MOLECULAR AND CELLULAR BIOLOGY, Aug. 1993, p. 4679-4690 Vol. 13, No. 8 0270-7306/93/084679-12$02.00/0

Molecular Cloning, Expression, and Characterization of the Human Mitogen-Activated Kinase p44ekl DAVID L. CHAREST 1,2 GUY MORDRET,1'2 KENNETH W. HARDER,1 FRANK JIRIK,1 AND STEVEN L. PELECH .2* Biomedical Research Centre and Department ofMedicine, University ofBritish Columbia, Vancouver, British Columbia, Canada V6T IZ3,1 and Kinetek Biotechnology Corporation, 7600 No. 1 Road, Richmond, British Columbia, Canada V7C 1T62 Received 7 August 1991/Returned for modification 11 November 1991/Accepted 18 May 1993

p44ek is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erkl cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erkl cDNA clone shared -96% predicted amino acid identity with partial sequences of rodent erkl cognates, and the erkl gene was assigned to human chromosome 16 by hybrid panel analysis. Human erkl expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erkl) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphory- lation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erkl protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotrans- ferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein- tyrosine kinase p56kk catalyzed phosphorylation of GST-Erkl at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erkl was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erkl. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erkl in which Lys-71 was converted to Ala by site-directed mutagenesis.

