Isolation and characterization of microtubule-associated protein 2 (MAP2) kinase from rat brain

Molecular Bram Research, 14 (1992) 43-50 © 1992 Elsevier Science Pubhshers B.V. All rights reserved. 0169-328X/92l$05 00

43

BRESM 70420

Isolation and characterization of microtubule-associated protein 2 (MAP2) kinase from rat brain N. Carolyn Schanen* and Gary Landreth* Departments of Neurology and Neurosctences, AIzhetmer Research Laboratory, Case Western Reserve Untverstty Medteal School, Cleveland, OH 44106 (USA)

(Accepted 24 December 1991) Key words Microtubule-associated protein 2 klnase, Protein phosphorylatlon

Mxcrotubule-assoclated protein 2 (MAP2) kinase has been isolated and characterized from rat brain. The enzyme has an apparent Mr of -42,000 and its pI is 4.9. MAP2 was the preferred substrate, but it also phosphorylated myelin basic protein (MBP), histone V-S, tubuhn and the PC12 protein substrate pp250. The enzyme is &stinct from protein kmase C, cAMP-dependent kinase and the calclum/calmoduhndependent kmases, as specific mhlbltors of these kmases did not affect MAP2 phosphorylation The addmon of the relatwely non-speofic protein kmase inhibitor H7 (20/~M) had a modest inhibitory effect The enzyme was active m both 5 mM Mn 2+ and Mg2+, and displayed Kms for MAP2, MBP, and ATP of 56 nM, 254 nM, and 4 ~M, respectively This enzyme, which represents a low abundance protein m whole brain, is analogous to the MAP2 kmase observed m growth factor-stimulated cell lines

INTRODUCTION Microtubule-associated protein 2 ( M A P 2 ) kinase is an important serine/threonine kinase involved in signal transduction in a variety of cell types. The enzyme is rapidly activated in response to a wide range of extracellular stimuli 1"15"17"2°'27"31'35"36. M A P 2 kinase and its homologues have also been t e r m e d extracellular response kinases ( E R K ) s. The physiological function of M A P 2 kinase is presently unknown; however, its identified substrates include cytoskeletally associated proteins 26'43. Protein phosphorylation plays a critical role in the regulation of neuronal function, thus, it is not surprising that brain represents a particularly rich source of protein kinases lB. M A P 2 Kinase has recently been identified in rat brain where it is rapidly but transiently activated following electroconvulsive t r e a t m e n t 42, and activation of N M D A receptors 54. Activation occurs within 2 min of depolarization and appears to involve phosphorylation on tyrosine residues 5. In the PC12 p h e o c h r o m o c y t o m a cell line, M A P 2 kinase ts activated by exposure of the cells to nerve growth factor ( N G F ) , e p i d e r m a l growth factor ( E G F ) , and 12O - t e t r a d e c a n o y l phorbol-13-acetate ( T P A ) , but not by calcium or dibutyryl c A M P 26"2s. M A P 2 kinase is activated by phosphorylation on serme, threonine and ty-

rosine residues. The tyrosine phosphorylation of this enzyme suggests that it may be a substrate for the r e c e p t o r tyroslne kinases 4'37"32 , although o t h e r mechanisms have been p r o p o s e d including autophosphorylation 2'55. Further, M A P 2 kinase will phosphorylate and activate $6 kinase II, indicating it is likely to play a fundamental role in the cascade of kinase activations involved in intraceUular signal transduction 44 Boulton et al. have purified M A P 2 kmase from insulin-treated Rat1 cells and obtained hmited sequence information allowing the synthesis of oligonucleotide probes 7's. Screening of a rat brain c D N A library yielded a partial M A P 2 kinase clone (termed E R K 1 ) as well as two additional clones encoding related protein kinases E R K 2 and ERK39. The E R K 1 clone represents the M A P 2 kinase gene 34 . E R K 1 and its homologues are expressed at high levels in the brain relative to other tissues 9. The presence of M A P 2 kinase in brain suggests a possible role for this enzyme in the immediate response of neurons to extracellular stimuli. MATERIALS AND METHODS Matertals

NGF was prepared by the method of Smith et al. 41. Radlolabeled ATP was synthesized using 32p1from ICN (Irvine, CA, USA) and Gamma Prep A (Promega, Madison, WI, USA) Chromato-

* Present address. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA Correspondence G Landreth, Alzhelmer Research Laboratory, E504, Case Western Reserve Umvers,ty Medical School, 2119 Abington Road, Cleveland, OH 44106, USA Fax (1) (216) 368-3079.

44 focussing reagents were from Pharmacia (Plscataway, NJ, USA) The Fractogel TSK 55F resin and the Butyl TSK 650C were from Merck, Inc (Rahway, NJ, USA) IP20 was obtained from Peninsula Labs (San Mateo, CA, USA) and H7 was supplied by Seikagaku (RockviIle, MD, USA) Purified Walsh Inhibitor was a gift of Drs A Nairn and R Nichols (Rockefeller University) MBP was a girt from Dr M Wlese All other reagents were obtained from S~gma (St Louis, MO. USA)

