues established that the NGF-stimulated PC12 cell ation of the activatorwas tested by incubation of MAP kinase "
MAP Kinase Activator in PC12Cells
7060 Pi
+
p-Ser
+
p-Thr
+
p-Tyr
+
I
Origin+ I
WT
ERKP
KM
FIG.7. Phosphoamino acid analysis of wild t y p e (WT), recombinant ( E R K 2 ) .and kinase-defective( K M ) MAP kinases after activation by MAP kinase activator. WT, EKKZ, and KM MAP kinases were incuhated with MAP kinase activator, and phosphoamino acidswereanalyzed as describedunder“Experimental Procedures.” After electrophoresis on thin layer cellulose,phosphorylated amino acids were located by autoradiography. The position of phosphotyrosine (p-Tyr),phosphothreonine (p-Thr),phoaphoserine (p-Ser), inorganic phosphate (Pi), and the point of sample application (Origin) are indicated. Other labeled spots represent partially hydrolyzed phosphopeptides.
phorylationisrequired for the expression of MAP kinase activator activity. Treatment of the activator with the tyrosine-specific phosphatase rrbPTP-1 alsoled to its inactivation. Only 4% of the MAPkinaseactivatoractivityremainedafter 30 min of incubation withPTP-1 (300units/ml). In control experiments in which the activity of PTP-1 was inhibited by the presence of 2 m M Na3V04, there was no loss in the activityof activator (Fig. 8). The specificity of PTP-1 for phosphotyrosine indicated that the expression of MAP kinase activator activity involved tyrosine phosphorylation. Taken together, these results indicate that the MAP kinase activator must he phosphorylated a t serine/threonine and tyrosinefor expression of enzyme activity. Detection of Phosphotyrosine Phosphotylation of the MAP Kinase Actiuator-To determine whether MAP kinase activator was tyrosine-phosphorylated in response toNGF in PC12 cells, we prepared theMono Q fractions from the NGFtreated or untreated control cells. Fractions containing the MAP kinase activator activity were separated on 10% SDSPAGE and then transferred to an Immobilon P membrane andimmunoblotted withmonoclonal anti-phosphotyrosine antibodies. The anti-phosphotyrosine antibodies stronglyrecognized a protein band whose molecular mass on SDS-PAGE was -50 kDa in Mono Q fractions containing MAP kinase activator activity (Fig. 9R). The anti-phosphotyrosine immunoreactivity of the protein was dramatically stimulated on NGF treatment of the cells. The peak immunoreactivity was detected in fraction 14, coincident with the peak MAP kinase activator activity as determinedby MRP phosphorylation in the presenceof ERK2 (Fig. 9A ). The observation of the NGFof an appropriatelysized stimulated tyrosine phosphorylation protein in a position coincident with MAP kinase activator activity, coupled with the rrPTP-1 sensitivity of the activator, provides strong evidence for the critical involvementof tyrosine phosphorylation in MAP kinase activator activation. DISCUSSION
The cellularmechanismsthrough which NGF elicits its biological actions has recentlybeen the focus of much attention following the discovery that the trk proto-oncogenewas capable of binding NGF, resulting in activation of its intrinsic tyrosine kinase activity (5, 6). NGF triggers a signal transductioncascadein which MAPkinasesare a prominent component. The potential downstream targetsor effectors of Raf-1, S-611 kinase, and c-jun MAP kinase action include (40-42). It is now clear that p21” is an obligatory interme0 3 01 0 20 TIME (min) diateinthispathway,asras-dominant negative mutants FIG.8. Inactivation of MAP kinase activator by phospha- abolish the ability of NGF to stimulate MAP kinase activity af. (43) haveprovided evidence that pp60”” tase. The peak MAP kinase activator fraction from Mono Q chro- (12,13). Kremer