The mitogen-activated protein (MAP) kinases (also re- implies the existence of specialized functions for the distinct ferred to as extracellular signal-regulated kinases, or ERKs) isoforms or their differential regulation. are shared components in the intracellular signal transduc- Immunoblotting studies with antibodies generated against tion pathways of very diverse extracellular mediators (for MAP kinase peptides have revealed additional, potentially recent reviews, see references 2, 4, 14, 49, 50, 73, 75). The related MAP kinase isoforms in extracts from sea star, frog, first MAP kinase to be reported was a 42-kDa kinase avian, and mammalian cells (5, 63, 64). One of these is a (p42'aPk, also known as ERK2) that exhibited increased human 40-kDa MBP kinase that appears to physically asso- phosphotransferase activity toward microtubule-associated ciate with the human epidermal growth factor receptor (64). protein 2 (MAP-2) following mitogenic stimulation of quies- Another is a 46-kDa protein that has been tentatively desig- cent (Go) mammalian cells with insulin or epidermal growth nated p46e"*4 (5). Kyriakis and Avruch (35) have reported the factor (31, 54). A second MAP kinase was identified as a purification of an activated 54-kDa MAP-2 kinase from the myelin basic protein (MBP) kinase (p44PPk) that underwent livers of cycloheximide-treated rats. This kinase exhibits activation during meiotic maturation of sea star oocytes (51). -50% amino acid identity with rat p42maPk within its cata- Subsequently, an additional insulin-activated MAP kinase lytic domain (3a). Furthermore, 40- to 46-kDa MAP kinase- (p44") from a rat hepatoma cell line was purified and like with about 46 to 51% amino acid identity with characterized (6). cDNA sequence data for rat p42naPk and rat p44e?*l have been uncovered by genetic studies with p44cr*l revealed -90% amino acid identity between these yeast cells. These kinases, encoded by the Saccharomyces MAP kinase isoforms (7) and -70% amino acid identity with cerevisiae FUS3 (19) and KSSJ (15) genes and the partial sequences of sea star p44""k obtained by protein Schizosaccharomyces pombe spkl (76) gene, have been microsequencing (53). Enzymological analyses of highly implicated in the pheromonal control of yeast mating. Other purified preparations of p42mapk,p44ek*l, and p44mPk from MAP kinase homologs encoded by the MPKJ (39) and various sources also supports their relatedness (6, 26, 55, HOGI (10) genes appear to function in additional signal 65). However, there is a high degree of conservation of the transduction pathways in S. cerevisiae. With such a diver- primary structures for p42mnaPk (,95% amino acid identity sity of MAP kinase homologs that appear to be regulated between human [24, 47], rat [7], mouse [30], and frog [25, independently, subtle differences in their activation require- 53]) and p44erk (296% amino acid identity between human ments are anticipated. [24, 47], rat [7, 41], mouse [16], and guinea pig [43]). This Mammalian and frog MAP kinases are activated by -45- kDa kinases that catalyze their tyrosyl and threonyl phos- phorylation (18, 37, 42, 45, 59, 67, 69). cDNA sequencing studies have revealed that these vertebrate kinases are * Corresponding author. highly related to the yeast BYRI- and STE7-encoded protein 4679 4680 CHAREST ET AL. MOL. CELL. BIOL. kinases, which are required for pheromone-induced mating jected to PCR with the Perkin-Elmer Cetus ampliTaq DNA (3, 17, 33, 68, 78). Genetic and biochemical studies with S. polymerase and thermal cycler. The PCR reaction was cerevisiae have indicated that p56ste7 acts upstream of the performed for three cycles at low primer-annealling temper- MAP kinase p40t'3 in pheromone signalling (20, 23, 74). atures (30 s at 95°C, 60 s at 37°C, and 120 s at 72°C) and then The two key regulatory phosphorylation sites in p42maPk at a higher primer-annealling temperatures for 35 cycles (30 (ERK2) have been identified as neighboring threonyl and s at 95°C, 90 s at 55°C, and 120 s at 72°C). A final elongation tyrosyl residues; Thr-183 and Tyr-185 in mouse p42maPk (37, step (5 min at 72°C) ensured the synthesis of complete 48) and Thr-188 and 1yr-190 in the frog cognate (52). The double-stranded fragments. An aliquot of the reaction was equivalent sites were also phosphorylated within a nearly electrophoresed through a 1% low-melting-point agarose gel, full-length mouse recombinant glutathione S-transferase- and the bands were visualized by ethidium bromide staining Erkl (GST-Erkl) fusion protein that was incubated with a before excision and purification (Bio 101 Geneclean kit). The murine 45-kDa MAP kinase kinase (1). Phosphorylation of purified fragment was blunt end ligated into EcoRV restric- these sites, located just upstream of the conserved protein tion endonuclease-digested Bluescript plasmid (Stratagene) kinase subdomain VIII region implicated in substrate bind- and sequenced by the dideoxynucleotide chain termination ing (27), might function by relieving inhibition of substrate method (60) with a Pharmacia sequencing kit. The partial access to the active site of these MAP kinases. Sea star erkl clone (A431-400-8) thus identified was excised from the p44mPk is activated in vivo in concert with its tyrosyl and Bluescript plasmid and used to probe a Hep G2 cDNA X seryl phosphorylation (61, 62), but it appears to lack the ZAP library from Stratagene Cloning Systems by standard regulatory threonyl phosphorylation site (53). This MAP procedures (40). Double-positive phage clones were en- kinase isoform could also be stimulated in vitro by the riched, isolated, and then rescued as Bluescript plasmids murine protein-tyrosine kinase p561ck in the absence of from the phage vector according to the manufacturer's detectable threonyl phosphorylation (22). Finally, in the instructions (Stratagene). DNA sequencing of both sense putative MAP kinase encoded by the rat erk3 gene, the and antisense strands was performed on double-stranded regulatory threonyl and tyrosyl phosphorylation sites have plasmid DNAs with a combination of cDNA fragment sub- not been conserved (7). Consequently, the various MAP cloning, T7 and T3 primers (Stratagene), as well as erkl- kinase isoforms may differ markedly in their requirements specific oligonucleotide primers (synthesized on an Applied for phosphorylation-dependent activation. Biosystems DNA Synthesizer model 300A). Because of the Here, we report the complete amino acid sequence of the difficulties associated with sequencing GC-rich regions and human homolog of the erkl-encoded protein kinase, the secondary structure, both T7 DNA polymerase (Pharmacia) incomplete sequence of which has been previously published and Sequenase 2.0 (United States Biochemical) sequencing (24, 47). The human erkl protein was expressed in bacteria kits were used. and characterized with respect to its enzymatic activity, its Nucleotide and predicted amino acid sequence searches of immunoreactivity with MAP kinase-specific antibodies, and GenBank, EMBL and Swiss data banks were kindly per- its potential regulation by other protein kinases. formed by Allen Delaney on the Sun UNIX system with (A preliminary account of these findings has been pre- locally developed software (A. Delaney, Biomedical Re- sented previously [11].) search Centre). MAP kinase comparisons were performed with Justin Hein's (29) software Align. MATERIALS AND METHODS In vitro mutagenesis. The regulatory tyrosine at residue 204 and lysine at residue 71 in Erkl were separately altered Cell culture and RNA isolation. Two human cell lines were according to the procedure of 0 et al. (46). The oligonucle- used, A431 and HepG2 (American Type Culture Collection). otide used to mutagenize the tyrosine to a glutamic acid A431 cells were maintained in Dulbecco's modified Eagle contained two base changes (sense strand, GGC TTC CTG medium (5% calf serum, 5% fetal bovine serum and 5 mM ACG GAG GAGi GTG GCT ACG CGC TGG). The oligonu- 2-mercaptoethanol), while the Hep G2 cells were cultured in cleotide used to mutagenize the lysine to an alanine con- Eagle minimal essential medium (10% fetal bovine serum, tained two base changes (sense strand, ACT CGC GTG GCC Earle's salts, and 5 mM 2-mercaptoethanol). Total cellular ATC GCG AAG ATC AGC CCC TCC). RNA was isolated from approximately 108 cells by acid Chromosomal assignment. Nylon membranes containing guanidinium thiocyanate-phenol-chloroform extraction (12). panels of HindIII-digested hamster-human hybrid cell line Cloning of human erkl. Approximately 1 ,g of total RNA DNA (BIOS Corporation) were probed with the partial from A431 cells was used as a template for cDNA synthesis. A431-400-8 erkl cDNA clone to obtain the chromosomal The Superscript reverse transcriptase (Bethesda Research localization of the human erkl gene. After processing ac- Laboratories) reaction with nucleotide triphosphates was cording to manufacturer's specifications, the membranes carried out in a total volume of 20 RI of polymerase chain were exposed to autoradiography film at -70°C for 7 days by reaction (PCR) buffer A (50 mM KCl; 10 mM Tris-Cl, pH 8.3 using image-intensifying screens. at 20°C; 1.5 mM MgCl2; 0.01% gelatin) (10 min at 23°C, and Bacterial expression of human erkl. To clone erkl in the then 45 min at 42°C). The potential degeneracy of erkl- pGEX-2T expression vector (Pharmacia), the Bluescript/ specific coding and complementary oligonucleotides (sense ERK-1 recombinant plasmid was used as a template to strand of subdomain II, GTG GCT/C ATC AAG AAG ATC amplify the human erkl by PCR. Two oligo- AGC CCC TTC GAG CAT; antisense strand of subdomain nucleotides with EcoRI restriction sites based on 5' end VII, CTC AGG GTC AGC AAT CCG GGC AAG GCC coding region (sense strand, GA CTC GAA TTC CC ATG G/AAA G/ATC; and antisense strand of subdomain IX, GCG GCG GCG GCG GCT CAG GGG) and on the 3' end GCA GCC CAC AGA CCA GAT GTC A/GAT GGA T/CTll coding region (antisense strand, CT GAG GAA TTC CTA GGT GTA) was reduced by codon selection on the basis of GGG GGC CTC CAG CAC TCC GGG CTG GAA) were human codon utilization (38). A paired combination of sense combined with 1 ,ig of erkl cDNA template, nucleotide and antisense oligonucleotides were added to the single- triphosphates, Vent PCR buffer B [10 mM KCl, 10 mM strand cDNA reaction mixture, and the mixture was sub- (NH4)2SO4, 20mM Tris-HCl (pH 8.8 at 25°C), 2 mM MgSO4, VOL. 13, 1993 CHARACTERIZATION OF HUMAN p44er* 4681