PmtBcatton o] MAP2 l~ma~e Frozen brains from young (< 1 month old) Sprague-Dawley rats (45 g) were homogemzed in Lysls buffer [(20 mM TnsCl, pH 7 4. 1 mM EGTA, 100 tiM sodium orthovanadate, 20 mM p-mtrophenyl phosphate (PNP). 1 mM phenylmethylsulfonyl fluoride (PMSF)] The homogenate was clarified by centrifugatlon at 60,000 × g for 30 mm at 4°C Phosphatase lnhlbltors (20 mM PNP, and 100 tiM sodium ortho~anadate) were included in the buffers used for chromatography and all procedures were performed at 4°C The soluble traction was bound to a DEAE-52 ion exchange column (2 5 × l0 cm) in lysls buffer The column was washed with lysis buffer containing 100 mM NaCI, and the enzyme was eluted using a 100500 mM NaC1 gradient in lysls buffer at a flow rate of 60 ml/h, 2 ml tractions were collected Once the elutlon profile for MAP2 kinase was determined, batch elunon was performed by washing the resin In 100 mM NaCI and subsequent collection of the enzyme upon elution in 250 mM NaCI MAP2 klnase obtained following ion exchange chromatography was brought to 0 75 M in NaC1 and loaded onto a butyl TSK-650C (1 ~ 1 5 cm) column at a flow rate of 30 ml/h, 1 ml fractions were collected The column was washed sequentmlly with lysis buffer containing 0 5 M NaCI (without PNP), then 0 2 M NaC1 (without PNP) The enzyme was eluted in salt-free lysls buffer containing 10% ethylene glycol Peak fractions from the butyl TSK elutlon were pooled, brought to 20 mM in lmldazole, pH 7 4, and loaded onto a PBE94 chromatofocussmg column (1 × 5 5 cm) The column was developed with Polybuffer 74 at pH 4 0 at a flow rate of 60 ml/h, 1 ml fracuons were collected The pH of the column fractions was rapidly adjusted to 7 2-7 4 using 1 0 M Tns HC1, pH 8 0 Peak fractions were then used tor characterization of the enzyme Gel filtration of MAP2 kmase was performed application ot the peak butyl fraction (0 45 ml) to a Fractogel TSK HW 55F gel illtrauon column (1 × 45 cm) which had been previously calibrated with Blue Dextran (Vo), bovine serum albumin (BSA, M~ = 68 kDa), and soybean trypsin inhibitor (STI, M~ = 20 1 kDa) The column was developed at 30 ml/h with 20 mM Trls (pH 7 4, 4°C), 1 mM EGTA, 50/.tM sodium orthovanadate and 0 5 ml fractions were collected Ahquots (50 ~tl) of the indicated fractions were assayed for MAP2 kmase activity

rated radioactivity determined by Cerenkov counting The MAP2 klnase preparations used for characterization of the enzyme were purified by sequential chromatography on DEAE-52, Butyl TSK and PBE94 chromatofocussmg columns

Pmparauon o f M A P substtate Microtubules were prepared exactly as described by Vallee 5" MAPs were eluted from the microtubules in 350 mM NaC1 and the soluble MAPs heated to 100°C for 5 mm, then centrifuged at 10.000 × g for 10 mm 23 The concentration of MAP2 m the preparation was determined by densltomemc scanning ot Coomasslestained gels MAP2 represented approximately 5~ of the total protern m the MAP substrate preparation Determination o)protein ~oncetlttatlott Protein concentration was determined by the method ot Bradford using BSA as a standard 1" RESULTS

Isolation of MAP2

kmase

T h e c h r o m a t o g r a p h i c s t e p s u s e d to isolate M A P 2 kinase from bram were based on previously described prot o c o l s d e v e l o p e d t o p u r i f y t h e e n z y m e f r o m P C t 2 cells 27. B r a i n c o n t a i n s a large n u m b e r o f p r o t e i n k l n a s e s , several o f w h i c h will p h o s p h o r y l a t e MAP251; t h u s , it was expected that M A P 2 kinase would represent a relatively m i n o r c o m p o n e n t o f t h e total k i n a s e activity. T h e r e f o r e , the purification o f M A P 2 k m a s e f r o m b r a i n w a s s u b j e c t to a n u m b e r o f difficulties. First, t h e very h i g h levels o f p r o t e i n k l n a s e activity significantly c o m p l i c a t e d i s o l a t i o n o f a q u a n t i t a t i v e l y m i n o r activity such as M A P 2 k i n a s e . M o r e o v e r , in the b r a i n , only t h e b a s a l activity o f t h e enz y m e was m e a s u r e d unlike clonal cell lines w h e r e a h o r m o n a l l y - s t l m u l a t e d activity c o u l d b e f o l l o w e d

Finally,

the cell t y p e s a n d b r a i n r e g i o n s e x p r e s s i n g M A P 2 k m a s e was u n k n o w n . L i m i t e d e x p r e s s i o n in specific cell t y p e s w o u l d result in d i l u t i o n o f t h e activity in w h o l e b r a i n . Previous data indicated that M A P 2

klnase from a

n u m b e r o f cell t y p e s c o n s i s t e n t l y e l u t e s f r o m D E A E - 5 2 ion e x c h a n g e resin at 250 m M NaC127 36. T h u s , it was c h o s e n as the initial resin e m p l o y e d for i s o l a t i o n o f t h e

~,e~tern blot analysts Column fractions obtained from a butyl TSK column were pooled m groups of three and the proteins precipitated with 5% trichloroacetic acid using 10 l~g of bovine serum albumin as a career The proteins were separated on 10% SDS-PAGE and transferred to an Immobllon membrane The blot was incubated with an anti-MAP kmase antibody (Zymed Inc , San Francisco, CA) followed by detection using the enhanced chemiluminescence method exactly as described by the manufacturer (Amersham) The antibody detects both MAP2 kmase (ERK1) and ERK2

e n z y m e . P a s s a g e o f clarified b r a i n h o m o g e n a t e o v e r t h e

Phosphol ylatton a~says Ahquots of column fractions were incubated in a 61) Ftl reaction mixture with 10 itg MAP substrate or 5 l~g MBP in 20 mM Tns pH 7 4, 1 mM EGTA, 2 mM MnC12, and 10 ,uM 32p-ATP (100-200 cpm/fmol) for 40 rain at room temperature The reaction was stopped by the addition of Laemmh electrophoresls sample buffer, then heated to 100°C for 5 min Samples were separated by SDSPAGE on 3-12% or 12% gels as described by Laemmliz5 and autoradlograms obtained Substrate bands were excised and mcorpo-

