the of biological chemistry vol. 266, no. 23, issue 15, pp ... · the calcium phosphate technique....

THE JOURNAL OF BIOLOGICAL CHEMISTRY IC> 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 266, No. 23, Issue of August 15, PP. 15266-15276,1991 Printed in U. S. A.

Isolation and Characterization of Two Growth Factor-stimulated Protein Kinases That Phosphorylate the Epidermal Growth Factor Receptor at Threonine 669”

(Received for publication, February 6, 1991)

Ingrid C. Northwood$, Fernando A. GonzalezS$, Markus Wartmann$((, David L. RadenSll, and Roger J. Davis$V From the llHoward Hughes Medical Institute, Program in Molecular Medicine, and the $Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Bio2. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. How- ever, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 OC using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLTB6’PSGEAPN&ALLR). The Km(app) for ATP was 40 2 5 p~ (mean 2 S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 2 40 p~ (mean f S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr“’ (ERT) kinase.

It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at ThrS6’ observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.

~ ~~~~ ~~~

The cell-surface receptor for epidermal growth factor (EGF)’ is a 170-kDa transmembrane glycoprotein. The bind-

~~

*This work was supported in part by Grants CA39240 and GM37845 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a postdoctoral fellowship from the National Science Foundation.

1) Recipient of a postdoctoral fellowship from the Swiss National Science Foundation.

The abbreviations used are: EGF, epidermal growth factor; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; ERT kinase, EGF protein kinase; HEPES, 4-(2-hydroxyethyl)-l-piperazi- neethansulfonic acid; MAP2 kinase, microtubule-associated protein 2 protein kinase; MBP, myelin basic protein; NaDodSO,, sodium dodecyl sulfate.

ing of EGF to the extracellular domain of the receptor causes an increase in the protein-tyrosine kinase activity of the receptor cytoplasmic domain (1). Substantial evidence has been accumulated demonstrating that the function of the EGF receptor is regulated by phosphorylation (1,2). Protein kinase C phosphorylates the EGF receptor at Thr654 and causes an inhibition of the EGF receptor protein-tyrosine kinase activity (2-11). A second site of EGF receptor phosphorylation, T h P 9 (2, 12, 13), has been reported to regulate receptor internalization and substrate phosphorylation on tyrosine residues (14). Phosphorylation of the EGF receptor at Thr654 and T h P Y therefore represent physiologically significant mechanisms of regulation of EGF receptor signal transduction.

Protein kinase C accounts for the phosphorylation of the EGF receptor at T h P 4 (5 , 6, 8). However, the mechanism of phosphorylation of the receptor a t Thr66y is not understood (13). This is because the primary sequence surrounding Thr669 (Pro-Leu-Thr-Pro) is unusual for a site of protein phosphorylation (15). Treatment of cultured cells with EGF, platelet- derived growth factor, phorbol ester, or serum causes a marked increase in the phosphorylation state of the receptor at Thr669 (13). The increased phosphorylation at Thr6‘jY can be accounted for by the stimulation of a protein kinase activity detected in cell extracts (13). The rapid kinetics and marked extent of activation of this protein kinase activity by growth factors suggest that it is relevant to the process of signal transduction (13, 16).

The purpose of the experiments presented here was to perform a biochemical analysis of the enzymes that account for the phosphorylation of the EGF receptor a t ThP9 . We report here the identification of two distinct protein kinases: 1) the MAP2 kinase and 2) a novel enzyme that we designate EGF receptor Thrfi6’ (ERT) kinase.

EXPERIMENTAL PROCEDURES

Materials“[y-:”P]GTP and [“’Plphosphate were obtained from Du Pont-New England Nuclear. [y-:”P]ATP was prepared using a y - Prep A kit (Promega Biotec) according to the manufacturer’s direc- tions. The synthetic peptide LRRASLG and myelin basic protein were from Sigma. The synthetic peptides KRELVEPLTPSGE APNQALLR, PTPSAPSPQPKG, AKAKKTPKKAL, RRLIEDAE- YAARG, and IVQQFGFQRRASDDFKLTD were obtained from the Peptide Synthesis Core Facility, University of Massachusetts Medical School, The peptides YTRFSLAR (17) and KRTLRR (6) were obtained from Applied Biosystems, Inc. and Peninsula Laboratories,

RRREEETEEE, and KKRPQRATSNVFS were obtained from Dr. Inc., respectively. The synthetic peptides RRLSSLRA, RKRSRKE,

peptide RRTASFSESRRADEV was obtained from Dr. A. Bradford G. Johnson (National Jewish Center, Denver, CO). The synthetic

(University of Massachusetts Medical School). The synthetic peptide

15266

Phosphorylation of the EGF Receptor at Thr66g 15267

PRPASVPPSPSLSRHSSP was from Dr. T. Miller (University of Massachusetts Medical School). The synthetic peptide FKNI VTPRTPPPSQGKGRG and MAP2 kinase isolated from Swiss 3T3 cells were provided by Dr. T. W. Sturgill (University of Virginia). Okadaic acid was from Moana Bioproducts (Honolulu, HI).

Plasmid Construction-Oligonucleotide-directed mutagenesis of Lys7" to Arg was carried out using a 17-mer oligonucleotide (5'- gct atc agg gaa tta ag-3') according to Zoller and Smith (18) using methods described previously (19). The mutation was confirmed by sequencing using "S-dATP, dideoxynucleotide triphosphates, and Sequenase (20). The mutated EGF receptor cDNA was then cloned as a 4-kilobase XbaI-Hind111 fragment into the plasmid pX (2). This expression vector contains the murine dihydrofolate reductase gene as a selectable marker and allows the expression of the EGF receptor cDNA using the SV40 early promoter and polyadenylation signals (2). This plasmid was designated pXER(Arg'").

Solutions-Buffer A contained 50 mM @-glycerophosphate (pH 7.3), 1.5 mM EGTA, 0.1 mM sodium orthovanadate, 1 mM dithiothreitol, 10 pg/ml leupeptin, and 1 mM benzamidine. Buffer B contained 50 mM (3-glycerophosphate (pH 7.3), 1 mM EGTA, 0.1 mM sodium orthovanadate, and 1 mM dithiothreitol. Buffer C contained 25 mM Tris (pH 7.5), 250 mM NaC1, 40 mM p-nitrophenyl phosphate, 2 mM EGTA, 1 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride. Buffer D contained 20 mM HEPES (pH 7.5), 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium pyrophosphate, 50 p~ sodium orthovanadate, and 10 pg/ml leupeptin. Buffer E contained 20 mM HEPES (pH 7.5),0.5 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM sodium pyrophosphate, 50 p M sodium orthovanadate, and 2 mM MgC1,. Buffer F contained 20 mM imidazole (pH 7.5), 0.1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 50 p~ sodium orthovanadate, and 1 mM MgCI,. Buffer G contained 50 mM HEPES (pH 7.4), 50 mM NaC1, 0.1% Triton X-100, 10% glycerol, and 5 mM MgC12. Buffer H contained 50 mM HEPES (pH 7.4), 50 mM NaCl, 0.1% Triton X-100, 10% glycerol, and 10 mM EDTA.

Tissue Culture-KB cells were obtained from the American Type Culture Collection and were maintained in suspension culture using Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Chinese hamster ovary cells expressing [Arg7"]EGF receptors were obtained after transfection with the plasmid pXER(Arg'") using the calcium phosphate technique. After 3 days, the cells were passaged and selected using a-minimal essential medium supplemented with 5% dialyzed fetal bovine serum and 1 p M amethopterin. Stable colonies were isolated using cloning rings and were screened for the expression of EGF receptors by measuring the cell-surface binding of "'I-EGF at 4 "C.