0.1% Triton X-100, 100 ,ug of bovine serum albumin per ml) performed with purified recombinant erkl protein (-1 ,ug), in a final volume of 50 ,ul, and the mixture was subjected to 50 mM [y-32P]ATP (9,000 cpm/pmol), and kinase assay PCR with 10 U of Vent DNA Polymerase (New England buffer E (25 mM sodium 3-glycerophosphate, 20 mM MOPS Biolabs) and a Perkin-Elmer Cetus thermal cycler. The PCR [pH 7.2], 10 mM MnCl2 and/or 10 mM MgCl2, 2 mM NaF, 1 reaction was performed for 30 cycles (45 s at 95°C, 90 s at mM dithiothreitol, 1 mM sodium vanadate, 10 ,ug of aproti- 55°C, and 120 s at 72°C). The reaction mixture was electro- nin per ml, 10 ,ug of soybean trypsin inhibitor per ml, and 5 phoresed through a 1% low-melting-point agarose gel, and ,ug of leupeptin per ml) for 30 min in a final volume of 40 ,ul. the expected 1.2-kb amplified band stained with ethidium The reaction was terminated by addition of 30 ,ul of 4 x bromide was excised, purified (Sephaglass Bandprep Kit, concentrated sodium dodecyl sulfate (SDS) sample buffer F Pharmacia), digested with EcoRI, and subcloned into pGEX (125 mM Tris-HCI [pH 6.8], 4% SDS, 0.01% bromophenol 2T. SmaI restriction endonuclease digests were performed blue, 10% mercaptoethanol, and 20% glycerol) and boiling for verification of the proper orientation. for 5 min. Following SDS-polyacrylamide gel electrophore- The GST-Erkl fusion protein was produced in Escherichia sis (36), the were transferred onto a polyvinyl coli and purified by glutathione-agarose chromatography by difluoride membrane (Millipore). For phosphorylation of the method of Smith and Johnson (70). The cleaved protein MBP and MBP peptides, the incubations contained -0.8 ,ug was produced by incubation of the affinity-purified GST- of recombinant Erkl protein, 1 mg of MBP per ml or 2 mM Erkl immobilized on the glutathione-agarose beads with 40 MBP peptide, and 50 p,M [y-32P]ATP (-2,000 cpm/pmol) in U of thrombin in TEC buffer C (50 mM Tris-HCl [pH 8.0], the kinase assay buffer. The other details of the MAP kinase 150 mM NaCl, 6 mM CaCl2) at 25°C for 1 h. Subsequently, assay have been described (65). Protein was measured by the the beads were washed three times with TEC buffer C, and method of Bradford (9) with bovine serum albumin as a the supernatants were retained. The combined supernatants standard. were diluted (1:1) with buffer D (20 mM) morpholinepro- Purified GST-Erkl, A71-GST-Erkl, E204-GST-Erkl, or panesulfonic acid [MOPS, pH 7.2], 25 mM 3-glycerophos- p44erkl (1 to 3 ,ug) was incubated for 15 to 30 min at 30°C in phate, 10 mM ethylene glycol-bis(3-aminoethyl ether)- the absence or presence of various other kinases (-2.5 ,ug of N,N,N',N'-tetraacetic acid [EGTA], 2 mM EDTA, 100 ,uM p60c-src, p65t/Vabl or p56"k or -20 ,ug of partially purified sodium orthovanadate, 1 mM NaF, 1 mM dithiothreitol) and sheep MAP kinase kinase) and 50 ,uM [.y-32P]ATP (9,000 concentrated by chromatography on a MonoQ column. The cpm/pmol) in a final volume of 80 ,ul. The reactions were purified (95%) recombinant Erkl protein eluted between 160 terminated with 30 p,l of 4x SDS sample buffer F, and the and 300 mM NaCl. The peak fractions were pooled and mixture was boiled for 5 min prior to SDS-polyacrylamide stored at -70°C. gel electrophoresis. The electrophoresed proteins were Antibodies, protein kinases, phosphatases, and peptides. transferred for 3 h at 400 mA in transfer buffer G (25 mM The following rabbit polyclonal antibodies were prepared as Tris, 192 mM glycine, 20% methanol) onto a polyvinyl previously described: affinity-purified anti-p44mt (62), anti- difluoride membrane (Millipore). MAP kinase bands visual- mpk-I against sea star p44mPk subdomain I sequence GLAY ized by autoradiography were excised for counting or phos- IGEGAYGMV(C) (referred to as anti-GEGA in reference phoamino acid analysis. 53), affinity-purified anti-erkl-III against rat p44erkl subdo- Electrophoresis. SDS-polyacrylamide gel electrophoresis main III sequence PFEHQTYCQRTLREIQILLGFRHEN was performed on 1.5-mm-thick gels by using the buffer VIGIRDILRAP(GGC) (63), and affinity-purified anti-erkl- system described by Laemmli (36). An 11% separating gel CT against rat p44er* C terminus sequence (CGG)PFTFD and a 4% stacking gel were used. Samples were boiled for 5 MELDDLPKERLKELIFQETARFQPGAPEAP (63). Affin- min upon addition of 4x-concentrated SDS sample buffer F ity-purified rabbit polyclonal erkl-NT antibodies were also and electrophoresed for 17 h at 10 mA. The separating gel produced against the human p44erk N-terminal sequence was silver stained by the method of Merril et al. (44). EPRRTEGVGPGVPGEV by the procedures outlined else- Two-dimensional peptide mapping was performed after where (63). The erkl-NT, erkl-III and erkl-CT antibodies excision of radiolabelled Erkl proteins from dried SDS- are now commercially available through Upstate Biotechnol- polyacrylamide gels. The gel slice containing the Erkl ogy Inc., as is the antiphosphotyrosine mouse monoclonal protein was reswollen in a methanol-acetic acid solution, antibody 4G10. Immunoblotting with these antibodies was washed in 50% methanol several times, and dried again. The performed as described previously (63). gel was incubated with solution of 50 mM ammonium p44mPk was purified to near homogeneity from maturing bicarbonate containing 100 jig of trypsin (tolylsulfonyl phe- sea star oocytes (65). Purified recombinant murine p56" nylalanyl chloromethyl ketone [TPCK] treated; Sigma) at from Sf-9 cells was generously provided by J. Watt and R. 37°C for 12 h, and then the supernatant was dried in a Speedy Aebersold (Biomedical Research Centre). Murine recombi- Vac. The tryptic peptides were washed three times with nant bacterially expressed p65t/,bl and purified human distilled water, once with the first-dimension buffer, and platelet p60csPC were gifts from Jean Wang (University of finally resuspended in 2 ml of the same buffer plus 0.5 ml of California, La Jolla) and Fred Hall (University of California, 0.5% phenol red and spotted on a cellulose plate (Merck). Los Angeles), respectively. Purified CD45 protein tyrosine Electrophoresis in the first dimension was performed at 750 phosphatase from human spleen was provided by Nicholas V in 10% acetic acid-1% pyridine, and thin-layer chroma- Tonks (Cold Spring Harbor Laboratory), and partially puri- tography was carried out in the second dimension in 1-buta- fied protein phosphatase 2A from human erythrocytes was nol-acetic acid-water-pyridine (10:3:12:15, by volume). donated by David Brautigan (Brown University, Provi- Phosphoamino acid analysis of phosphorylated GST-Erkl dence, R.I.). and p44e1*l was performed by digestion of the appropriate All of the short polypeptides used in this study were radiolabelled protein band in 300 ,ul of constant boiling HCl provided by the laboratory of I. Clark-Lewis (Biomedical at 105°C for 1 h. The acid-hydrolyzed sample was dried Research Centre). The MBP Thr-97 site peptides have been under vacuum (Speedy Vac), washed with water, redried, described previously (13). and then resuspended in 5 pl of electrophoresis buffer Phosphorylation experiments. Autophosphorylation was containing pyridine-acetic acid-water (1:10:189, vol/vol/vol). 