It was n o t p o s s i b l e to d e t e r m i n e t h e d e g r e e o f purifica-

DEAE-52

r e s i n m the a b s e n c e o f salt y i e l d e d a large

a m o u n t o f c o n t a m i n a t i n g e n z y m e activity w h i c h utilized t h e M A P 2 s u b s t r a t e . F u r t h e r w a s h i n g o f t h e c o l u m n in 100 m M N a C l r e m o v e d t h e m a j o r i t y o f t h e c o n t a m i n a t ing p r o t e i n . M A P 2 k l n a s e was e l u t e d u s m g a l i n e a r NaC1 gradient (0.1-0 5 M)

A p e a k o f k i n a s e activity e l u t e d

at 250 m M N a C l w h i c h i n c l u d e d M A P 2 k i n a s e (Fig. I A ) tion f r o m t h e i o n - e x c h a n g e c h r o m a t o g r a p h y s t e p , as t h e e l u a n t c o n t a i n e d o t h e r c o n t a m i n a t i n g k i n a s e a c u v i t l e s as evidenced m subsequent chromatography. The second chromatographic step utthzed for isolation ol M A P 2 k i n a s e was t h e h y d r o p h o b i c i n t e r a c t i o n r e s i n , butyl T S K - 6 5 0 - C . T h e p r e s e n c e o f P N P significantly di-

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Fig. 1 Chromatographic isolation of MAP2 kinase from rat brain A. 1on exchange chromatography Clarified extracts were prepared from rat brain and apphed to a DEAE-52 column The column was developed with a NaCl gradient (100-500 mM). Ten itl ahquots of column fractions were assayed for MAP2 kinase activity as described in Materials and Methods ( ~ ) Protein concentration is indicated (4~) B' hydrophobic interaction chromatography. The pooled 250 mM NaC1 eluant obtained from DEAE-52 chromatography was brought to 0 75 M with NaCl and apphed to a butyl Toyopearl TSK-650C column The enzyme was eluted by washing the column with 15 ml of lysls buffer contaimng 10% ethylene glycol. Ahquots of indicated fractions were assayed for MAP2 kmase activity (~) The protein concentration of the fractions is in&cated (0). Inset magmficatlon of area indicated by the arrow demonstrating a symmetric peak of MAP2 kmase activity C. chromatofocussing chromatography Peak fractions obtained following butyl TSK chromatography were pooled, brought to 20 mM m imidazole (pH 7.4), and apphed to a PBE94 chromatofocussing column equlhbrated to pH 7.4 (4°C). The column was developed using Polybuffer 74 (pH 4 0, 4°C) Aliquots of column fractions were assayed for MAP2 kinase activity (~) Fraction pH is indicated (~I~) D gel filtration chromatography The peak fraction (0 45 ml) of the butyl TSK elutlon was made 10% m Glycerol then loaded onto a Fractogel 55F gel filtration column which had been previously calibrated with Blue Dextran (Vo), bovine serum albumin (BSA: M~ = 68 kDa), and soybean trypsin inhibitor (STI; Mr = 20 1 kDa) orthovanadate and 0.5 ml fractions were collected.

minished binding of M A P 2 kinase to this resin, thus, PNP was omitted from the buffers prior to elution of the enzyme. W h e n the pooled D E A E - 5 2 eluant was passed over butyl resin, a large a m o u n t of enzyme activity was present in the u n b o u n d fraction coincident with a large peak in protein. Subsequent washes in 0.2 M NaC1 removed greater than 90% of the contaminating protein. Elution of a small, sharp peak of M A P 2 kinase activity was accomplished by washing the resin in Lysls buffer containing 20 m M PNP and 10% Ethylene Glycol (Fig. 1B) in a position coincident with the elution of MAP2 kinase from PC12 cells 27.

D e t e r m i n a t i o n of the isoelectrlc point for MAP2 kinase was achieved by application of peak butyl TSK fractions to the chromatofocusslng resin (PBE 94) at pH 7.4. The column was developed in Polybuffer, pH 4, and MAP2 kinase eluted at pH 4 9 (Fig. 1C). The enzyme was unstable near its pI, thus it was important to rapidly adjust the fraction pH to 7.2-7.6. Peak fractions of enzyme obtained following this chromatographic step were used for further characterization of MAP2 klnase. To determine the apparent size of the MAP2 kinase enzyme, an aliquot of a peak butyl TSK fraction was subjected to gel filtration which revealed an apparent M r

46

68 45

29

Km for ATP was determined to be 4 ktM (Fig 4C). These K m values are similar to those reported for the PCI2 enzyme 27'3~. However, the K m values for MAP2 reported by Ray and Sturgfl136 for the insulin-stimulated enzyme from adipocytes were 4-12 /~M These differences are thought to be due to varmtlon m the determination of MAP2 concentration within the substrate preparation 27

Acm'ators and mhtbttor~ A variety of protein kmase inhlbltors have been char-

41 44 47 50 53 56 59 62 Fig 2 MAP kmase lmmunostamlng. Column fractions obtained from TSK butyl chromatography were pooled m groups of three and the proteins TCA preopltated The proteins were transferred to Immobllon membranes and lmmunostalned with a anU-MAP klnase antibody The fraction numbers correspond to those of Fig IB The band present in fraction 50 has an apparent M~ = 42 kDa and the minor band below it with slightly lower M,. ot approximately 40-41 kDa