Assay of Protein Kinase Actiuity-Protein kinase assays were performed using a modification of the procedure that we have previously described (13, 16). The synthetic peptide substrate used to determine ThrMy peptide kinase activity was Lys-Arg-Glu-Leu-Val-Glu-Pro- Leu-Thr"g-Pro-Ser-Gly-Glu-Ala-Pro-Asn-Gln-Ala-Leu- Leu-Arg. (Experiments designed to characterize substrate specificity were performed with alternative synthetic peptide substrates or myelin basic protein.) Assays were performed using 25 mM HEPES (pH 7.41, 10 mM MgC12, 50 p M [y-"'PIATP (10 pCi/nmol), 1 mg/ml synthetic peptide in a final volume of 25 @I. The reactions were terminated after 20 min at 22 "C by the addition of 10 p1 of 45% formic acid containing 25 mM ATP. Control experiments demonstrated that the phosphorylation reaction was linear with time for 30 min. The phosphorylated synthetic peptide was isolated by applying 25 p1 of the reaction mixture onto Whatman P-81 phosphocellulose paper and washing the filters twice in 1 M acetic acid, 4 mM sodium pyrophosphate. Radioactivity was quantitated by measuring Ceren- kov radiation with a Beckman liquid scintillation counter. Nonspe- cific incorporation of radioactivity was determined in incubations without the synthetic peptide.

Analytical Ion-exchange and Phenyl-Superose Chromatography- KB cells were seeded in 150-mm plates and grown to confluence. The medium was replaced with serum-free medium, and the cells were incubated at 37 "C for 30 min. The cells were treated without and with 10 nM EGF or 0.2 mM sodium orthovanadate for defined times. The medium was then aspirated, and the monolayers were washed rapidly at 4 "C with 5 ml of buffer A. The cells were collected by scraping with 2 ml of buffer A at 4 "C and lysed by sonication for 10 s. The extracts were then centrifuged at 100,000 X g for 20 min at 4 "C, and the supernatant fraction was used for anion-exchange chromatography using a Mono-Q HR5/5 column (Pharmacia LKB Biotechnology Inc.) at 4 "C. The column was equilibrated with buffer B, and the cell extracts were loaded at a flow rate of 0.5 ml/min. The

column was then washed with 10 ml of buffer B, and the bound proteins were eluted with a gradient of 0-0.4 M NaCl in buffer B (80- ml total volume). Fractions (1 ml) were collected, and the protein kinase activity in each fraction was measured.

Phenyl-Superose chromatography of fractions eluted from the Mono-Q column was performed as described by Ray and Sturgill(21). The samples were diluted with an equal volume of buffer C and were loaded onto a phenyl-Superose HR5/5 column (Pharmacia LKB Biotechnology Inc.) equilibrated with buffer C. The column was washed with 14 ml of buffer C, and bound proteins were eluted with a 20-ml gradient of decreasing concentration of NaCl (250 to 25 mM) and an increasing concentration of ethylene glycol (0-60%). Fractions (2 ml) were collected, and the protein kinase activity was measured.

Purification of ERT Kinase-Exponentially growing KB cells (10 liters) were treated with 200 p~ sodium vanadate for 2 h at 37 "c. The cells were then harvested by centrifugation at 5000 rpm for 5 min at 4 "C. The cells were resuspended in 50 ml of buffer D (4 "C) and homogenized using a Polytron (Brinkmann Instruments) three times for 5 s. The homogenate was centrifuged at 100,000 X g for 30 min at 4 "C. The supernatant (containing 200-300 mg of protein) was incubated with 2 ml of CM-cellulose (Whatman) for 30 min and subsequently filtered through a nylon membrane (0.2 pm, Nalgene) at 4 "C. The filtrate was loaded onto a Mono-Q HR10/10 column (Pharmacia LKB Biotechnology Inc.). The flow-through fraction was discarded, and the column was washed with 40 ml of buffer E. Proteins bound to the column were eluted at a flow rate of 2 ml/min with a linear gradient of 0-450 mM NaCl in buffer E (300-ml total volume). Fractions (8 ml) were collected and assayed for protein kinase activity. The peak fractions were pooled and dialyzed (Spectra-pore, 50-kDa pore size) against 4 liters of buffer F for 4 h at 4 "C.

The dialyzed sample was diluted with an equal volume of buffer F and loaded onto a Mono-P HR5/20 column (Pharmacia LKB Bio- technology Inc.) at a flow rate of 0.2 ml/min. The column was washed with 20 ml of buffer F. Bound proteins were then eluted at a flow rate of 0.2 ml/min with a linear gradient of 0-500 mM NaCl in buffer F (120-ml total volume). Fractions (4 ml) were collected and assayed for protein kinase activity. The peak fractions were pooled and diluted with 4 volumes of buffer F.

The diluted sample was pumped onto a Mono-!? HR5/5 column (Pharmacia LKB Biotechnology Inc.) at a flow rate of 0.2 ml/min. The column was washed with 5 ml of buffer F, and the bound proteins were eluted with a linear gradient of 0-100 mM MgC12 in buffer F at a flow rate of 0.2 ml/min (25-ml total volume). Fractions (1 ml) were collected and assayed for protein kinase activity.

Analysis of Protein Concentration-The protein content of fractions was measured using the dye binding assay (Bio-Rad) and bovine serum albumin as standard. Fractions eluted from the Mono-P HR5/5 column used at the last step of purification contained a very low concentration of protein that was below the level of detection of the Bio-Rad protein assay. The concentration of protein in these fractions was estimated after polyacrylamide gel electrophoresis by silver staining and comparison with bovine serum albumin.

NaDodS04-Polyacrylamide Gel Electrophoresis-Electrophoresis was performed as described (22). The gels were fixed and stained with the Bio-Rad silver staining kit.

In Situ Detection of Protein Kinase Activity after NaDodS04- Polyacrylamide Gel Electrophoresis-Detection of protein kinase activity in polyacrylamide gels was performed using the method of Kameshita and Fujisawa (23) as modified by Gotoh et al. (24). The substrate for phosphorylation by protein kinases (0.5 mg/ml MBP) was included in the separating gel prior to polymerization. NaDodSOl was removed after electrophoresis by washing the gel with 20% (v/v) 2-propanol in 50 mM Tris (pH 8.0). The proteins in the gel were denatured by incubation with 6 M guanidine HC1, 50 mM Tris (pH 8.0), 5 mM 2-mercaptoethanol. Renaturation was obtained by incubation of the gel in buffer containing 0.04% Tween 40 at 4 "C for 14- 16 h. Protein kinase activity was detected by incubating the gel at 22 "c for 60 min with 40 mM HEPES (pH 8.0), 2 mM dithiothreitol, 0.1 mM EGTA, 5 mM MgC12, 50 p~ [y-"PIATP ( 5 pCi/ml). The protein kinase reaction was terminated, and the gel was washed extensively with 5% (w/v) trichloroacetic acid, 1% (w/v) sodium pyrophosphate. The incorporation of phosphate was then analyzed by autoradiography of the dried gel.

Phosphorylation of [Arg7"lEGF Receptor by ERT Kinase-Chinese hamster ovary cells expressing the [Arg7"]EGF receptor were seeded in 35-mm dishes and grown to a density of 1 X IO6 cells/well. The cells were washed three times with serum-free medium and then incubated at 37 "C for 30 min. The cells were then solubilized in 1 ml

15268 Phosphorylation of the

of lvsis huffer (10% glvcerol, 1% Tri ton X-100, 10 mM EGTA, 500 mM NaCI, 10 yg/ml leupeptin, antl 25 mM HEPES ( p H 7.4)). Insol- uhle material was removed hv centrifugation for : 1 0 mi, a t 100.000 X g and 4 "C. The supernatants (0.8 ml) were added to 20 yI of protein A-Sepharose prehound to I pg of a monoclonal anti-EGF receptor antihodv (MIOR.1). 'The samples were then incuhated for 90 min at 4 "C. The immunoprecipitates were washed twice in lysis huffer and once in huffer G . The heads were then resuspended in 30 PI of buffer G at 4 "C. Purified ERT kinase (-10 ng) or huffer (10 P I ) was added to the immunoprecipitates. Phosphorylation was initiated hv the addition of 10 yl of 100 pM [y-,'.'P]ATP (10 yCi/nmol). After 20 min a t 22 "C, the reaction was terminated hy the addition of 1 ml of huffer H. The immunoprecipitates were then collected I ) v centrifugation and analvzed hv polyacrylamide gel electrophoresis and autoradiographv.