4682 CHAREST ET AL. MOL. CELL. BIOL. Phosphoserine, phosphothreonine, and phosphotyrosine were isolated and rescued as plasmids, designated p26ap-3, standards (1 ,ug each) were electrophoresed alongside the was selected for sequencing, as the 1.9-kb insert corre- sample. Electrophoresis was performed for 45 to 60 min at sponded in size to the full-length mRNA from rat. 1,000 V with cooling. The p26a3-3 insert cDNA was 1,850 bp in length, exclud- Partial purification of sheep platelet MAP kinase kinase. ing the origin of the poly (A)' region, and was predicted to About 3 mg of cytosolic protein extract from untreated sheep encode a single 1,136-bp open reading frame. A polyadenyl- platelets or platelets exposed to 200 nM phorbol myristate ation signal (AUAAA) preceded the poly (A)' tail. The 5' acetate (PMA) for 5 min at 30°C were loaded on a MonoS nucleic acid and complete predicted amino acid sequences of (HR 5/5, 1 ml) column after dilution (1:5) in buffer H (20 mM the p26a,B-3 insert are shown in Fig. 1. The complete cDNA morpholineethanesulfonic acid [MES, pH 6.5] plus compo- sequence is identical in the region of overlap with the partial nents of buffer D except MOPS). Proteins were eluted with cDNA clones for human erkl that have been reported (24, a 0 to 0.8 M NaCl linear gradient. Fresh glutathione-agarose 47). Alignment of rat and human erkl sequences revealed 10 beads carrying GST-Erkl were prepared the same way as amino acid differences in the region of overlap, yielding an the purification of the fusion protein, except that they were overall identity of approximately 96% between these two washed and equilibrated with buffer H. The MAP kinase species (Fig. 1B). One of these residues, leucine 228 in rat kinase assay was performed as follows: 20 ,l of GST-Erkl erkl, is deleted from human erkl and from mapk in Xenopus beads was preincubated with 40 pl of the MonoS fractions species and rodents (Fig. 1B). and 50 ,uM of unlabelled ATP in a final volume of 70 ,ul. The As the partial cDNA sequences reported for human erkl beads were then washed three times with buffer D containing (24, 47) lacked both the initiating AUG and 5' untranslated 0.1% Triton X-100. Finally, the beads were incubated for 20 sequences, it was important to establish that the erkl cDNA min at 30°C with 1 mg of MBP per ml, 50 ,M [-y-32P]ATP that was isolated did in fact encompass the complete protein (2,000 cpm/pmol), and kinase assay buffer E in a total coding sequence. Two possible AUG codons at nucleotides volume of 50 ,lI. At the end of the assay, 25 ,ul of the 73 and 160, respectively, were identified. Both satisfy crite- supernatant was applied to 1.5-cm2 pieces of phosphocellu- ria identified (34) for efficient translation initiation. Interest- lose P81 paper (Whatman), which were washed extensively ingly, both AUG sites share nucleotide identity at position with 1% phosphoric acid prior to scintillation counting. +4 as well as in positions -1 through -6 with respect to the Nucleotide sequence accession number. The accession num- AUG, raising the possibility that either site might be func- ber given the human erkl cDNA sequence from the EMBL tional. Data Library is X60188. As no termination codons were found in the first 72 nucleotides of the cDNA 5' to the first AUG, the possibility of an even more 5' initiation site remained. However, the RESULTS AND DISCUSSION results of primer extension analysis make this unlikely. An Oligonucleotide design for PCR and amplification of a end-labelled 26-base oligonucleotide primer that corre- partial cDNA encoding a MAP kinase. p44er* was originally sponded to cDNA sequence coordinates 148 to 174 was used isolated as an activated MAP-2 kinase from insulin-treated in a primer extension reaction on Hep G2 mRNA. This Rat 1 (HIRc B) cells that were transfected with human procedure resulted in the identification of a single-stranded insulin receptor (6). To clone the cDNA for the human cDNA species of approximately 185 bases in length (data not homolog of p44erkl, it was necessary to identify cell lines in shown). This was approximately 10 bases beyond the ex- which this MAP kinase isoform was expressed. We have pected size as predicted by the erkl cDNA that was isolated. described the presence of p42maPk and a p44ef*l-ike protein However, an initiating AUG located within 10 bases of the 5' in the human epidermoid carcinoma A431 cell line on the cap is very rarely seen, as a longer stretch of 5' untranslated basis of their immunoreactivity with MAP kinase antibodies sequence appears to be required for efficient ribosome (64). Similarly, we detected p44e!*' as an insulin-activated binding and translation initiation (34). The primer extension MBP kinase in the human hepatocellular carcinoma line Hep results also suggested the presence of a fixed site for G2 (data not shown). translational initiation in the Hep G2 cells, as no heteroge- PCR was exploited for the cloning of a partial cDNA for neity in the primer extension products was observed. The human erkl from RNA derived from A431 cells. Three predicted molecular mass of the 379-amino-acid-residue pro- segments within the conserved kinase catalytic subdomains tein encoded by the human erkl cDNA with the AUG codon (i.e., II, VII, and IX [27]) of the rat erkl sequence were at nucleotide 73 was 43 kDa. Antipeptide antibodies de- selected for the design of oligonucleotide primers (8). Am- signed to recognize the C terminus of rat erkl were found to plifications conducted with two pairs of these primers immunoblot a 43-kDa protein in insulin-stimulated Hep G2 yielded bands of 400 and 500 bp, respectively, corresponding cells (data not shown). to the distance separating these primers in the rat erkl Further support for the assignment of cDNA nucleotide 73 nucleic acid sequence (8). The predicted translated sequence AUG as the correct initiation site for human erkl can be of these two PCR fragments revealed only two amino acid inferred from studies of other MAP kinase isoforms. The differences compared with the same intervals in rat erkl first six amino acid residues (MAAAAA) originating with the (data not shown). putative initiating methionyl residue of human erkl were Isolation and sequencing of a full-length cDNA putatively identical to the predicted N-terminal sequences of the mapk encoding the human erki protein. A probe consisting of the isoform from mice, rats, and Xenopus species (Fig. 1B). putative human erkl 500-bp fragment isolated by PCR was Also, Marquardt and Stabel (41) have recently inferred a utilized to screen -300,000 plaques of a bacteriophage similar, but not identical, initiating sequence for rat erkl lambda ZAP cDNA library prepared from Hep G2 cell (i.e., NAAAAAAP). The occurrence of five or more con- poly(A)+ RNA. Initial low-stringency screening yielded 26 secutive alanyl residues is rare outside the MAP kinase recombinants, only 4 of which were able to bind the 32p_ family, although it has been described for the acidic 80- to labelled partial erkl cDNA probe under more stringent 87-kDa protein kinase C substrate MARCKS isolated from conditions on further screening. One of the four clones that bovine (72) and rat (21) brain. Interestingly, Northern (RNA) VOL. 13, 1993 CHARACTERIZATION OF HUMAN p44e?* 4683