= 42,000 (Fig 1D), an apparent M~ identical to that found for this enzyme in a n u m b e r of cell lines t'27 37 MAP2 klnase is a very low abundance protein in whole brain and was not detectable by silver staining of concentrated chromatographic fractions (data not shown)

hnmunostatnmg The identity of the enzymatic actlwty as MAP2 kinase was verified through use of a recently available antibody to the M A P kinases. Fractions containing MAP2 kinase actlvlty possess a 42 kDa protein detected with the M A P kinase antibody. The peak fractions contained MAP2 kmase (ERK1) with only a minor amount of E R K 2 present (Fig. 2) MAP2 kinase is distmguished from ERK2 by the difference in molecular weight, E R K 2 migrated w~th a slightly greater mobility (M~ = 41.000)

Substrate spectficttv MAP2 kinase prepared by sequential ~on exchange, hydrophobic interaction, and chromatofocussing chromatography was incubated with a variety of substrates including the endogenous substrate for the PC12 enzyme, pp25026 28. as well as MAP2, taxol-polymenzed tubulin, casein, MBP. and histones II-S, V-S, and VIII-S MAP2 kinase was found to efficiently phosphorylate pp250, MAP2, MBP. tubulin and histone V-S, while histones II-S and VIII-S were not phosphorylated by MAP2 klnase. Phosphorylatlon of casein was inconsis-

acterxzed which show varying degrees of speoficity for particular enzymes The brain MAP2 kinase was distinct from A-klnase as the addition of the Walsh inhibitor, or its specific peptlde mhlbltor, IP20 It, did not affect the phosphorylatlon of MAP2. Trifluoroperizine (TFP) which inhibits the calclum-calmoduhn (CAM) kinases 24 also had no effect on MAP2 phosphorylation. Further, the assays were performed m 1 m M E G T A which effectively inhibits the activity of calcium-dependent kinases, including protein klnase C (PKC). Addition of the inhibitor. H7 ( 2 0 / , M ) , caused a diminution m activity of approximately 35%, which is consistent with the behavior of MAP2 klnase from PC12 and T-cells 27 29 H7 is a relatively non-specific mhibitor, inhibiting C A M kinases. PKC (K, = 6/~M) and A-klnase (K, = 3 /~M)lg: thus, the concentration ol H7 used was sufficient to com-

-250

_56 -54

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tently observed (Fig. 3).

Enzyme kmetlc~ Kinetic analysts of the enzyme revealed a high affinity for its protein substrates with a Km for MAP2 of 56 nM (Fig. 4A) and K~ for MBP of 254 nM (Fig. 4B): The

F~g 3 Substrate specificity of MAP2 klnase. MAP2 kmase was mcubated with l0 pg of the indicated substrates except 5 ~g for MBP m otherwise standard assay condmons Protems were separated on 3-12~ or 12~ polyacrylamlde gels Tub, tubuhn, Cas, casein, MBP, myelin basic protem, HII-S, HV-S and HVIII-S. hlstone~ II-S V-S and VIII-S, respectively

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The MAP2 kinase isolated in these preparations demonstrated a unique cation preference. When MBP was used as the substrate, the enzyme was maximally active at 5 mM Mn 2+, with a subsequent fall in activity at higher Mn 2+ concentrations. It required 10 mM Mg 2+ to achieve the activity seen with 5 mM Mn 2+. However, when MAP2 was used as a substrate, the dependence on Mn 2+ was less apparent. The enzyme was maximally activated in 5 mM Mn 2+, but its activity in Mg 2+ roughly paralleled the activity in Mn 2+ (Fig. 6).

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Fig. 5 Activators and lnhlbitors of rat brain M A P 2 kinase M A P 2 klnase was incubated with M A P 2 (10 /~g) m standard reaction buffer in the presence or absence of c A M P (10/~M), T P A (50 ng/ ml), T F P (50 ~M), H-7 (20/~M), Walsh inhibitor (83/tg/ktl), or IP20 (20 nM) Data shown are m e a n s of duplicate assays (_+ range).

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Fig 4 Kinetic properties of M A P 2 kinase. M A P 2 klnase was combined with M A P 2 (6 25-100 nM, A) or M B P (0.1-10 p M , B) in otherwise standard assay conditions and phosphorylatlon carried out for 40 rain at room temperature Alternatively, using M A P 2 as a substrate, the concentration of A T P in the reaction mixture was varied ( 1 - 1 0 0 0 / t M ) in otherwise standard reaction conditions (C). Data points represent values of trlphcate assays Similar results were seen m two independent experiments.

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Fig 6. Cation dependence of rat brain M A P 2 / M B P klnase activity M A P 2 kinase activity was combined with either M A P 2 (10 pg, triangles) or M B P (5 pg, & a m o n d s ) substrate were added together with the indicated concentration of Mn 2+ (closed symbols) or Mg 2+ (open symbols) in otherwise standard reaction conditions The radioactivity incorporated into M A P 2 and M B P was measured by excision of the substrate band and Cerenkov counting