RESULTS

In preliminary experiments, we investigated the Thr"""peptide kinase activity present in homogenates prepared from tissues and cultured cell lines. Very low levels of Thr""!' peptide kinase activity were detected in extracts prepared from human placenta or rat liver. The low activity present in these tissue extracts is likely to he a result of a requirement for acute growth factor treatment t.o increase Thr""" peptide kinase activity (13, 16). These tissue extracts therefore did not pro- vide a suitable source of the enzyme that could he used for purification. We therefore examined the possibility of using cultured cells as starting material. Extracts prepared from human tumor cells (A431, HeLa, and KR) were ohserved to have a higher level of Thr""" peptide kinase activity compared with fihrohlasts (data not shown). Highest activity was ohserved in experiments using KR cells. It was observed that EGF treatment of KR cells caused a marked, hut transient increase in Thr""" peptide kinase activity (13, 16). KB cells were therefore used in further experiments.

Init,ial biochemical characterization of the Thrrir.!' peptide kinase activities present in extracts prepared from EGF- treated KB cells was performed by analytical ion-exchange chromatography using a Mono-Q column. Two resolved Thr"'"' peptide kinase activities were obtained (Fig. 1A ). Samples corresponding to these peaks of Thr""'' peptide kinase activity were subjected to NaDodSO.,-polyacrylamide gel electrophoresis. We then renatured the proteins in t,he gel and measured the in situ protein kinase activity using MRP as a substrate. Fig. 2 shows that protein kinases with apparent molecular masses of 42-43 and 45-46 kDa were observed in fractions corresponding to the first and second peaks of Thr';';!' peptide kinase activity elut,ed from the Mono-Q column, respectively. Thus, two distinct enzymes account for the Thr""" peptide kinase activity ohserved in extracts prepared from EGF- treated KR cells.

Identification of M A P 2 Kinase as the First Peak of T h P ' Peptide Kinase Activity Eluted during Mono-Q Chromatogra- p h y o f K B Cell Extracts-A possihle candidate enzyme that could account for Thr""!' pept,ide kinase activity is the MAP2 kinase descrihed by Ray and Sturgill (21). This is because the site of phosphorylation of MRP hy the MAP2 kinase (Pro- Arg-Thr-Pro) is similar to the sequence surrounding EGF receptor T h F ' , Pro-Leu-Thr-Pro (13, 25). Furthermore, the size of the 42-43-kDa protein kinase present in the first peak of Thr""" peptide kinase activity e k e d from the Mono-Q column (Fig. 2) is similar to the purified MAP2 kinase (21, 26). To test the hypothesis that the MAP2 kinase is a Thr""" peptide kinase, we performed three experiments, as follows.

1) MRP is a substrate for the MAP2 kinase (25). We therefore compared the elution profiles of MRP kinase and Thr""" peptide kinase activities during Mono-Q ion-exchange chromatography of cell extracts. Fig. 1A demonstrates that the Thr';"!' peptide and MRP kinase activities exhihited elution

EGF Receptor at Thr""!'

/I

Fracllon

I 700. E

B.Orthovanad&

1 5 0 ~ EGFR Thr MI) - MRP

n,

100' ,. ...-- 0 4

o I O 7- 30 40 x Go i n m Fraclron

FIG. 1. A n 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 o f T h r " " " p e p t i d e k inases . Kt3 cells were grown t o contlurnrc~ on four lTd)-nlm cl~shc~s. The cells were treated with 10 n M E(;F for 5 min id 1 or with 0.2 1nsf sodium orthovanadate for 2 h ( H ) at :I7 " ( I . Soluhle cxtrnct.; prcpnrrtl from the K H cells were fractionated hy analytirnl irm-exch;tngcs chromatography using a Mono-Q column as desrrihrtl under "Kxper- imental I'rocedures." The concentration o f sodium chloride in thr eluate is indicated ( - - -). Protein kinase activity in fractions elutrd from the column was measured using thc synthrtic peptide KREI,VEPI,'T'"'"PS~EAPNqAI,I,II antl \'lI31' as suhstrntes. f,'f;/.'/{, E(;F receptor.

1 2 3 4 5 6

- 47 LDa

+ 33kD. - 24 LDm - I 6 k D a

FIG. 2. In situ d e t e c t i o n o f p r o t e i n k i n a s e a c t i v i t i e s a f t e r po lyac ry lamide ge l e l ec t rophores i s . ( ' ons rcu t ivc fract ions elutcd from the Mono-Q column (Fig. 1.4 1 were ponlrd nnd rr~nrentrntetl (-30-fold) with a Centricon-:lO (Amiron (*nrp.). The snmples i25 pll were electrophoresed on a polvacrylarnide gel rontnining > I R l ) (0.:

mg/ml). After renaturation of the proteins in the gel, prntein kinnw activity was determined using [ y - .'P]ATl' as descrihed r~ndrr "Ex- perimental Procedures." Shown is an autoradiograph of the dried cel. I'restained molecular mass standards were ohtnincd from t<i~)-J<d. l ane 1 , fractions 20 and 21; lnnc 2, frnctinns 22 antl 23; h n 6 - :I, fractions 24 antl 25; lnnc 4 , fractions 26 and 27; lonv 5 , frrlrtinns 28 and 29; lnnr 6 , fractions 30 and 31.

profiles that were nearly identical. The coelution of Thr"" peptide and MAP2 kinase activities during Mono-Q ion- exchange chromatography suggests that these activit ies are closely associated.

2) Efficient purification of the MAP2 kinase can he achieved by phenyl-Superose Chromatography (21, 26) . The first peak of Thr'.'"" peptide kinase activity eluted from the Mono-Q column (Fig. 1A ) was therefore applied to a phenvl- Superose column. The protein kinase activity was ohserved to hind to the column and was eluted using a decreasing gradient of NaCl in the presence of ethylene glycol (Fig. 3 , upper). The MAP2 kinase activity was detected using XTRP as a suhstrate (25). Significantly, Thr"'"' peptide kinase activity coeluted with MRI' kinase activity (Fig. 3 , u p p w ) .

Phosphorylation of the EGF Receptor a t T h P 9 15269

p 80 eeahl & 60 -t MBP

- EGFR Thr-669

Fraction

$ 6 0 / a - EGFR Thr-669 - -C MBP

FIG. 3. Phenyl-Superose chromatography of Threes peptide kinases. Two peaks of T h P 9 peptide kinase activity were resolved by Mono-Q chromatography of extracts prepared from EGF-treated cells (Fig. lA). Fractions corresponding to the first peak (upper) and the second peak (lower) of protein kinase activity eluted from the Mono-Q column were further analyzed by phenyl-Superose chromatography as described under "Experimental Procedures." The concentration of sodium chloride in the eluate is indicated (- - -). Protein

using the synthetic peptide KRELVEPLTG9PSGEAPNQALLR and kinase activity in fractions eluted from the column was measured

MBP as substrates. EGFR, EGF receptor.