et matography was dialyzed and then incubated for the indicated times is a potential upstream regulatorof ~ 2 1 ’ ”action in PC12 cells at 30 “C with 10 milliunits/ml PPZA, 300 units/ml PTP-1, or 50 in response to NGF, a t least with respect to the capacity of units/ml alkaline phosphatase as describedunder“Experimental these cells to extend neurites. Procedures.” Phosphataseswere inactivated by okadaic acid (PPZA, The molecular mechanisms subserving the activation of the m), Na3V04 (PTP-1, 0 ) or by centrifugation (alkaline phosphatase, A).MAP kinase activator was then tested for its ability to reactivate MAP kinases have come under increasing scrutiny (44) and the inactive MAPkinase. In controlexperiments, activator was have been proven to be substantially more sophisticated than incuhatedwithPPZA + okadaic acid (A) or PTP-1 + Na3VOI (0) originally believed. It was initially presumed that a very simultaneously. limited number of intervening steps might existbetween the hormone receptor and MAP kinase activation. However, the with PP2A (10 milliunits/ml). PP2A inhibited the ability of recognition that expression of enzymaticactivity required that the enzyme be phosphorylatedonbothtyrosineand the activator to stimulate MAP kinase activity. More than 50% of activity was lost within 5 min of incubation, with a threonine residues was interpreted as being indicative of the convergence of multiple signal transduction pathways acting complete loss of activity within 30 min. In control experiuponMAPkinase(16). Although thisinterpretation was ments,approximately 90% of theactivityremainedwhen wholly activator was treated with PP2A in the presence of okadaic conceptuallyappealing,theavailabledataarenot consistent with this hypothesis. Subsequently, it was found acid. Thesedatademonstratethatserine/threoninephos-
PC12 MAP inActivator Kinase
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previously reported findings. When Mono Q column fractions from NGF-treated PC12cells were screened for the activities able to reactivate inactive MAP kinase I (Fig. 1, peak I ) . the activator consistently eluted before the MAP kinase I peak as a sharp peak a t 0.03-0.05 M NaCI. We have never detected any MAP kinase activating activity in any other fractionsnor in fractions derived from untreated control cells. The MAP kinase activator described by Gomez and Cohen (9) eluted as a broad peak on a Mono Q column and eluting immediately before and overlapping with the MAP kinase I peak. We did not detect activator activity elutingin the flow-through fractions, as reported by Ahn et al. (22, 14) using PC12 or 3T3 10 1 2 13 1 4 15 16 17 Fraction No. cells. Theapparent M, = 50-60.000 for theMAP kinase B ( K , activator reported here is similar to that reported by both Ahn et af. (14)and Gomez and Cohen (9).The recently I characterized MAPkinaseactivatorexhibitssimilarchro49.5 matographic behavior, but has an apparent size of 45 kDa (23. I I 24, 45, 52). 1 0 1 2 1 3 1 4 1156 17 is itself a phosphoprotein whose The MAP kinase activator Fraction No. activity is regulated through phosphorylation. We report here C )aDkf - I MW that treatment of the MAP kinase activator with PP2A or rrbPTP-1results in thecomplete loss of activity.These I findings demonstrate that the activator requires phosphorylation a t bothserine/threonineandtyrosine residues.It is notable that these data differ from those of Comez andCohen 10 12 13 14 15 16 17 (9), who found that PP2A was effective in inactivating the Fraction No. MAPkinaseactivator,whereasthethreeproteintyrosine FIG.9. Stimulation of tyrosine phosphorylation of MAP ki- phosphatases, CD45, T-cellphosphatase,and LAR, were nase activator by NGF in vivo. PC12 cells were treated with or of theMAP kinase without NGF (50 pglml) for 5 min, and the extracts were prepared without effect. ThePP2Asensitivity and chromatographed on a Mono Q column. A, the Mono Q column activator was also verified by others (25. 