48 DISCUSSION The data presented here demonstrate the presence of a M A P 2 kinase in rat brain which appears to be homologous to the growth factor-stimulated M A P 2 kinase ldennfied in variety of cell lines ~'27~29'31'35 and verifies that the activity can be isolated from tissue as well. The enzyme from brain behaves similarly on a n u m b e r of chromatographic resins and has an identical molecular weight and isoelectric point relative to the enzyme prepared from PC12 cells Immunostaining of the brain enzyme confirmed that the activity was M A P 2 klnase ( E R K 1 ) and distinct from E R K 2 (Fig. 2) F u r t h e r m o r e , It demonstrates similar specificity and affinity for M A P 2 and ATP relative to the PC12 enzyme 27'3~ The brain M A P 2 klnase differs somewhat in its cation preference, being less sharply d e p e n d e n t on Mn 2+ than the PC12 enzyme, but was clearly active in low manganese concentrations The M A P 2 klnase activity is distinct from PKC, A-klnase and the C A M klnases, as specific inhlbitors of these enzymes did not inhibit M A P 2 phosphorylatlon. It was m o d e r a t e l y inhibited by the relatively non-specific protein klnase inhibitor, H7. consistent with the behavior of the enzyme isolated from PC12 and T_cells27 2~. The activity was also distract from casein kinase II, which will not phosphorylate M A P 2 ~3. The principal difficulty attending the isolation of this enzyme from brain is that only the basal, unstimulated enzyme activity could be measured. In different biological systems, a diverse group of extracellular stimuli activate M A P 2 kinase. Mitogenic stimulanon of actively dividing 3T3 fibroblasts results in M A P 2 klnase acnvation ~, yet in post-mitotic adipocytes, the enzyme is activated by insulin which p r o m o t e s glucose uptake by these cells 35. Secretogogue stimulation of cultured chromaffin cells induces M A P 2 kinase activanon coincident with catecholamine release ~5 In rat brain, M A P 2 klnase IS regionally activated following electroconvulsive treatment and activation of N M D A receptors42 54 Thus, M A P 2 kinase activation is implicated m cell growth and division, as well as the specialized activlnes of differentiated cells. M o r e o v e r , both mitogenic and neurotrophic stimulation of PC12 cells (i.e. with E G F and NGF, respectively), activate M A P 2 kinase through different cell-surface receptors and apparently different pathways 26 2v,31. The ability of this enzyme to respond to such diverse stimuli is evidence that it plays a fundamental biologic role in intracellular signal transduction Activation of M A P 2 kinase consistently involves tyroslne phosphorylation of the enzyme, although phosphorylatlon of other residues is required for expression of full enzymatic activity. The identity of the other res-

ldues p h o s p h o r y l a t e d vanes between activating hgands, msuhn stimulates phosphorylatlon of tyrosine and threonlne ~. while N G F stimulates phosphorylatlon of tyroslne, threonine and serlne ~ 32 M o r e o v e r , its phosphorylatlon on multiple residues implies that it represents a point of convergence of signalling pathways The physiologically relevant substrates for M A P 2 klnase are not clear The only definitive in vivo substrate is the PC12 protein, pp250, which may be a M A P by virtue of its size and cytoskeletal association 26 25 In vitro, M A P 2 klnase phosphorylates M A P 2 , M B P and $6 klnase II The latter enzyme is activated by its phosphorylatlon It has been suggested that $6 klnase II also represents an in VlVO substrate, placing M A P 2 kinase within a cascade of kinase activations which stimulates an increase in protein synthesis in response to extracellular stimuli 3 44 W h e t h e r M A P 2 is an in VlVO substrate for M A P 2 klnase has not been d e t e r m i n e d M A P 2 is not expressed in undifferentiated PC12 cells 6 and as M A P 2 kinase is only transiently acnvated in response to NGF, it is unlikely that it represents an in vivo substrate for the enzyme in this model system H o w e v e r , phosphorylatlon of the endogenous PCI2 substrate, pp250, occurs coincident with a loss of mlcrovllh, and ruffling of membrane5~2 26 If pp250 were indeed a MAP, it would be reasonable to suggest that these changes in the cell surface could reflect changes in stability of the underlying cytoskeleton due to phosphorylatlon. In fact, M A P 2 kinase has been d e m o n s t r a t e d to slow microtubule polymerization in Xenopu.s interphase extracts, presumably by phosphorylatlng M A P 2 and decreasing its ability to direct microtubule assembly ~6 M A P 2 may be an in vivo substrate for the enzyme in brain where M A P 2 is a prominent protein involved in d e v e l o p m e n t and maintenance of the cytoskeleton 3°. The function of M A P 2 is controlled by phosphorylatlon 2I 20 4~ While M A P 2 represents a good substrate for PKC, A-kinase, C A M kinase II, and tyrosine kinases in vitro, it has been d~fficult to discern which are the relevant species In vivo 22 ~3 45 4. 52. M A P 2 kinase exhibits an extremely high affinity for M A P 2 ( K m =- 56 nM) which is greater than that r e p o r t e d for the other classes of proteln kinases implicated in the regulation of M A P 2 function (Km range 0.2-5 f~M) 4~ 46,q2 53 Considering the relative abundance of the M A P 2 substrate in brain, this high affinity lends credence to the hypothesis that M A P 2 is a substrate for the enzyme in brain A n o t h e r serlne/ threonine kinase which phosphorylates high molecular weight M A P s , t e r m e d H M K , has been identified in PC12 c e l l s 4'47 H M K phosphorylates M A P 1 2 in vivo and M A P 2 m vitro and appears to be similar to the PC12 M A P 2 klnase in many respects It demonstrates similar 5ubstrate specificity and cation preference, however,

49 H M K is p r e f e r e n t i a l l y a c t i v a t e d in P C 1 2 cells by N G F

cdc2 kinase and two yeast s e r i n e / t h r e o n i n e kinases which

and F G F , b u t n o t E G E

mediate

M o r e o v e r , it has a p r o l o n g e d

antagonistic

cell-cycle

responses

to

phero-

a c t i v a t i o n t i m e c o u r s e r e l a t i v e to M A P 2 kinase 4v. A l -

m o n e s ~. T h e e n z y m e with which it shares m o r e h o m o l -

t h o u g h t h e s e q u e n c e of H M K has n o t b e e n d e t e r m i n e d ,

ogy (52%), F U S 3 , is crucial for the p h e r o m o n e - i n d u c e d

it is possible that it r e p r e s e n t s o n e m e m b e r of a family

w i t h d r a w a l f r o m the cell-cycle p r i o r to c o n j u g a t i o n . Fur-

of r e l a t e d kinases.