Fraction

3) The coelution of ThrS9 peptide and MBP kinase activities during consecutive steps of chromatography on Mono-Q and phenyl-Superose columns suggested that a single protein kinase, the MAP2 kinase, may account for the observed phosphorylation. We therefore obtained a sample of purified MAP2 kinase isolated from Swiss 3T3 cells (21, 25) from Dr. T. W. Sturgill. This purified enzyme was tested for kinase activity using the T h P 9 synthetic peptide (KRELVEPLT'j6' PSGEAPNQALLR) and MBP as substrates. Phosphorylation of these substrates (1 mg/ml) was performed for 10 min at 22 "C in an incubation mixture containing 50 JLM [Y-~'P]ATP (10 pCi/nmol). It was observed that the incorporation of radioactivity into the synthetic peptide was 45 f 6% of the incorporation observed for MBP (mean f S.D., n = 3). Thus, the Thr'j6' synthetic peptide therefore represents an excellent in vitro substrate for the purified MAP2 kinase.

Together, these data strongly support the hypothesis that the MAP2 kinase accounts for the first peak of T h P 9 peptide kinase activity eluted during Mono-Q chromatography of extracts prepared from EGF-treated cells (Fig. lA).

Identification of ERT Kinase as the Second Peak of Thr'j6' Peptide Kinase Activity Eluted during Mono-Q Chromatogra- phy of Cell Extracts-Two peaks of ThrS9 peptide kinase activity were eluted during Mono-Q chromatography of extracts prepared from EGF-treated cells (Fig. L4). The first peak of activity was identified as the MAP2 kinase. It was therefore possible that the second peak of activity represented a modified form of the MAP2 kinase. Consistent with this hypothesis was the observation that this Thr'j'j' peptide kinase activity coeluted with MBP kinase activity (Fig. lA). How- ever, we obtained experimental evidence that this second peak of Thr'j6' peptide kinase activity was distinct from the MAP2 kinase. 1) In contrast to the MAP2 kinase, the second peak of T h P 9 peptide kinase activity eluted from the Mono-Q column did not bind to phenyl-Superose (Fig. 3, lower). 2) The major protein kinase present in this fraction exhibited

an apparent molecular mass of 45-46 kDa during polyacrylamide gel electrophoresis (Fig. 2). This is significantly larger than the purified MAP2 kinase (21, 26).

Together, these data indicate that the second peak of Thr6" peptide kinase activity eluted from the Mono-Q column is distinct from the MAP2 kinase. The identity of this Thr'jfi9 peptide kinase is unknown. We have therefore designated this enzyme ERT kinase. A major goal of further studies was the biochemical characterization of the ERT kinase.

Purification of ERT Kinase from KB Cells-The transient nature of the growth factor-stimulated ERT kinase activity represented a logistical problem for harvesting large cultures of stimulated cells that could be used as the starting material for the purification of the ERT kinase. We therefore investigated alternative methods for stimulating ERT kinase activity. Phorbol ester, orthovanadate, and okadaic acid were found to stimulate ERT kinase activity. The increase caused by phorbol ester and okadaic acid was transient. However, a relatively stable increase (-3-6-fold) in the measured T h P 9 peptide kinase activity was observed after the treatment of KB cells with 200 p~ orthovanadate for 1.5-3 h. Analytical Mono-Q ion-exchange chromatography (Fig. 1B) demonstrated that the major Thr'j'j' peptide kinase activity detected in orthovanadate-treated KB cells was accounted for by the ERT kinase.2 This stable increase in ERT kinase activity enabled the harvesting of large quantities of KB cells for protein purification. We therefore used KB cells incubated for 2 h with orthovanadate for the purification of the ERT kinase."

Ten liters of exponentially growing KB cells were harvested by centrifugation and were lysed in homogenization buffer. The inclusion of phosphatase inhibitors (pyrophosphate and orthovanadate) in the homogenization buffer was found to be critical for maintaining the stability of ERT kinase activity during later chromatographic steps. The homogenate was centrifuged at 100,000 x g to remove insoluble material. Generally 200-300 mg of protein was obtained in the soluble supernatant fraction, and little ERT kinase activity could be detected (Table I). The sample was then applied to CM- cellulose. The flow-through fractions were collected and found to contain significant ERT kinase activity (Table I). These fractions were pooled and applied to a Mono-Q HR10/10 ion- exchange column. The bound proteins were eluted with a linear gradient of NaC1. A major peak of ERT kinase activity eluted at -180 mM NaCl (Fig. IA). The peak of activity was usually preceded by a prominent shoulder that was sometimes resolved as a distinct peak of protein kinase activity. If phosphatase inhibitors were omitted from the homogenization buffer, the overall activity eluted from the column was greatly reduced.

After chromatography on the CM-cellulose and Mono-Q columns (steps 1 and 2), a substantial increase in ERT kinase activity was detected (Table I). As a loss of activity would normally be anticipated during chromatography, it is likely that the increase in protein kinase activity was caused by the removal of an inhibitor. Thus, the level of ERT kinase activity detected in crude fractions probably represents a large under- estimation of the amount of enzyme present.

The fractions corresponding to the main peak of ERT kinase activity eluted from the Mono-Q column at 180 mM

The effect of orthovanadate to cause only a small increase in MAP2 kinase activity and a large increase ERT kinase activity contrasts with EGF action to cause a marked increase in the activity of both protein kinases.

Jeno et al. (43) have previously described the use of orthovanadate to stimulate large cultures of Swiss 3T3 cells for the purification of the activated ribosomal S6 protein kinase.

15270 Phosphorylation of the EGF Receptor at ThP9

TABLE I Purification of ERT kinase from human tumor KB cells

The purification of ERT kinase activity from cytosolic extracts of KB cells is summarized. The methods used are described in detail in the text. No significant ERT kinase activity was detected in the initial supernatant fraction obtained after centrifugation of the KB cell homogenate. The apparent -fold purification was therefore calculated based upon the activity measured after step 2 (CM-cellulose chromatography). The increased recovery of ERT kinase activity during purification suggests that an endogenous inhibitor of the enzyme was removed during purification. It is therefore very likely that the protein kinase activity present in the early steps of the purification is significantly underestimated. At the last step of purification, the level of protein recovered was low and could not be reliably detected using the dye binding assay (Bio-Rad). The level of protein was therefore estimated by polyacrylamide gel electrophoresis, silver staining, and comparison with a bovine serum albumin standard. The estimate of protein (and consequently the calculated specific activity and -fold purification) is therefore subject to a possible systematic error.

mg % mg/min -fold nmolf

1. Soluble extract 229 ND" ND ND 2. CM-cellulose 171 100 0.005 1 3. Mono-Q HR10/10 4.8 830 1.5 300 4. Mono-P HR5/20 0.28 330 10 2000 5. Mono-P HR5/5

Fraction 21 0.005 48 86 17,000 Fraction 22 0.001 30 260 52,000

" ND. not detected.

NaCl (Fig. 4A) were pooled and dialyzed against a low ionic strength buffer. The use of a dialysis membrane with a 50- kDa pore size allowed the removal of low molecular mass substances from the ample.^ The dialyzed protein kinase activity was then applied to a second ion-exchange column (Mono-P HR5/20).5 After washing the column, ERT kinase activity was eluted with a linear gradient of NaCl. A single peak of activity was observed to be eluted from the column at 290 mM NaCl (Fig. 4B).