45). The capacity of fractions (10 pl) from NGF (solid bars) or nontreated control cells rrbPTP-1 to inactivate the MAP kinase activator may he a (htched bars) were tested for MAP kinase activator activity using consequence of a restricted substrate specificity exhibited by the MBP phosphorylation assay as described under the "Experimen- this enzyme. Alternatively, rrbPTPmay express low levels of tal Procedures," except that recombinant ERK2 (1 p g ) was substituted for inactive wild type MAP kinase. R, the peak Mono Q column serine/threonine phosphatase activity, although this has not fractions (1 ml) obtained after NGF treatment of PC12 cells were been reported and we consider it unlikely. Our observation of trichloroacetic acid-precipitated and subjected to 10% SDS-PAGE. rrbPTP inactivation of the activator was unexpected, and Proteins were transferred to lmmobilon P membrane. Membranes serial repetitions of this experiment lead us to conclude that wereprobedwith monoclonal anti-phosphotyrosine antibody. Im- tyrosine phosphorylation is a critical regulator of the MAP munoreactive proteinswere detected by the enhanced chemilumines- kinase activator. This finding is furthersupported by our cencemethod as described under"ExperimentalProcedures." C, Mono Q column fractions from untreated control PC12 cells were finding that NGF treatmentof the PC12 cells resulted in an processed and probed by monoclonal anti-phosphotyrosineantibody increased tyrosine phosphorylation of a protein band having as described above. An arrow denotes the electrophoretic mobility of a molecular mass of 50-60 kDa eluting in fractions coincident the tyrosine phosphorylatedproteins having a molecular mass of SO with MAP kinase activator activity on Mono Q chromatogkDa. raphy.Indeed,MAPkinaseactivatorhas beenshown to undergo autophosphorylation on tyrosine, serine, and threothat a soluble MAPkinase"activator" was generatedon nine residuesin EGF-stimulated A431 cells (23) and from hormone treatment of 3T3 cells (14). Further, the discovery Xenopus oocytes (45), suggesting the importance of tyrosine that MAP kinases had the capacityto autophosphorylate on phosphorylation in expression of MAP kinase activator activboth threonine and tyrosine residues has contributed to the ity. MAPkinase was notreactivated following incubation controversy surrounding the mechanisms through which these with Mg-ATP alone, indicating that the autophosphorylation enzymes are activated. of tyrosine and serine/threonineresidues occurs at levels that The original observation that a MAP kinase "activator" are insufficient to result in significant elevation of the enzycould be detected was that of Ahn et al. (14). They found that matic activity. MAP kinase could be activated upon combination of inactive NGF very rapidly stimulated the generationof MAP kinase MAP kinase with other fractions from hormone-treated cells. activator that was detected within 30 s of NGF exposure. Moreover, inactivation of MAP kinase by removal of either Importantly, our results are consistentwith an in oiuo role of threonine or tyrosine residues was reversed with the regen- the activator, as its activation precedes the development of eration of the doubly phosphorylated species. Subsequently, MAP kinase activity,which was maximal at 5 min (8. 