t h e r , F U S 3 has b e e n i m p l t c a t e d as an e n z y m e i n v o l v e d

T h e r e c e n t c l o n i n g of M A P 2 kinase (also t e r m e d extracellular r e s p o n s e kinase-1, E R K 1 ) a l l o w e d the d e t e c tion of a n u m b e r of r e l a t e d m R N A

transcripts, all of

in the initial signalhng r e s p o n s e to e x t r a - c e l l u l a r stimuli in yeast 14. M A P 2 k m a s e r e p r e s e n t s a relatively low a b u n d a n c e

which are highly e x p r e s s e d in b r a i n 7"9. T h e M A P 2 kinase

p r o t e i n m w h o l e brain, h o w e v e r , it m a y be only ex-

c h a r a c t e r i z e d h e r e c o r r e s p o n d s to E R K 1 species b a s e d

p r e s s e d m specific cell-types as it is o n l y regionally acti-

o n its c h r o m a t o g r a p h i c b e h a v i o r and i m m u n o l o g i c crossreactivity 9. T h e E R K 1 g e n e p r o d u c t is u b i q u i t o u s l y ex-

v a t e d in r e s p o n s e to e l e c t r o c o n v u l s i v e t r e a t m e n t and N M D A r e c e p t o r agonists 42"54. A l t h o u g h it is not k n o w n

p r e s s e d t h r o u g h o u t the b r a i n and by b o t h n e u r o n s and

w h e t h e r M A P 2 kinase is a c t i v a t e d in brain by n e u r o -

astocytes. T h e E R K 2 and E R K 3 g e n e p r o d u c t s h a v e not

trophic factors, N G F

yet b e e n e n z y m a t i c a l l y c h a r a c t e r i z e d and their p r o t e i n

cells 2v'3L. T h u s , it is possible that M A P 2 kinase p r o v i d e s

stimulates the e n z y m e in PC12

substrates are u n k n o w n . T h e s e e n z y m e s h a v e a distinc-

a m e a n s of c o u p l i n g a variety of extra-cellular stimuli to

tive r e g i o n a l and t e m p o r a l p a t t e r n of e x p r e s s i o n in the

m e t a b o l i c r e g u l a t i o n and M A P 2 function.

brain. M A P 2 kinase a p p e a r s to b e well c o n s e r v e d e v o l u t i o n arily with h o m o l o g s in e c h i n o d e r m 38, a m p h i b i a n 16, and m a m m a l i a n cells 27'29'35. M A P 2 kinase ( E R K 1 ) and related family m e m b e r s b e a r s e q u e n c e similarity to the

REFERENCES 1 Ahn, N G , WeIlel, J.E., Chan, C.P. and Krebs, E G , Idennficatlon of multiple epidermal growth factor-stimulated protein serme/threonme klnases from Swiss 3T3 cells, J Bzol C h e m , 265 (1990) 11487-11494. 2 Ahn, N., Seger, R., Brathen, R , Dlltz, C., Tonks, N. and Krebs, E , Multiple components in an epidermal growth factorstimulated protein kmase cascade, J Bzol. Chem , 266 (1991) 4220-4227 3 Ahn, N G. and Krebs, E . G , Evidence for an epidermal growth factor-stimulated protein kmase cascade m Swiss 3T3 cells, J Btol Chem , 265 (1990) 11495-11501. 4 Aletta, J . M , Lewis, S.A., Cowan, N.J and Greene, L A , Nerve growth factor regulates both the phosphorylatlon and steady-state levels of m~crotubule-associated protein 1 2 (MAP1 2), J Cell Biol , 106 (1988) 1573-1581 5 Anderson, N,G., Mailer, J L., Tonks N K and Sturglll, T.W., Requirements for integration of signals from two &stmct phosphorylation pathways for activation of MAP klnase, Nature, 343 (1990) 651-653. 6 Black, M.M., Aletta, J.M. and Greene, L.A., Regulation of microtubule composition and stability during nerve growth factor-promoted neurite outgrowth, J Cell B l o l , 103 (1986) 549557. 7 Boulton, T G , Yancopolous, G D., Gregory, J.S., Slaughter, C., Moomaw, C , Hsu, J and Cobb, M , An msuhn-stlmulated protein kinase similar to yeast kmases revolved m cell cycle control, Sctenee, 249 (1990) 64-67 8 Boulton, T , Gregory, J and Cobb, M., Purification and properties of extracellular s~gnal-regulated kmase 1, an msuhn-st~mulated mlcrotubule-assooated protein 2 kinase, Btochemlstry, 30 (1991) 278-286 9 Boulton, T , Nye, S , Robbms, D , Ip, N , Radziejewska, E , Morgenbesser, S., DePmho, R , Panayotatos, N , Cobb, M and Yancopoulos, G., ERKs. a famdy of protein serine/threorune kmases that are acnvated and tyrosme phosphorylated m response to Insuhn and NGF, Cell, 65 (1991) 663-675

Acknowledgements We would hke to thank Cindy Glttlnger for her assistance m preparation of figures for this manuscript This work was supported by a grant from the National Science Foundation (BNS-96302)