The peak fractions from the second ion-exchange column were diluted and loaded onto a third ion-exchange column (Mono-P HR5/5). A linear gradient of MgCl, was used to elute the ERT kinase activity from this column. A single peak of activity was eluted at 65 mM MgClz (Fig. 4C). The proteins eluted from the third ion-exchange column were analyzed by NaDodS0,-polyacrylamide gel electrophoresis. The gels were fixed, and proteins were visualized by silver staining (Fig. 5). It was observed that the peak of ERT kinase activity correlated with the presence of a 46-kDa protein. Several other proteins eluted immediately prior to the ERT kinase activity (Fig. 5). However, the 46-kDa protein was the major protein species in fraction 22 eluted from the column (Fig. 5 ) . This evidence suggests that the 46-kDa protein represents the

The use of dialysis tubing with a pore size of 50 kDa represents a significant step in the purification of the ERT kinase because it allowed the removal of the majority of low molecular mass proteins. However, as the molecular mass of the ERT kinase estimated by polyacrylamide gel electrophoresis is -46 kDa (Fig. 5), it is surprising that this enzyme is retained by dialysis tubing with a 50-kDa pore size. Two hypotheses can be proposed to account for this observation: 1) the ERT kinase may not have a simple globular structure; or 2) the ERT kinase may exhibit an abnormal electrophoretic mobility on NaDodSOI-polyacrylamide gels. At present, insufficient information is available to distinguish between these hypotheses. ' The ERT kinase activity obtained after Mono-Q chromatography

of extracts prepared from orthovanadate-treated cells did not bind to phenyl-Superose. A phenyl-Superose column was therefore not employed for the purification of the ERT kinase.

700 1 I1000

-I 200 LW -0 10 20 30 40"

1200, Fraction

.T 100

1000.

800.

600-

400 -

200-

- C.

0 : . . . 0 5 10 15 20 25 30

Fraction

FIG. 4. Purification of ERT kinase. Ten liters of KB cells growing in suspension were treated with 0.2 mM sodium orthovanadate for 2 h at 37 "C. The cells were collected by centrifugation and homogenized. The extract obtained was centrifuged at 100,000 X g. The supernatant was collected and incubated with CM-cellulose. ERT kinase activity present in the flow-through fractions was further purified by chromatography on a Mono-Q HR10/10 column ( A ) . The peak fractions were dialyzed and then applied to a Mono-P HR5/20 column and eluted with a linear gradient of NaCl ( B ) . Fractions containing ERT kinase activity were subsequently applied to a Mono- P HR5/5 column and eluted with a linear gradient of MgC12 (C). In each panel, the salt (NaCI or MgCIz) concentration is indicated

peptide substrate (KRELVEPLT6'j9PSGEAPNQALLR) as described (- - -). Protein kinase activity (0) was determined using a synthetic

under "Experimental Procedures." The data shown were obtained in a single representative experiment.

enzyme that accounts for ERT kinase activity. To test the hypothesis that the 46-kDa protein and the

ERT kinase are identical, we performed four experiments. 1) Nondenaturing polyacrylamide gel electrophoresis of the purified ERT kinase was performed. The major 46-kDa protein detected by silver staining was found to correspond to ERT kinase activity measured using minced gel slices (data not shown). 2 ) The purified ERT kinase was analyzed by sucrose density gradient centrifugation. The sedimentation of the 46- kDa protein was found to correspond closely to the ERT kinase activity (data not shown). 3) The 46-kDa protein and the ERT kinase both eluted at pH 4.9 during chromatofocusing using a Mono-P HR5/20 column (data not shown). 4) Analytical gel filtration chromatography indicated that the ERT kinase activity eluted with an apparent molecular mass of -45 kDa (Fig. 6). Together, these data strongly support the hypothesis that the 46-kDa protein and the ERT kinase are identical. Confirmation of this conclusion will require the molecular cloning of the 46-kDa protein.

Characterization of ERT Kinase Actiuity-In initial studies, we examined the possible autophosphorylation of ERT kinase

Phosphorylation of the EGF Receptor at ThP9 15271

Fraction Numbers

17 18 19 20 21 22 23 . .-.

116-Wa

c 97-Wa

c BB-LD.

c 46W.

"" 17 18 19 a0 21 22 23

Fraction Numbers FIG. 5. Analysis of fractions eluted from Mono-P HR5/5

column by polyacrylamide gel electrophoresis. Fractions eluted from the Mono-P HR5/5 column were collected, and ERT kinase activity was assayed using a synthetic peptide substrate (KRELVEPLl?'PSGEAPNQALLR) as described under "Experi- mental Procedures." An aliquot of each fraction was analyzed by NaDodS04-polyacrylamide gel electrophoresis. After fixation, the gel was stained with silver (Bio-Rad) and dried. The data obtained in a representative experiment are presented.

.r( CI c 0 - 4 ?

3-1

0 10 20 30 40

Fraction FIG. 6. Analytical gel filtration chromatography of ERT

kinase. Aliquots of ERT kinase activity purified by Mono-P HR5/5 chromatography were analyzed by gel filtration chromatography using a Superose-12 column (Pharmacia LKB Biotechnology Inc.). The flow rate was 0.3 ml/min, and 0.5-ml fractions were collected. Protein

substrate (KRELVEPLT"'PSGEAPNQALLR) as described under kinase activity was assayed in each fraction using a synthetic peptide

"Experimental Procedures." Protein was measured by monitoring the absorbance at 280 nm. Cross-linked blue dextran and molecular mass standards (bovine serum albumin (66 kDa), ovalbumin (43 kDa), and chymotrypsinogen (25 kDa)) were obtained from Pharmacia LKB Biotechnology Inc. Similar results were obtained in three separate experiments.

activity eluted from the Mono-P HR5/5 column. The purified ERT kinase was incubated with 50 FM [r-"P]ATP (50 pCi/ nmol) for 20 min a t 22 "C, and the products of any phosphorylation reaction were investigated by polyacrylamide gel electrophoresis and autoradiography. No protein phosphorylation was detected (data not shown). The purified protein

kinase therefore does not autophosphorylate under the standard assay conditions. The data further indicate that the purified protein kinase preparation does not contain any significant contamination by substrates.

In experiments designed to characterize the activity of the purified ERT kinase, we used a peptide substrate based on the primary sequence of the EGF receptor, KRELVEP- LT'"PPSGEAPNQALLR. The time course of phosphorylation of the synthetic peptide was observed to be linear for >30 min at 22 "C (Fig. 7A), and the extent of phosphorylation observed was dependent on the amount of enzyme added to the assay (Fig. 7B). However, at 30 and 37 "C, rapid thermal inactivation of ERT kinase activity was observed (data not shown). Therefore, further experiments designed to characterize the ERT kinase activity were performed at 22 "C.

To examine the effect of reductant on the activity of the purified ERT kinase, we investigated the effect of the addition of 1 mM dithiothreitol to the assay buffer. It was observed that dithiothreitol caused no significant change in the level of protein kinase activity detected. Dithiothreitol was therefore not included in the standard buffer used to assay ERT kinase activity.

To determine the optimal assay conditions for measuring the activity of the purified ERT kinase, we investigated the effect of salt concentration. Low concentrations (0-100 mM) of NaCl or Na2P04 caused no significant change in the rate of phosphorylation of synthetic peptide substrates (data not shown). However, higher concentrations (>lo0 mM) caused a marked decrease in protein kinase activity (data not shown). Divalent cations (Me or Mn2+) were required for protein kinase activity (Fig. 8). It was found that ERT kinase activity was greater in the presence of low concentrations ( 4 mM) of MnZ+ compared with M$+ (Fig. 8). However, maximal protein

0 15 30 45 60

Roteln Klnaae (ne)

FIG. 7. Time course and dose response of ERT kinase activity. Aliquots of ERT kinase purified by Mono-P HR5/5 chromatography were used to investigate the time course ( A ) of phosphorylation in assays employing the synthetic peptide KRELVEPLT""SSG- EAPNQALLR (1 mg/ml) and 50 p~ [y-'"PIATP (10 pCi/nmol) as substrates. The effect of enzyme concentration was examined in assays terminated after 20 min of incubation ( B ) . The incorporation of radioactivity into the synthetic peptide was determined as described under "Experimental Procedures." Similar results were obtained in three separate experiments.