A GGG GGGlGOI GGAG C1C GCA GCG GGA GTG GAG ATG GCG GCG GCG GCG GCT CAG OGG GGC GGG GGC GGG GAG CCC 2

CGT AGA ACC GAG GGG GTC GGC CCG GGG GTC CCG GGG GAG GTG GAG ATG GTG AGG GGG B hu,man ERK1 rat erkl rat erklV mouse/rat mapk Xenopus mapka Xenopug mapk human ERK1 rat erkl rat erklV mouse/rat mapk xenopue mapka Xenopus mapk( human ERK1 rat erkl rat erk1V mouse/rat mapk Xenopus mapkoz xenopus mapkj human BRK1 rat erkl mouse/rat mapk xenopus mapka Xenopua mapkI d:k.. ,&-': 379 human ERK2 NK.KIK-17- m rat erkl 369 mouse/rat mapk 358 Xenopus mapka 361 Xenopus mapkj3 361 FIG. 1. (A) Nucleotide sequence and predicted N-terminal amino acid sequence of human erkl; (B) aligned sequences of human erkl, rat erkl, rat erk1 pseudo gene (n), rat and mouse mapk, and Xenopus mapkot and mapkl. Residues that are identical with human erkl are shaded; dashes indicate spaces introduced to improve sequence alignments; residues that are identical or conserved in at least 62 of a collection of 65 protein kinase sequences are shown in white; putative threonyl and tyrosyl phosphorylation sites are asterisked. Alignments were generated with J. Hein's (29) program Align.

blot and Western blotting (immunoblotting) analyses have panel, the human erkl gene was localized to chromosome 16 also revealed high levels of p44erl in rat brain (5, 7). (data not shown). No discordances were observed. Following the N terminus polyalanyl stretch, p44e"l fea- Expression and immunological detection of a GST-Erkl tures five consecutive glycyl residues. Such a string of glycyl fuision protein. We undertook the expression of full-length residues has also been described in the sequence of the human erkl as a GST fusion protein by using the pGEX-2T protein kinase encoded by the human rafB gene (71), but its vector, which also introduced a thrombin cleavage site functional significance is unclear. As can be seen in Fig. 1B, immediately upstream of the erkl sequence. Crews et al. (16) the chief difference between p44erkl and p42maPk is the previously reported that nearly full-length mouse erkl ex- presence of an insert of 14 to 19 residues near the N terminus pressed as a GST fusion protein by using the pGEX-3X of p44erkl. The second in-frame translational initiation site of expression vector was quite active as a kinase both before human erkl aligns precisely with the methionine start posi- and after cleavage of the GST portion with factor Xa. tions of p4Q't3 (19) and p43ksl (15) from S. cerevisiae. This Expression of the recombinant human GST-Erkl protein methionine is replaced by a valyl residue in rat p43erkl (8). in E. coli was monitored with a panel of antipeptide antibod- Both methionyl residues are also absent in a rat pseudo gene ies based upon the erkl unique region near the N terminus of with erkl sequences, which further lacks conserved ATP human erkl (anti-erkl-NT [Fig. 2A]), the kinase subdomain binding residues (7). Taken together, it is highly unlikely that III (anti-erkl-III [Fig. 2C]) and C terminus (anti-erkl-CT the second AUG is used to initiate translation of the p44ekl [Fig. 2D]) regions of rat erkl, and the kinase subdomain I mRNA. region of sea star p44mPk (a-mpk-I [Fig. 2B]), as well as by By using the partial human erkl 500-bp fragment as a antibodies raised against purified sea star p44mPk (Fig. 2E). probe and a commercially available human-hamster hybrid All of these antibodies detected a protein of the predicted 4684 CHAREST ET AL. MOL. CELL. BIOL.

1 2 3 4 5 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 A B C B -1 06K CD 1 -u 1 -L -80K GST-erkl - o r I NT at- m _ m -50K p44mPk- 2 3 4 1 2 3 4 0 C D _ -50K _.- I C 33K 48 M erkl -U - ---.n;-. . _erkl1 -L 28K 4. C I ------33K STO Mn2+ Mg2+ Mg2+ +Mn2+ anti-erkl -NT anti-mpk-l anti-erki-III 1 2 3 4 5 12 3 4 12 3 4

anti-erkl-CT anti-p44mPk anti-PY 4Gl 0 FIG. 2. Immunoreactivity of recombinant p44erkl to MAP kinase Mn2+ Mg2+ Mn2+ Mn2+ Mg2+ Mg2+ antibodies. Isopropyl-1-D-thiogalactopyranoside-induced bacterial GST-erkl erkl-U erkl-L erkl-U erkl-L lysates from Bluescript control plasmid (lane 2), lysates from FIG. 3. Autophosphorylation of recombinant p44erkl. Incubation isopropyl-p-D-thiogalactopyranoside-induced GST-Erkl plasmid of GST-Erkl (A and C) and its thrombin-cleaved Erkl products, (lane 3), 0.5 ,ug of purified sea star p44mPk (lane 4), and 1 ,ug of erkl-U (U = upper band) and erkl-L (L = lower band) (B and D), thrombin-cleaved and purified GST-Erkl (lane 5) were subjected to with [y- 2P]ATP (9,000 cpm/pmol) in the presence of either 10 mM SDS-polyacrylamide gel electrophoresis. Western blotting was per- MnCl2 (lanes 2) or 10 mM MgCl2 (lanes 3) separately or together formed with anti-erkl-NT antibodies (A), anti-mpk-I antibodies (B), (lanes 4). Autoradiograms are shown in panels A and B, and anti-erkl-III antibodies (C), anti-erkl-CT antibodies (D), anti- immunoblots with the antiphosphotyrosine antibody 4G10 are p44fPk antibodies (E), and antiphosphotyrosine monoclonal anti- shown in panels C and D. Phosphoamino acid analysis was per- body 4G10 (F). Lanes 1, migrations of the prestained marker formed on the excised bands of GST-Erkl (E) and its cleavage proteins: bovine serum albumin, ovalbumin, carbonic anhydrase, products (F) that were autophosphorylated in the presence of either soybean trypsin inhibitor, and lysozyme. Since the prestained Mn2+ or Mg2e. Positions of the prestained molecular weight stan- standard did not photograph well, the positions of the markers are dards are indicated with radioactive ink (A and B, lanes 1) and with also indicated with bars. bars (C and D, lanes 1). Migrations of the phosphoamino acid standards are indicated in panels E and F.