10 Bradford, M., A rapid and sensitive method for the quantltanon of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Btochem , 72 (1976) 248-254 11 Cheng, H.C., Kemp, B., Pearson, R., Smith, A , MIsconl, L , Van Patten, S and Walsh, D., A potent synthetic pept~de inhJbltor of the cAMP-dependent protein klnases, J Btol Chem , 261 (1986) 989-992 12 Connolly, J . L , Greene, L . A , Vlscarello, R R and Riley, W D , Rap~d, sequennal changes in surface morphology of the PC12 pheochromocytoma cells m response to nerve growth factor, J. Cell B t o l , 82 (1979) 820-827 13 Dlaz-Nldo, J , Serrano, L., Mendez, E. and Avda, J , A casem kinase II-related activity is revolved m phosphorylatlon of mlcrotubule-assoclated protein MAPIB during neuroblastoma cell differentiation, J Cell B t o l , 106 (1988) 2057-2065 14 Elaon, E , A , Gnsafi, P L and Fmk, G R , FUS3 encodes a cdc2+/CDC28-related klnase required for the transmon from mitosis into conjugation, Cell, 60 (1990) 649-664. 15 Ely, C.M , Oddle, K.M., Lltz, J S , Rossomando, A.J., Kannet, S B., Sturglll, T W and Parsons, S , A 42-kD tyroslne kinase substrate hnked to chromaffin cell secretion exhibits an assooated MAP kinase actlwty and ~s highly related to a 42-kD mltogen-stImulated protein m fibroblasts, J Cell B t o l , 110 (1990) 731-742. 16 Gotoh, Y., NIshlda, E , Matsuda, S , Shllna, N., Kosako, H , Shlokawa, K , Aklyama, T , Ohta, K and SakaL H , In vitro effects on mlcrotubule dynamics of purified Xenopus M phaseactivated MAP kmase, Nature, 349 (1991) 251-254 17 Hanekom, C., Nel, A , Glttinger, C , Rheeder, A and Landreth, G , Complexmg of the CD-3 subumt by a monoclonal antibody activates a m~crotubule-associated protein 2 (MAP2) serane kmase m Jurkat cells, Btoehem J , 262 (1989) 449-456. 18 Hemmlngs, H C , Nalrn, A C , McGumness, T L , Hugamr, R L and Greengard, P, Role of protein phosphorylanon in neuronal signal transductaon, FASEB J , 3 (1989) 1583-1592. 19 Hldaka, H , Inagakl, M , Kaeamoto, S and SasakL Y , Isoqmnollnesulfonam~des, novel and potent mhlbltors of cyclic nucle-

50

2i)

21

22

23

24

25

26

27

28

29

31)

31

32

33

34

35

36

37

otlde dependent protein klnase and protein kinase C, Btochetntstry, 24 (1984) 5(136-5044 Hoshl, M , Nlshlda, E and Sakal, H , Activation of a Ca z+lnhlbltable protein klnase that phosphorylates mlcrotubule associated protein 2 m vitro by growth factors, phorbol esters, and serum m qmescent cultured human fibroblasts, J Btol Chem , 263 (1988) 5396-5401 Jameson, L , Frey, T , Zeeberg, B • Dalldorf• F and Caplow, M , Inhibition of mlcrotubule assembly by phosphorylation ot mlcrotubule assocmted proteins, Biochemistry, 19 (1980) 24722479 Kadowak~, T , Fujlta-Yamaguchl, Y , Nlshlda, E , Takaku, F , Aklyama, T , Kathurla, S , Akunama Y and Kasuga, M , Phosphorylat~on of tubuhn and mlcrotubule associated proteins by the purified Insulin receptor kmase, J Btol Chem , 260 (1985) 4016-4020 Klm, H , Binder, L and Rosenbaum, J L , The perlo&c association of MAP2 w~th brain mlcrotubules in vitro. J ('ell B t o l , 80 (1979) 266-276 Kuo, J • Schatzman, R , Turner, G and Mazzei. G Phosphohpld-sensltlVe Ca2+-dependent protein kmase a major protein phosphorylatlon system, Mol Cell E n d o c r t n o l , 35 (1084) 6573 Laemmh, U K , Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) ~80-685 Landreth, G E and Relser• G D , Nerve growth factor and epidermal growth factor-stimulated phosphorylatlon of a PC12 cytoskeletally-assoclated protein m SltU, J Cell B t o l , 100 (1985) 677-683 Landreth, G E , Smith, D S , McCabe, C and Glttlnger, C , Characterization of a nerve growth factor-stimulated protein klnase m PC12 cells which phosphorylates mlcrotubule-assoclated protein 2 and pp250, J Neurochem • 55 (1990) 514-523 Landreth, G E and Williams, L K , Nerve growth factor stimulates the phosphorylatlon of a 250 kDa cytoskeletal protein in cell-free extracts of PC12 cells• Neurochem R e ~ , 12 (1987) 943-950 Letterler, J F• Llem, R K H and Shelanskl, M L , Interaction between neurofilaments and mlcrotubule-assoclated proteins a possible mechamsm for lntraorganellar bridging. J Cell Bud 95 (1982) 982-986 Matus, A , Mlcrotubule-assocmted proteins their potential role m determining neuronal morphology, A n n u Rev Neuto~ct , 11 (1988) 29-44 Mlyasaka, T , Chao, M , Sherllne, P and Sattlel, A , Nerve growth factor stimulates a protein klnase in PC-12 cells that phosphorylates mlcrotubule-assoclated protein 2, J Btol C h e m • 265 (1990) 4730-4735 Nel. A E , Hanekom• C , Rheeder• A , Wlllams• K , Pollack, S • Katz, R and Landreth, G E • Stimulation of MAP2 klnase actlvJty in T lymphocytes by antl-CD3 or antl-Tl monoclonal antibody is partially dependent on protein kmase C• J l m m un o l , 144 (1990) 2683-2689 Nlshlda, E , Hoshl, M , Mlyamata, Y , Sakal, H • Kadawakl, T , Kasuga, M , Saljo, S , Ogawara, H and Aklyama, T , Tyrosme phosphorylatlon by the epidermal growth factor receptor induces functional alterations in mlcrotubule associated protein 2, J BIol Chem , 262 (1987) 16200-16204 Payne, M , Rossomando, A , Martmo, P , Erlckson, A , Her, J , Shabanowltz• J , Hunt, D , Weber, M and Sturgdl, T . Identification of the regulatory phorylaUon sites in pp42/mitogen activated protein klnase (MAP kmase), E M B O J , 10 (1991) 885892 Ray, L and Sturglll, T , Rapid stimulation by lnsuhn of a serlne/threonme klnase in 3T3-L1 adlpocytes that phosphorylates mlcrotubule assocmted protein 2 in wtro, Proc Natl A c a d 3Cl USA, 84 (1987) 1502-1506 Ray, L and Sturglll, T , Characterization of an insulin-stimulated mlcrotubule-assocmted protein kmase, J Blol Chern , 263 (1988) 12721 - 12727 Ray• L and Sturglll, T , lnsuhn-stlmulated mlcrotubule-assocl-