15272 Phosphorylation of the EGF Receptor at Thr6'j9 800- - Magnesium - Manganese 600-

400-

200-

O* 0 5 10 15 20

Concentration (mW

FIG. 8. Effect of divalent cation concentration on ERT kinase activity. The purified ERT kinase eluted from the Mono-P HR5/5 column was dialyzed against 25 mM HEPES, 0.1 mM EGTA (pH 7.4) to remove divalent cations. The effect of different concentrations of MgC12 or MnCI2 in the assay buffer on protein kinase activity was examined. The activity of purified ERT kinase was assayed in a final volume of 25 pl containing 25 mM HEPES (pH 7.4), 50 p~ [-y-'"P]ATP (10 pCi/nmol), 1 mg/ml synthetic peptide (KRELVEPLT""!PSGEAPNQALLR), and different concentrations of MgCly or MnC12. The reactions were terminated after 20 min a t 22 "C, and the incorporation of radioactivity into the synthetic peptide was determined as described under "Experimental Procedures." Similar results were obtained in three separate experiments.

kinase activity was observed in the presence of 10 mM M e . In contrast, little ERT kinase activity was found in the presence of 10 mM MnZ+ (Fig. 8).

The addition of Ca'+ to crude fractions containing ERT activity caused a marked inhibition (>95%) of protein kinase activity (data not shown). We therefore investigated the ef- fects of Ca'+ on the activity of the purified ERT kinase. It was observed that the addition of CaC12 (0.1-1.0 mM) caused no significant change in the level of ERT kinase activity detected (data not shown). This observation suggests that the inhibition of ERT activity observed in crude fractions is an indirect action of Ca'+ (e.g. Ca'+-stimulated proteolysis).

The kinetic parameters for synthetic peptide phosphorylation were estimated by measurement of the initial rate of phosphorylation in the presence of different concentrations of substrates in a standard assay buffer containing 10 mM MgCl? and 25 mM HEPES (pH 7.5) a t 22 "C. GTP was not found to be a significant substrate for the ERT kinase (Fig. 9). In contrast, ATP caused rapid peptide phosphorylation. The Km,Rpp, for ATP was determined to be 40 k 5 PM (mean f S.D., n = 3) (Fig. 9), and the Km(app) for the synthetic peptide substrate (KRELVEPLTfifi9PSGEAPNQALLR) was 260 k 40 PM (mean f S.D., n = 3) (data not shown). The Vmax(app) was estimated to be 400-900 nmol/mg/min a t 22 "C in three separate experiments.

Characterization of Substrate Specificity of Isolated ERT Kinase-The ERT kinase purified from KB cells was identified by its activity to phosphorylate the synthetic peptide KRELVEPLT"'PSGEAPNQALLR. This synthetic peptide corresponds to the primary sequence of the EGF receptor surrounding the site of phosphorylation, ThrGfig. As this peptide represents a model substrate, it was important to determine whether the purified protein kinase could account for the phosphorylation of the EGF receptor a t Thrfi6'.

To determine whether the full-length EGF receptor was a substrate for phosphorylation by the isolated ERT kinase, we used a protein-tyrosine kinase-defective EGF receptor that contained a point mutation (substitution of LysT2I with Arg) within the ATP-binding site. The rationale for this approach was that the [Arg"']EGF receptor can be used as a substrate for phosphorylation by exogenous protein kinases in the absence of autophosphorylation of the receptor on tyrosine residues (Fig. 10). Marked phosphorylation of the [Arg2']

TJ 2000

IO00 GTP >200

, L _ 100 200 300

Nucleotide Concentration

FIG. 9. Effect of ATP and GTP concentrations on ERT kinase activity. The activity of purified ERT kinase (-10 ng) was assayed using different concentrations of [-y-"P]ATP and [-y-"'PI GTP. The specific activity of ATP and GTP was 10 pCi/nmol. The synthetic peptide KRELVEPLT""!PSGEAPNQALLR (1 mg/ml) was used as a substrate for phosphorylation. The assays were terminated after 20 min of incubation a t 22 "C, and the incorporation of radioactivity into the synthetic peptide was determined as described under "Experimental Procedures." The results are presented as the mean of the data obtained in three separate experiments. The K,,, values for ATP and GTP were determined by curve fitting to the Michaelis- Menten formalism. This was achieved by weighted nonlinear regres- sion employing "robust" methods for the modification of residuals (44).

ThrW9 Protein ~ f n a s e + -

0 c EGF Receptor

FIG. 10. Phosphorylation of EGF receptor by ERT kinase in vitro. The protein-tyrosine kinase inactive [Arg"']EGF receptor was isolated by immunoprecipitation. The immunoprecipitates were incubated in the absence and presence of ERT kinase (-10 ng) purified by chromatography on a Mono-P HR5/5 column. Phos- phorylation was initiated by the addition of 20 p M ATP (10 pCi/ nmol) for 20 min a t 22 "C. The immunoprecipitates were then washed and electrophoresed on a polyacrylamide gel in the presence of 0.1% NaDodS04. An autoradiograph of the dried polyacrylamide gel is presented. Similar results were obtained in three separate experiments.

EGF receptor was observed after the addition of the purified ERT kinase (Fig. 10). We conclude that the EGF receptor is a substrate for the purified ERT kinase.

In further experiments, we investigated the ability of the purified ERT kinase to phosphorylate potential synthetic peptide substrates. Table I1 shows that peptides demonstrated to be substrates for protein kinase C, cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase, casein kinase 11, myosin light chain kinase, ribosomal S6 protein kinase, and the c-raf-1 protein kinase were not significantly phosphorylated by the purified ERT kinase. As expected, a protein-tyrosine kinase substrate (RR-SRC) also was not phosphorylated by the ERT kinase (Table 11). Together, these data suggest that the purified ERT kinase has a relatively restricted substrate specificity.

A distinguishing feature of the primary sequence surrounding the phosphorylation site ThrGfi" is the proximity of 2 proline residues (&-Leu-Thr'"-&). Four protein kinases have been reported that phosphorylate sites on proteins that are adjacent to proline. These are the cdc2 protein kinase

15273 Phosphorylation of the EGF Receptor at Thr'j'j' TABLE I1

Substrate specificity of ERT kinase isolated from human tumor KB cells Aliquots of ERT kinase activity purified by Mono-P HR5/5 chromatography (fraction 22) were employed to

characterize the substrate specificity. Each synthetic peptide was used in the phosphorylation assay at a concentration of 1 mg/ml. Incubations were performed with 50 IM [y-"P]ATP (10 pCi/nmol) at 22 "C for 20 min. The reactions were terminated, and the incorporation of radioactivity into the synthetic peptides was determined as described under "Experimental Procedures." The data presented are the means of three separate experiments and are normalized to the incorporation observed using the synthetic peptide KRELVEPLT669PSGEAPNQALLR as a substrate (100%).

Substrate Putative kinase Sequence Ref. Kinase activity

% KRELVEPLTPSGEAPNQALLR EGF receptor, T h P ERT kinase 13,16 100.0

Transferrin receptor, SerZ4 PKC" YTRFSLAR 17 1.0 EGF receptor, T h P 4 PKC" KRTLRR 6 0.4 Kemptide cAMP kinase LRRASLG 45 1.5 ATP citrate-lyase cAMP kinase RRTASFSESRRADEV 46 1.3 Ribosomal protein S6 RSK RRLSSLRA 47

PTPSAPSPQPKG 1.0

Tyrosine hydroxylase PDPK 29 1.0 Glycogen synthase (sites 3) PDPK PRPASVPPSPSLSRHSSP 28, 29, 32, 33, 48 9.7

AKAKKTPKKAL Histone H1 cdc2lPDPK 27,29 0.1 RR-SRC c-src RRLIEDAEYAARG 49 0.0 cGMP substrate cGMP kinase RKRSRKE 50 0.2 Casein kinase I1 substrate Casein kinase I1 RRREEETEEE 51

IVQQFGFQRRASDDFKLTD 0.2

RAF substrate c-raf-1 52 0.8 MLCK peptide MBP FKNIVTPRTPPPSQGKGRG

0.4 MLCK KKRPQRATSNVFS 53, 54 MAP2 (ERK-I) 25 7.8

a PKC, Drotein kinase C: RSK, ribosomal S6 kinase; PDPK, proline-directed protein kinase; MLCK, myosin light chain kinase.