29, 46) Gomez and Cohen (9) have reported similar findings in NGFand isclosely correlated with the tyrosine phosphorylationof treated PC12 cells. After completion of the present studies,a the enzyme (11). Activator levels have returned to nearbasal number of reports describing isolation of MAP kinase acti- levels after 15 min of NGFtreatment.Theseresultsare vator thatpossess as an intrinsic protein kinase activity have consistent with thoseof Seger et af. (23) butdiffer from those been reported (22-25). reported by Gomez and Cohen (9). The latter study found The partial purification of the activatorisolated from NGF- MAP kinase activatorin PC12 cells following 15 min of NGF treated PC12 cells by ion exchange and gel filtration chro- treatment. It is unclear whether the activity they have charmatographyreportedhereare,ingeneral,consistentwith acterized represents the samespecies or if multiple activator
'I
I
7062
M AActivator P Kinase
in PC12 Cells
species are generated. It is also possible that the activator is to regulation by other protein kinases andoccupies an interprogressively modified as a function of time of exposure to mediate position in the cascade of protein kinase activation NGF, resulting in different susceptibilities to phosphatase ultimately mediating thespecific effect of the growth factors. inactivation. Acknowledgments-We thank Dr. Jack Dixon and Dr. Mark The MAP kinases are members of a newly discovered class of dual specificity proteinkinases (47). Dual specificity Mumby for generously providing us with phosphatases and Dr. Mekinases undergo autophosphorylation at serine/threonine, as lanie Cobb for ERK2 and K52R clones. well as at tyrosine residues, and can phosphorylate other Addendum-While this manuscript was under review, Erikson et substrates at the same residues (48,49). These enzymes share al. (53-55) reported the purification and cloning of MAP kinase/ERk sequence homology with the ~ 3 4 " protein ~ " ~ kinase (18, 47- kinase (MEK1). MAP kinase activator isolated from PC12cells cross50). We haveclearly demonstratedthattheMAPkinase reacts with a monoclonal antibody to MEK1.' activator is a protein kinase thatdirectly phosphorylates the REFERENCES MAP kinases. However, we can not rule out the possibility 1. Halegoua, S., Armstrong, R. C., and Kremer,N. E. (1991) in Nerve Growth that there are other species that also serve to activate the Factor (Bothwell, M., ed), pp. 119-170, Springer-Verlag, 2. Greene, L. A,, and Tischler, A. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 8 3 , MAP kinases. The existence of a n "autokinase-enhancing 2714-2718 .- . - .- factor" was suggested by L'Allemain et al. (51), which causes 3. Dichter, M. A.A., Tischler, A. S., and Greene, L. A. (1977) Nature 2 6 8 , 501-504 the phosphorylation of the wild type ~ 4 2 " ' but ~ ~ not of a 4. Aletta, J., Rukenstein, A., and Green, S. (1987) Methods Enzymol. 1 4 7 , kinase-defective mutant. They also observed a higher level of 207-216 5. Hempstead, B. L., Martin-Zanca, D., Kaplan, D. R., Parada, L. F., and phosphorylation of the wild type ~ 4 2 " "than ~ ~ the kinaseChao, M. V. (1991) Nature 350,678-683 defective K52R mutant, suggesting the possible existence of 6. Kaplan, D.R., Hempstead, B.L., Martin-Zanka, D., Chao, M.V., and Parada, L. F. (1991) Science 252,554-558 an autokinase-enhancing factor. Surprisingly, we have also 7. Boulton, T. G., Nye, S. H., Robhins, D. J., Ip, N. Y., Radziejewska, E., noted the higher levels of phosphorylation in wild type and Morgenbesser, S. D., De Pinho, R. A., Panayotatos, N., Cobb, M. H., and Yancoooulos. G. D. (1991) Cell 65.663-675 ERKB than of the K52R mutant, which could conceivably be 8. Landret6, G. E . , Smith, D.' S., McCabe, C., and Gittinger, C. (1990) J. the result of such a n autokinase-enhancing factor. However, Neurochem. 