38

39

40

41 42

43

44

45

40

47

48

49

50

5i

52

53

54

55

ated protein klnase is phosphorylated on tyrosme and threonlne m rico, Ppoc Natl A c a d Sct USA, 85 (1988) 3753-3757 Sanghera, J S , Paddon, H B , Bader S A and Pelech, S L Purification and characterization of a maturatlon-actwated myelm basic protein klnase from sea star oocytes, J Btol Chem . 265 (1990) 52-57 Schanen-Kmg, C , Nel, A Williams, K and Landreth, G • Nerve growth factor stimulates the tyroslne phosphorylatlon ot MAP2 klnase in PC12 cells, Neuron• 6 (1991) 915-922 Selden, S C and Pollard, T D , Phosphorylatlon of mlcrotubule-assoclated proteins regulates their interaction with actln filaments. J Btol Chern. 258 (1983) 7064-7071 Smith• A , Varon• S and Shooter, E M , Multiple forms of nerve growth factor protein, Btochermstpy, 7 (1968) 3259-3268 Stratton, K R , Worley, P F , Lltz, J S , Parsons, S J , Huganlr, R L and Baraban, J M , Electroconvulswe treatment induces a rapid and transient increase in tyroslne phosphorylatJon of a 40-kllodalton protein associated with mlcrotubule associated protein 2 kmase activity, J Neutochem , 56 (1991) 147-152 Sturgfll• T W and Rat• L B • Muscle proteins related to microtubule associated protein-2 are substrates for an lnsuhn-st~mulatable klnase, Btochem Btoptlv~ Re,s C o m m u n , 134 (1986) 565-57 I Sturglll, T . Ray, L , Erlkson, E and Mailer, J • Insuhn-stlmulated MAP2 klnase phosphorylate~ and activates ribosomal protein $6 klnase II. Nature• 334 (1988) 715-718 Theurkauf, W E and Vallee, R B • Molecular characterization of the cAMP-dependent protein klnase bound to mlcrotubule assocmted protein 2• J Blol C h e m . 258 (1982) 3284-3290 Theurkauf, W E and Vallee• R B , Extensive cAMP-dependent and cAMP-independent phosphorylatlon of mlcrotubule associated protein 2, J Btol Chem , 258 (1983) 7883-7886 Tsao. H , Aletta, J M and Greene. L A . Nerve growth factor and fibroblast growth factor selectively activate a protein klnase that phosphorylates high molecular weight mlcrotubule-assoclated proteins, J Blol Chem • 265 (1990) 15471-15480 Tsuyama, S , Bramblett, G T , Huang K P and Flavm, M , Calcmm/phosphohpld-dependent klnase recognizes s~tes m microtubule assocmted protein 2 which are phosphorylated in hvmg brain and are not accessible to other kmases, J Btol Chern , 261 (1986) 4110-4116 Tsuyama. S , Terayama, Y and Matsuyama, S , Numerous phosphates o/ mlcrotubule-assoclated protein 2 in living rat brain, J Biol Chern . 262 (1987) 10886-10892 Vallee, R A taxol-dependent procedure for the isolation ol mlcrotubules and mlcrotubule assocmted proteins (MAPs)• J Cell Btol • 92 (1982) 435-442 Walaas, S I and Nalrn, A C , Multlslte phosphorylatlon of mlcrotubule-assoclated protein 2 (MAP2) in rat brain peptlde mapping d~stlngulshes between cyclic AMP, calclum/calmoduhn and calclum/phosphohpld-regulated phosphorylatlon mechanisms. J Mol Neuro~'ct, 1 (1989) 117-127 Yamamoto, H , Fukanaga• K . Goto, S . Tanaka, E and Mlyamoto, E , Ca :+, calmoduhn-dependent regulation of mlcrotubule formation ~ta phosphorylat~on of mlcrotubule-assoclated protein 2, tau factor, and tubuhn, and comparison with cychc AMP-dependent phosphorylatlon, ,l N e u r o c h e m , 44 (1985) 759-768 Yamauchl• T and Fuj~sawa. H • Phosphorylatlon of mlcrotubule associated protein 2 by calmoduhn-dependent protein klnase (klnase II) which occurs only m the brain tissues, Btochem Bloptlys Re~ C o m m u n , 109 (1982) 975-981 Badlng, H and Greenberg, M , Stlmulauon of protein tyroslne phosphorylatlon by N M D A receptor activation, Science, 253 (1991) 912-914 Seger, R , Ahn, N , Boulton, T , Yancopoulos, G , Panayotatos, N , Radzlejewska, E , Ericsson, L , Brathen, R • Cobb• M and Krebs, E , M~crotubule associated protein 2 klnases, ERK l and ERK 2, undergo autophosphorylatlon on both tyroslne and threonlne residues implications for their meehamsm of actwatlon, Proc Natl 4cad Sct USA, 88 (1991) 6142-6146