(27), glycogen synthase kinase-3 (28), proline-directedprotein kinase (29), and MAP2 kinase (25). Substrates for these protein kinases were tested in assays using the purified ERT kinase, as follows.

1) It was observed that a cdc2 protein kinase substrate based on the sequence of histone Hl(27) was not phosphorylated by the ERT kinase (Table 11). Thus, the specificity of the cdc2 protein kinase is different from that of the ERT kinase.

2) A synthetic peptide based on the sequence of glycogen synthase surrounding "sites 3" was phosphorylated by the ERT kinase (Table II).'j The rate of phosphorylation observed for this peptide was -10% of the rate measured for the Thr'j6' synthetic peptide (KRELVEPLT'j'j'PSGEAPNQALLR). It is therefore possible that there is some overlap between the substrate specificities of glycogen synthase kinase-3 and the ERT kinase.'j

3) Proline-directed protein kinase has been described as an enzyme that phosphorylates tyrosine hydroxylase at Sera (29). A synthetic peptide based on the sequence of tyrosine hydroxylase surrounding Ser' is a substrate for proline-directed protein kinase (29), but no significant phosphorylation of this peptide was observed in assays using the purified ERT kinase (Table 11). These data indicate that the specificity of the ERT kinase is distinct from proline-directed protein kinase.

4) The MAP2 kinase phosphorylates MBP at Thrg7 (25). A synthetic peptide based on the sequence of MBP surrounding Thrg7 is a substrate for phosphorylation by the MAP2 kinase (25). Protein kinase assays demonstrated that this peptide was also a substrate for the ERT kinase (Table 11). The rate of phosphorylation of the MBP peptide by the ERT kinase

' The synthetic peptide corresponding to glycogen synthase sites 3 is truncated and lacks the casein kinase I1 phosphorylation site. Phosphorylation of this casein kinase I1 site is required for phosphorylation by glycogen synthase kinase-3 (28, 32, 33). Thus, the peptide used is not a substrate for glycogen synthase kinase-3 (28, 32,33). The observed phosphorylation of the synthetic peptide by the purified ERT kinase demonstrates that the substrate recognition by glycogen synthase kinase-3 is distinct from that of the ERT kinase.

was -8% of the rate of phosphorylation of the Thr'j'j' synthetic peptide (KRELVEPLT6'jgPSGEAPNQALLR). These data indicate that the MAP2 and ERT kinases may exhibit similar substrate specificities.

DISCUSSION

Two Protein Kinases Account for Thr6'j9 Peptide Kinase Activity Detected in Cell Homogenutes

Ion-exchange chromatography using a Mono-Q column demonstrated that the Thr6'j9 peptide kinase activity detected in cell extracts (13) could be accounted for by two distinct enzyme activities (Fig. 1). The first peak of Thr'j'j' peptide kinase activity eluted from the Mono-Q column was identified as the MAP2 kinase (Figs. 1-3). The second peak of activity eluted from the Mono-Q column was identified as a novel enzyme that we designate ERT kinase. Initial biochemical characterization indicated that the ERT kinase was not a modified form of the MAP2 kinase because: 1) the ERT kinase (in contrast to the MAP2 kinase) did not bind to phenyl- Superose (Fig. 3) and 2) the molecular mass of the ERT kinase is significantly greater than that of the MAP2 kinase (Fig. 2). A major goal of the study described in this report was the isolation and biochemical characterization of the ERT kinase.

Purification of E R T Kinase We describe in this report the isolation of a protein kinase

(ERT) that phosphorylates the EGF receptor at Thr6'j9. The human tumor KB cell line was used as the source of the protein kinase. Considerable effort was expended in testing approaches employing affinity chromatography to facilitate the purification of the protein kinase. However, no successful procedure using immobilized substrates (peptides, bacterially expressed fragments of the EGF receptor, the purified EGF receptor, ATP, and ATP analogues) was achieved. The purification scheme described here consists of sequential steps involving ion-exchange chromatography and affinity elution.

15274 Phosphorylation of the EGF Receptor at Thr669

There are three factors that are important for the application of this method to the purification of the ERT kinase. First, it was found that the ERT kinase was retained by a 50-kDa cutoff dialysis membrane. Second, the elution profile of the ERT kinase was markedly different when chromatography was performed using a Mono-Q column compared with a Mono-P column. Third, it was observed that the concentration of Mg2+ used during chromatography significantly altered the ERT kinase elution profile. This effect may be related to the possibility that the ERT kinase is a Mg*+-binding protein.

The purified ERT kinase activity was observed to be unsta- ble, and a marked loss of activity was found during prolonged incubation at 4 "C. It was therefore important that all of the steps of the purification were performed rapidly. Furthermore, all experiments using the purified ERT kinase were performed within 5 h of isolation. Long-term storage of the purified ERT kinase could be achieved at -20 or -80 "C in the presence of several detergents or glycerol, but this was accompanied by marked losses of enzyme activity.

ERT Kinase Is Closely Correlated with a 46-kDa Protein

It was observed that the presence of a 46-kDa protein closely correlated with the level of ERT kinase activity detected (Fig. 5). The 46-kDa protein was the major protein present in fraction 22 eluted from the Mono-P HR5/5 column (Fig. 5). Sucrose density gradient sedimentation analysis (data not shown), chromatofocusing (data not shown), nondenaturing polyacrylamide gel electrophoresis (data not shown), and gel filtration chromatography (Fig. 6) of the purified ERT kinase activity further indicated a close correlation between the enzyme activity and the 46-kDa protein. The hypothesis that the ERT kinase is the 46-kDa protein is also consistent with the results of the kinetic analysis of the purified ERT kinase activity. It was found that the Vmax(app) at 22 "C was 400-900 nmol/mg/min in three separate experiments. This high specific activity indicates that it is unlikely that a minor protein contaminant that was undetected by silver staining could account for the measured protein kinase activity. Thus, the close correlation between the 46-kDa protein and the ERT kinase activity indicates that this protein may account for the enzyme activity. Current work in this laboratory directed toward the molecular cloning of the 46-kDa protein will allow this hypothesis to be rigorously tested.

Mechanism of Activation of ERT Kinase

Human tumor KB cells stimulated by treatment with the tyrosine phosphatase inhibitor orthovanadate were used as the source of the ERT kinase activity used for the purification reported here. The effect of orthovanadate to stimulate ERT kinase activity suggests that increased tyrosine phosphorylation may be relevant to the mechanism of activation of this enzyme. This is consistent with the action of several growth factor receptors with intrinsic protein-tyrosine kinase activity to increase ERT kinase activity (13, 16). Whether the ERT kinase is a physiological target of tyrosine phosphorylation is not clear. However, the requirement for the use of the phosphatase inhibitors (orthovanadate and pyrophosphate) during homogenization to stabilize ERT kinase activity suggests that phosphorylation of the enzyme may be relevant to the control of ERT kinase activity. Preliminary evidence indicates that the ERT kinase is inactivated by serine/threonine phosphatase type 2A.7 Thus, by analogy with the MAP2 kinase (30, 31), the ERT kinase could be regulated by both tyrosine as

F. A. Gonzalez, S. Jaspers, T. B. Miller, and R. J. Davis, unpub- lished observation.

well as serine/threonine phosphorylation. Substantial further work will be required to establish the mechanism of regulation of the ERT kinase.