55,514-523 9. Gomez, N., and Cohen, P. (1991) Nature 3 5 3 , 170-173 there may be a number of other factors causing the lower 10. Ray, L. B., and Sturgill, T. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 8 5 , levels of phosphorylation in K52R mutant. For example, the 3753-3757 11. Schanen-King, C., Nel, A., Williams, L. K., and Landreth, G. E. (1991) phosphorylating sites in bacterially expressed K52R are not Nmirnn . . -. . .. 6. 915-922 - - - --fully available for phosphorylation reaction due toa different 12. Thomas, S.-.M., DeMarco, M., D'Arcangelo, G., Halegoua, S., and Brugge, J. S. (1992) 6 8 , 1031-1040 folding of the protein. Alternatively, association of the acti- 13. Wood, K. W., Cell Sarnecki, C., Roberts, T. M., and Blenis, J. (1992) Cell 6 8 , vator with MAP kinasemay facilitate the phosphorylationof 1041-1050 N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, the enzyme by other protein kinases. The activation of MAP 14. Ahn. E. G. (1991) J. Bid. Chem. 266,4220-4227 kinase through its phosphorylation by other kinases has also 15. Gomez, N., Tonks, N. K., Morrison, C., Harmar, T., and Cohen, P. (1990) FEES Lett. 271,119-122 been demonstrated by Posada and Cooper (18) in T-lympho- 16. Anderson, N. G., Maller, J. L., Tonks, N. K., and Sturgill, T. W. (1990) cytes who found that p56Ick phosphorylated the enzyme on Nature 343,651-653 17. Ettehadieh, E., Sanghera, J. S., Pelech, S. L., Hess-Bienz, D., Watts, J., tyrosine residues. Similarly,Ettehadieh et al. (17)demonSastri, N., and Aebersold, R. (1992) Science 2 5 5 , 853-855 strated that the activation of the Xenopus MAP kinase was 18., Posada, J., and Cooper, J. A. (1992) Sclence 255,212-215 J., Rossomando, A. J., Her, J., DelVecchio, R., Weber, M. J., and accompanied by phosphorylation of the enzyme, at least in 19., Wu, Sturgill, T. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,9508-9512 20., Seger, R., Ahn, N. G., Boulton, T. G., Yancopoulos, G. D., Panayotatos, part, by an exogenouskinase.A number of recent reports N., Radziejewska, E., Ericsson, L., Bratlien, R. L., Cobb, M. H. (1991) have conclusively demonstrated that MAP kinase activator is Proc. Natl. Acad. Sei. U. S. A. 88,6142-6146 a dual specificity protein kinase(25, 51). The results reported 21. Crews, C., Alessandrini, A,, and Erikson, R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,8845-8849 here demonstrate that MAP kinase activator indeed is aMAP 22. Ahn, N. G., Robbins, D. J., Haycock, J. W., Seger, R., Cobb, M. H., and Krebs, E. G. (1992) J. Neurochem. 59,147-156 kinase kinase as indicated by the phosphorylation of the R., Ahn, N. G., Posada, J., Munar, E. S., Jensen, A. M., Cooper, J. kinase-defective mutant K52R (Fig. 6A). Furthermore, phos- 23. Seger, A,, Cobb, M. H., and Krebs, E. G. (1992) J. Biol. Chem. 2 6 7 , 143731A R A l phoamino acid analysis of the K52R is identical with that Na-clYky, S., Cohen, P., Wu, J., and Sturgill, P. (1992) EMBO. J. 1 1 , observed with the wild type MAP kinase andERKB (Fig. 7). 24. 2123-2129 Rossamando, A,, Wu, J., Weber, M. J., and Sturgill, T. W. (1992) Proc. 25. We were unable to detect the phosphorylation of a bacterially Natl. Acad. Sci. U. S. A. 89,5221-5225 expressed truncated MAP kinase, indicating that structural 26. Smith, A., Varon, S., and Shooter, E. (1968) Biochemistry 7 , 3259-3268 Kamps, M. P., and Sefton, B. M. (1989) Anal. Biochem. 176,22-27 27. features of thenative molecule are required forittobe 28. Gaun, K., Haun, R. S., Watson, S. J., Geahlen, R.L., and Dixon, J. E. phosphorylated. This is consistent with the findings of Mat(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1501-1505 Y., Nishida, E., Yamashita,T.,Hoshi, M., Kawakami, M., and suda et al. (52) who reported similar findings. We have also 29. Gotoh, Sakai, H. (1990) Eur. J. Biochem. 