size (-70 kDa) in lysates of bacterial cells transfected with fluorescence localization studies of MAP kinases in avian the erkl-pGEX-2T construct (Fig. 2, lanes 3), but not in cells (63). lysates from control cells containing only the pGEX-2T Autophosphorylation of GST-Erkl and its thrombin cleav- vector (Fig. 2, lanes 2). Each of these antibodies also reacted age products. Immunoblotting analysis of the GST-Erkl with a 43-kDa thrombin cleavage product of the GST-Erkl protein with the monoclonal antiphosphotyrosine antibodies protein (i.e., p44erkl) (Fig. 2, lanes 5, and data not shown) PY-20 (data not shown) and 4G10 (Fig. 2F, lane 3) indicated and purified sea star p44mPk (Fig. 2, lanes 4). The strong that the recombinant protein expressed in E. coli was detection of sea star p44mPk with the anti-erkl-NT antibody substantially tyrosine phosphorylated. This appeared to be indicated that this kinase is more closely related to p44erkl comparable to the degree of tyrosine phosphorylation de- than p42maPk. tected with purified active p44mpk, since both proteins also All three of the antipeptide antibodies generated against displayed similar relative levels of immunoreactivity to all of erkl sequences were found to immunoprecipitate up to 30% the MAP kinase antibodies, save for anti-erkl-III, which of the recombinant p44erkl in bacterial lysates in the pres- poorly recognized p44mPk (Fig. 2). ence of 1% Triton X-100. Anti-erkl-CT was about twofold- In vitro autophosphorylation of GST-Erkl immobilized on more effective than anti-erkl-III or anti-erkl-NT in this glutathione-agarose beads was also demonstrated by radio- regard (data not shown). We have previously shown that labelling with [,y-32PJATP (Fig. 3A). Thrombin cleavage of anti-erkl-CT and anti-erkl-III are also useful for immuno- the GST-erkl and similar incubation with [-y-32P]ATP gener- VOL. 13, 1993 CHARACTERIZATION OF HUMAN p44erk 4685

ated two antiphosphotyrosine antibody-reactive proteins of phosphorylate at Tyr-185 and to yield only one tryptic approximately 43 kDa (erkl-L) and 44 kDa (erkl-U) (Fig. phosphopeptide (58). 3D). Both species were radiolabelled with [y- 2P]ATP (Fig. Robbins and Cobb (56) have detected serine, tyrosine, and 3B). For all three forms, the autophosphorylation reaction prominent threoninephosphorylation of p44erkl immunopre- was more effectively supported by Mn2+ than by Mg2+, and cipitated from either 32P-labelled nerve growth factor-treated both divalent metal cations together were no better than PC12 cells or insulin-treated Rat 1 HIRcB cells. Two- Mn2' alone (Fig. 3A and B). Threefold-more phosphate was dimensional tryptic peptide maps of the immunoprecipitated incorporated into erkl-L and erkl-U after a 1-h incubation at p44erkl showed as many as eight phosphopeptides. How- 30°C in the presence of 15 to 30 mM Mn2+ than in the ever, a caveat about the results of these experiments is that presence of comparable concentrations of Mg2'; half-maxi- the MAP kinase antibodies used may have also immunopre- mal activation of autophos horylation of both species was cipitated other related -44-kDa MAP kinases besides achieved with -8 mM Mn + and -7 mM Mg2+ (data not p44ek*l, for example, p46e?*4. One of these phosphopeptides shown). The maximum level of autophosphorylation as was threonine and tyrosine phosphorylated. Phosphopeptide measured by 32p incorporation into GST-Erkl and its throm- mapping of autophosphorylated recombinant p44erkl was not bin-cleaved products was only about 0.01 mol of P per mol. performed in that study, but Alessandrini et al. (1) obtained This is comparable to the stoichiometry of autophosphory- only one tryptic phosphopeptide which was tyrosine phos- lation (0.005 to 0.02 mol of P per mol) reported for E. phorylated with their recombinant murine GST-Erkl pro- coli-expressed rat p42maPk (57, 66). However, this additional tein. Our results with human GST-Erkl and the cleaved tyrosine phosphorylation does not take into account the in p44erkl revealed multiple tyrosine autophosphorylation sites vivo prephosphorylation of GST-Erkl, which was at least and at least one serine autophosphorylation site. Site-di- fivefold fgreater. This becomes evident when the incorpora- rected mutagenesis of Ser-39 in rat p42mPk by Robbins et al. tion of3 P into erkl-U and erkl-L in lanes 2 and 3 of Fig. 3B (57) has indicated that this generally conserved serine resi- are compared with the corresponding antiphosphotyrosine due in MAP kinases is unlikely to be the major serine immunoblots shown in Fig. 3D. Despite a very marked autophosphorylation site. increase in the autophosphorylation of these cleaved prod- MBP phosphotransferase activity of GST-Erkl. The GST- ucts from GST-Erkl in the presence of Mn2+ compared with Erkl and its thrombin-cleaved forms were found to exhibit a that in the presence of Mg2+, this difference was not relatively high level of MBP phosphotransferase activity, up apparent from the corresponding Western blots with the to -2 nmol min-1 mg of -'. This is comparable antiphosphotyrosine antibody, which both detected the in with the results of Crews et al. (162 with recombinant mouse vivo and in vitro tyrosine phosphorylation. GST-Erkl (i.e., -7 nmol. min- mg of enzyme-1). Al- Phosphoamino acid analysis of the in vitro-autophosphor- though this is generally somewhat higher than documented ylated GST-Erkl, erkl-U, and erk-L confirmed that each of for bacterially expressed recombinant p42maPk, i.e., 0.5 to these species were predominantly tyrosine phosphorylated 0.8 nmol. min- mg of enzyme-1 (66, 79), Robbins et al. (Fig. 3E and F). However, in the presence of Mn2+, GST- (57) reported a similar degree of activity for histidine-tagged Erkl and erkl-U, but not erkl-L, were also partially serine p42P`aPk, i.e., 1.7 nmol. min-' mg of enzyme-l. Greater phosphorylated, with minor traces of threonine phosphory- MBP phosphotransferase activity for p44e compared with lation. The serine phosphorylation of erkl-U and its absence that for p42maPk could have reflected a higher basal rate in from erkl-L might account for the mobility differences of the absence of phosphorylation. However, treatment of these species on the SDS-polyacrylamide gel. Our finding GST-Erkl with the tyrosine-specific protein phosphatase contrast those of Crews et al. (16), who found that recom- CD45 resulted in a 85% decrease in the MBP phosphotrans- binant mouse GST-Erkl autophosphorylated on both ty- ferase activity of GST-Erkl (Fig. 5A) concomitant with its rosine and threonine with minor (<5%) autophosphorylation tyrosine dephosphorylation (Fig. SC). By contrast, incuba- on serine by a similar analysis. Seger et al. (66) with purified tion of GST-Erkl with a preparation of human erythrocyte rat p44erkl failed to detect any phosphoserine by autophos- serine/threonine protein phosphatase 2A (composed of a phorylation. Similarly, recombinant mouse p42mP' ex- catalytic subunit and a 60-kDa regulatory subunit) had pressed in E. coli was predominantly tyrosine phosphory- essentially no effect on its kinase activity (Fig. SB). Under lated, with minor threonine phosphorylation and no serine the conditions employed in this study, the phosphatase 2A phosphorylation evident (28). Furthermore, histidine-tagged did not substantially dephosphorylate phosphotyrosine in forms of human p44erkl and rat p42m4BPk were also reported to GST-Erkl (Fig. SD), although in a control experiment the exhibit minor serine autophosphorylation compared with same preparation readily dephosphorylated a synthetic pep- tyrosine and threonine autophospho?rlation (57). We have tide with the sequence Lys-Arg-Thr(P)-Ile-Arg-Arg (data not previously noted that sea star p44mP also autophosphory- shown). In a previous study by Crews et al. (16), treatment lates on serine (to 0.7 mol of P per mol), without detectable of GST-Erkl with a higher level of phosphatase 2A inhibited threonine phosphorylation (62). its MBP phosphotransferase activity by 88%, but this was Two-dimensional tryptic phosphopeptide mapping of au- also accompanied by tyrosine dephosphorylation. Further tophosphorylated GST-erkl (Fig. 4A) and its thrombin autophosphorylation of GST-Erkl for up to 2 h in the cleavage products (Fig. 4B) resolved at least seven major presence of Mn ATP correlated with a doubling of its MBP phosphopeptides. Phosphoamino acid analysis of the two kinase activity (data not shown). Since there was relatively major phosphopeptides, i.e., spots 1 and 2, revealed only little if any threonine autophosphorylation of the GST-Erkl, phosphotyrosine (Fig. 4C). Spots 3, 5, and 7 were also we conclude that tyrosine phosphorylation of GST-Erkl is principally tyrosine phosphorylated. In contrast, spots 4 and necessary and may be sufficient for a partial activation of this 6 were serine phosphorylated. There was little, if any, kinase. detectable threonine phosphorylation. These findings indi- Phosphorylation of GST-Erkl by protein kinases. We sus- cate the presence of multiple tyrosine phosphorylation sites pect that the elevated level of tyrosine phosphorylation of in p44erk . This in contrast to the situation with recombinant the recombinant GST-Erkl protein from bacteria reflected mouse p42T"Pk, which has been shown to exclusively auto- the absence or low levels of endogenous tyrosine protein 4686 CHAREST ET AL. MOL. CELL. BIOL.