Characterization of Substrate Specificity of ERT Kinase

An interesting aspect of the properties of the purified ERT kinase is the observation of a restricted substrate specificity. Thus, ATP was observed to be a good substrate for the ERT kinase, but little phosphorylation was observed in experiments using GTP as a substrate (Fig. 9). A similar restriction was observed in experiments using synthetic peptide substrates (Table 11). In an analysis of 15 peptides that have been characterized as substrates for protein kinases, significant phosphorylation was only detected for three substrate peptides: 1) the Thr'j6' synthetic peptide (13, 16) that is based on the primary sequence of the EGF receptor (KRELVEP- LT'j6'PSGEAPNQALLR) was phosphorylated. 2) A synthetic peptide based on the sequence of glycogen synthase surrounding sites 3 (32, 33) was phosphorylated by the ERT kinase.'j The rate of phosphorylation was -10% of the rate measured for the Thr'j6' synthetic peptide (Table 11). 3) A synthetic peptide based on the sequence surrounding Thrg7 in MBP was phosphorylated by the ERT kinase. The rate of phosphorylation of the MBP peptide was -8% of the rate of phosphorylation of the Thr6'j9 synthetic peptide by the ERT kinase (Table 11).

A similarity between these three substrates is the presence of proline residues in the primary sequence close to the sites of peptide phosphorylation. It is therefore possible that proline is an important determinant for substrate recognition by the ERT kinase.

Comparison of ERT Kinase with Other Protein Kinase Activities

The distinctive feature of the site of phosphorylation of the EGF receptor at Thr'j'j' is the proximity of 2 proline residues (=-Le~-Thr~~'-@). A comparison of the purified ERT kinase with other enzymes that have been found to contain a proline residue within their substrate recognition site is therefore warranted.

Glycogen Synthase Kinase-3"This protein kinase phosphorylates glycogen synthase at sites 3 (28, 32, 33). A peptide containing sites 3 of glycogen synthase was found to be phosphorylated by the ERT kinase (Table 11). This observation suggests that glycogen synthase kinase-3 and the ERT kinase may be similar protein kinases. However, there are three marked differences between the properties of these enzymes. First, the synthetic peptide containing sites 3 of glycogen synthase that was found to be a substrate for the ERT kinase (Table TI) is not a substrate for glycogen synthase kinase-3 (28, 32, 33). This is because the synthetic peptide lacks the site of phosphorylation by casein kinase I1 that is required for substrate recognition by glycogen synthase kinase-3 (28, 32, 33). The substrate specificities of the ERT kinase and glycogen synthase kinase-3 are therefore distinct. Second, glycogen synthase kinase-3 does not bind to DEAE columns and is eluted in the flow-through fractions (28, 32). In contrast, the ERT kinase binds efficiently to DEAE (data not shown), Mono-Q, and Mono-P columns (Figs. 1 and 4). Third, the ERT kinase is stimulated by treating cultured cells with EGF, platelet-derived growth factor, serum, or phorbol ester (13, 16). However, we are not aware of any reports of the stimulation of the activity of glycogen synthase kinase-3 by these agents. Together, these data indicate that glycogen synthase kinase-3 and the ERT kinase are distinct enzymes.

Proline-directed Protein Kinase-This protein kinase has

Phosphorylation of the

been identified as a nerve growth factor-stimulated activity that accounts for the phosphorylation of tyrosine hydroxylase at Ser" (29). Synthetic peptides based on the sequence of tyrosine hydroxylase and histone H1 have been reported to be phosphorylated by proline-directed protein kinase (29). However, these peptides were not found to be phosphorylated by the purified ERT kinase (Table 11). Thus, the substrate specificities of proline-directed protein kinase and the ERT kinase are distinct.

cdc2 Protein Kinase-ERT is acutely stimulated by the treatment of cultured cells with growth factors (13, 16). In contrast, the cdc2 protein kinase is active in mitotic cells (27). It is therefore unlikely that the cdc2 protein kinase (34 kDa) can account for ERT kinase activity. Further evidence to support this conclusion is provided by the analysis of the substrate specificities of these protein kinases. A synthetic peptide based on the sequence of histone H1 (AKAKKTPKKAL) has been reported to be a substrate for the cdc2 protein kinase (27). This synthetic peptide was not found to be a substrate for the purified ERT kinase (Table 11). We therefore conclude that the cdc2 protein kinase is distinct from the ERT kinase.

MAP2 Kinase-There is strong evidence demonstrating that the ERT kinase is distinct from the MAP2 kinase. This is because the MAP2 kinase (21,26) can be resolved from the ERT kinase by chromatography on Mono-Q (Fig. l), phenyl- Superose (Fig. 3), and gel filtration (Fig. 6) columns. However, although the ERT and MAP2 kinases are distinct, it is possible that they are related enzymes.

Recently, the molecular cloning of the rat MAP2 kinase (ERK-I ) has been achieved by Boulton et al. (34). Two additional cDNA clones (ERK-2 and ERK-3) were also isolated that correspond to enzymes that are homologous to the MAP2 kinase (34). The existence of a subfamily of MAPB- related kinases is consistent with previously reported biochemical evidence (23, 31, 35-42). In the context of this information, it is significant therefore that there are marked similarities between the substrate specificities of the ERT and MAP2 kinases. (Two examples of substrates that are phosphorylated by both enzymes are the EGF receptor at T h P 9 and MBP at Thrg7.) This similarity in substrate specificity suggests that the MAP2 and ERT kinases may be members of a subfamily of related protein kinases.' Further progress toward the identification of the relationships between these enzymes will require the molecular cloning of the ERT kinase.

Conclusions

The growth factor-stimulated phosphorylation of the EGF receptor at Thra9 can be accounted for by an increase in the activity of two distinct protein kinases: the MAP2 kinase and the ERT kinase. An important goal for further studies will be to establish the function of these enzymes during signal transduction by growth factor receptors.

Acknowledgments-Sharon Bowen, Erica Selva, and Nuria Girongs are thanked for assistance with the isolation and characterization of Chinese hamster ovary cells expressing [Arg7*']EGF receptors. Dr. T. Hunter and J. Meisenhelder (Salk Institute) are thanked for providing the monoclonal antibody M108.1. We thank Dr. T. W. Sturgill for the gift of the myelin basic protein peptide and MAP2 kinase

" If the ERT kinase is a MAPS-related kinase, it is likely that it is most closely related to protein kinase activities that have previously been described as: 1) MBP kinase I1 (31, 36-39) because of the similarity in the elution profile from a Mono-Q column or 2) a nerve growth factor-stimulated MAP2-related kinase (42) that has a similar molecular mass and a similar elution profile during chromatofocusing.

EGF Receptor at Thr6'j9 15275

purified from Swiss 3T3 cells. Dr. G. Johnson is thanked for providing synthetic peptides. The excellent secretarial assistance of Kim Gre- goire and Margaret Shepard is greatly appreciated.

Note Added in Proof-Recent progress toward the characterization of MAP-related kinases by cDNA cloning indicates that the MAP2 kinase corresponds to ERK-2 and that ERT may correspond to ERK- 1 or ERK-4 (Boulton, T. G., Nye, S. H., Robbins, D. J., Ip, N. Y., Radziejewska, E., Morgenbesser, S. D., DePinho, R. A., Panayotatos, N., Cobb, M. H., and Yancopoulos, G. D. (1991) Cell 65, 663-675; Boulton, T. G., and Cobb, M. H. (1991) Cell Regul. 2,357-371). The conclusion that EGF receptor Thr6'j9 is a substrate for phosphorylation by the MAP2 kinase has recently been reported (Takishima, K., Griswold-Prenner, I., Ingebritsen, T., and Rosner, M. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2520-2524).

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