193,661-669 screened the activator-containing fractionsfor activities that 30. Ca enter, G (1987) Annu. Reu. Biochem. 56,881-914 Ry%, R. E., and Greene, L. A. (1987) J. Neurosct. 7 , 3639-3653 would phosphorylate a synthetic peptide described by Ette- 31. 32. Sano, M., Nishiyama, K., and Katajima, S. (1990) J. Neurosci. 55. 427hadieh et al. (17) containing the MAP kinase phosphorylation 435 Landreth, G. E., and Williams, K. L. (1987) Neurochem. Res. 12,943-950 site. Wehave failedto detect an activity that would efficiently 33. 34. Miyasaka, T., Miyasaka, J., and Saltiel, A.R. (1990) Biochem. Biophys. Res. Commun. 168,1237-1243 phosphorylate this peptide, nor would the peptide inhibit the 35. Smith, D. S., Schanen-King, C. S., Pearson, E., Gittinger, C.K., and capacity of the activator to stimulate MAP kinase activity Landreth, G. E. (1989) J. Neurochem. 53,800-806 36. Koizumi, S., Contreras, M. L., Matsuda, Y., Hama, T., Lazarovici, P., and (data not shown). Guroff, G. (1988) J. Neurosci. 8, 715-721 In summary, these results shows that NGF rapidly stimu- 37. Lazarovici, P., Levi, B. Z., Lelkes, P. I., Koizumi, S., Fujita, K., Matsuda, Y., Ozato, K., and Guroff, G. (1989) J. Neumsci. Res. 2 3 , l - 8 lates a MAP kinase activator in PC12cells that is capableof 38. Berg, M. M., Sternberg, D. W., Parada, L. F., and Chao, M. V. (1992) J . phosphorylating and activating the MAPkinases. The MAP Biol. Chem. 2 6 7 , 13-16 G., Cohen, S., and Garber, D. L. (1981) J. Biol. Chem. 256,8197kinase activator is itself a protein kinase that is regulatedby 39. Swarup, oom tyrosine and serine/threonine phosphorylation, as treatment 40. Anderson, N. G., Li, P., Marsden, L. A., Williams, N., Roberts, T. M., and Sturgill, T. W. (1991) Biochep. J. 277,573-576 with PTP-1 and PP2A resulted in almost a complete loss in 41. Sturgill, T. W., Ray, L. B., Enkson, E., and Maller, J. L. (1988) Nature its ability to activate the MAP kinase. The mechanismb) by 3 3 4 , 715-718 which MAP kinase activator is activated in response to NGF C. Crews and R. Erikson, personal communication. remains unclear; however, it is probable that it too is subject O*"I
MAP Activator Kinase 42. Pulverer, B. J., Kyriakis, J. M., Avmch, J., Nikolakaki, E., and Woodgett, J. R. (1991)Nature 353,670-674 43. Kremer, N.E.,D’Arcangelo, G., Thomas, S. M., DeMarco, M., Brugge, J. S., and Halegoua, S. (1991)J. CeU BWl. 115,809-819 44. Thomas, G. (1992)Cell 68,3-6 45. Kosako, H.,Gotoh, Y., Matsuda, S., Ishikawa, M., and Nishida, E. (1992) EMBO J. 11,2903-2908 46. Mi asaka, T., Chao, M., Sherline, P., and Saltiel, A. R. (1990)J. BWl. &em. 265,4730-4735 47. Lindberg, R. A., Quinn, A, M., and Hunter, T. (1992)Trends Biochem. Sei. 17,114-119 48. Ben-David, Y.,Letwin, K., Tannock, L., Bernstein, A., and Pawson, T. (1991)478-480 EMBO J. 10,317-325
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49. Howell, B. W., Afar, D. E. H.,Lew, J., Douville, E. M. J. Icely, P.L. E., Gray, D.A., and Bell, J. C.(1991) MOL Cell. EWE. 12,568-572 50. Johnson, K. W., and Smith, K.A. (1991)J. Bid. Chem. 266, 3402-3407 51. L’Allemain, G.,Her, J., Wu, J., Sturgill, T. W., and Weber, M. J. (1992) Mol. Cell. Biol. 12.2222-2229 52. Matsuda, S., Kosako, H., Takenaka, K., Mariyama, K., Sakai, H.,Akiyama, T., Gotoh, Y.,and Nishida, E. (1992)EMBO J. 11,973-982 53. Alessandrini, A., Crews, C. M.,andErickson, R. L. (1992)Prm,Nuti. Acad, Sci. U.S. A. 89,8200-8204 54. Crews, C. M., and Erickson, R. L. (1992)Pmc.Nati. Acad. Sci. U. S. A. 89, &205~209 55. Crews, C.M., Alessandrini, A., and Erickson, R. L. (1992)Science 268,