,.,: G3

..

0 0 .Cat

- 4

- U

x .. a W) ......

< - Thin layer chromatography FIG. 4. Two-dimensional tryptic phosphopeptide mapping of au- < phosphate tophosphorylated GST-Erkl (A) and its thrombin cleavage products (B) after incubation with [y-32P]ATP (10,000 cpm/pmol) and trypsin digestion as described in Materials and Methods. The origin is indicated with an open circle. Each numbered spot in panel B was excised and subjected to phosphoamino acid analysis, and an autoradiogram is shown in panel C. <- Phospho-Ser * Phospho-Thr tides 1 and 2 as containing Thr-202 and Tyr-204 was achieved <-...Phospho-Tyr by the comigrations of these phosphopeptides on the two- dimensional phosphopeptide map generated with a synthetic peptide based on this sequence that was phosphorylated in vitro by p561ck (Fig. 6C). We cannot account for why phosphopeptides 1 and 2 were equally generated from both GST-Erkl and the synthetic peptide. There is only one tyrosine residue in the synthetic peptide, and phosphoamino acid analysis revealed only phosphotyrosine in phosphopep- tides 1 and 2 (Fig. 4C). It is possible that the trypsin .<- Origin

erkl tryptic spot number A B CD45 10

80 80 phosphatases that could dephosphorylate it. With the sub- -O- Control stantial tyrosine protein phosphatase activity present in .a 60 60 *-6 PP2A a. mammalian cells, it is unlikely that autophosphorylation of co 40 co 40 p44erkl on tyrosine in mammalian cells is sufficient to achieve 20 L0 appreciable activation of this MAP kinase. Therefore, we 0 0 explored the phosphorylation of p44erkl by other tyrosine 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Time (min) Time (min) protein kinases. D the 0 We have recently demonstrated tyrosine phosphory- 80 801r 'ST-erkl lation and activation of the sea star MAP kinase p44mPk by to baculovirus-expressed murine p561ck (22). Purified p561ck was IE-.C 5C c 501 also capable of phosphorylating both GST-Erkl and its O 2 5 10 2O 30 60 U 2 5 10U2 30 60 thrombin-cleaved product, p44erkl. Tryptic phosphopeptides Time (min) Time (min) 1 and 2 contained the major tyrosine autophosphorylating FIG. 5. Dephosphorylation and inactivation of recombinant site of GST-Erkl, i.e., Tyr-204, and this site among others p44e`1 by the protein tyrosine phosphatase CD45, but not by protein was phosphorylated by p561ck. This was confirmed by sev- phosphatase 2A. (A and C) GST-Erkl bound to glutathione-agarose eral approaches. Firstly, to identify potentially novel p56"ck beads was incubated for 0 to 30 min with -0.5 pLg of purified protein phosphorylation sites in GST-Erkl and reduce the autophos- tyrosine phosphatase CD45 before the addition of 1 mM sodium phorylation background, the GST-Erkl was preincubated orthovanadate. (B and D) GST-Erkl bound to glutathione-agarose with unlabelled ATP to incorporate phosphate into the major beads was incubated for 0 to 30 min with -4 U of human protein autophosphorylation sites prior to the inclusion of p56'ck and phosphatase 2A (1 U releases 1 nmol of phosphate min-' from 15 FM phosphorylase at 30°C) before the addition of 1 ,uM okadaic [.y-32P]ATP (Fig. 6B). There was still substantial [32P]phos- acid. The beads were washed, and half of the material was subse- phate from [.Y-3 P]ATP into tryptic phosphopeptide 2 but not quently assayed for MBP phosphotransferase activity (A and B). 1 after its addition with p561ck (Fig. 6B). Other sites were also The data are expressed as a percentage of the initial kinase activity evident, but apart from tryptic phosphopeptide 8, which was without preincubation with CD45 or phosphatase 2A. The remaining novel, these additional sites could have reflected autophos- half of the washed beads was subjected to Western blotting analysis phorylation. Secondly, confirmation of tryptic phosphopep- with the 4G10 antiphosphotyrosine antibody (C and D). VOL. 13, 1993 CHARACTERIZATION OF HUMAN p44erk 4687

TEY DeDtide + D56JCk E204-D.44erk I + D56/ck

1Thinlayer chromatograhy 0 FIG. 6. Two-dimnensional tryptic phosphopeptide mapping of recombinant p4-" phosphorylated by p56k The folowing autoradiograms are shown: (A) thrombin-cleaved Erkl subjected to autophosphorylating conditions for 20 mmn; (B) thrombin-cleaved Erkl treated with unlabeled ATP as in panel A and then incubated for an additional 20 min with [-y-32P]ATP in the presence of p56kk~; (C) high-pressure liquid chromatography-purified peptide (IADPEHDHTGFLTEYVATR) (TEY peptide) phosphorylated by p56kk and trypsin treated; (D) thrombin- cleaved Erkl-E204 mutant phosphorylated by p56kk. The spots are numbered as shown in Fig. 5B. The TEY peptide was subjected to the same tryptic digestion treatment as the cleaved Erkl protein before application on the cellulose plate. The migration of spot 6 was variable. Otherwise, similar results were obtained in at least three different sets of experiments. preparations used in these studies contained a contaminating There was no obvious activation of the MBP phos- protease that further cleaved the Tyr-204-containing tryptic photransferase activity of GST-Erkl by p56 ck in vitro (data phosphopeptide. Thirdly, phosphopeptides 1 and 2 were not not shown). The specific increase in phosphorylation at the evident in two-dimensional tryptic phosphopeptides of a Tyr-204 site may not have been sufficient to permit detection Glu-204 mutant GST-Erkl that was phosphorylated by p56'ck of a further stimulation of the MBP phosphotransferase (Fig. 6D). Since the autophosphorylating activity of the activity of GST-Erkl, since this site was already substan- Glu-204 GST-Erkl was severely compromised, the appear- tially phosphorylated in the bacterially expressed kinase. ance of radiolabelled phosphate in phosphopeptides 5 and 8 Measurement of a stimulation of the MBP kinase activity of could be directly attributed to p561Ck phosphorylation. Nev- GST-Erkl was also confounded by the strong phosphoryla- ertheless, the Tyr-204 site was a major site of p56t"" phos- tion of MBP by p56"'" and the ability of the p56Ikk to remain phorylation in GST-Erkl, because there was a substantial bound to GST-Erkl-agarose. Alternative synthetic peptide decrease in phosphorylation when the Glu-204 GST-Erkl substrates for MAP kinase were not helpful in this regard. was used as a substrate. In view of the ability of p561Ck to Previously, we reported that a panel of synthetic peptide phosphorylate the Tyr-204 site in vitro and the substantial analogs (e.g., Ala-Pro-Arg-Thr-Pro-Gly-Gly-Arg-Arg) of the activation of p44erkl in the absence of Thr-202 phosphoryla- Thr-97 phosphorylation site in bovine MBP were very effi- tion, it is possible that p56'kk could contribute directly to the cient substrates for sea star p44PPk (13). However, these physiological activation of this MAP kinase. same peptides were phosphorylated by GST-Erkl at 15-fold- The level of stoichiometry of phosphorylation of GST- lower rates than by sea star p44nPk, even though both Erkl by p56'kk based on 3 p incorporation in vitro was kinases produced comparable phosphorylation of MBP (data approximately 0.1 mol of P per mol, and this was distributed not shown). Even if phosphorylation by p561ck is insufficient at multiple sites. A slightly higher stoichiometry (-0.2 mol by itself for activation of p44rkl, it is possible that it may of P per mol) was achieved if the GST-Erkl was incubated prime the MAP kinase for further activation. For example, with CD45 protein-tyrosine phosphatase prior to the addition Haystead et al. (28) have found that recombinant tyrosine- of the p56"' and [_-32P]ATP (data not shown). It is possible phosphorylated p42""Pk is a 10-fold-higher affinity substrate that substantial phosphorylation of GST-Erkl by p561ck for phosphorylation and activation by a rabbit skeletal could not be achieved, because this tyrosine kinase is partly muscle MAP kinase kinase. inactivated by MAP kinase-catalyzed phosphorylation of To identify other tyrosine-specific protein kinases that Ser-59 in vitro (77). might also phosphorylate GST-Erkl, p6oc-src and p65tlv-abl 4688 CHAREST ET AL. MOL. CELL. BIOL.

1 2 3 4 5 kinase from phorbol ester-treated platelets was accompanied A C phosphate by a 5-fold enhancement of its MBP phosphotransferase GST-Erkl _ activity, whereas MAP kinase kinase from control platelets produced only a 1.5-fold effect (Fig. 7B, lanes 1 to 3). To confirm that the platelet MAP kinase activator was indeed a kinase, a mutated form of GST-Erkl that had cps substantially reduced catalytic activity was exploited as a B __ Phospho-Ser substrate. This GST-Erkl had an alanyl residue instead of __ Phospho-Thr lysine at residue 71, which has been implicated in ATP binding and shown to be critical for phosphotransferase MBP _P- activity in protein kinases (27). The Ala-71-GST-Erkl auto- 965 1499 4573 69 890 phosphorylated at 6% of the rate of Lys-71-GST-Erkl, and cps __Phospho_TyrPhospho-Tyr it exhibited only 7% of the MBP phosphotransferase activity GST-Erkl K71 K71 K71 A71 A71 MKK - U p - p (Fig. 7). Nevertheless, MAP kinase kinase from PMA- treated to FIG. 7. Phosphorylation of GST-Erkl by MvIAP kinase kinase platelets phosphorylated Ala-71-GST-Erkl -78% from sheep platelets. MAP kinase kinase (MKK) was partially of the level achieved with Lys-71-GST-Erkl. Furthermore, purified from untreated platelets (U-MKK) and from platelets ex- the MBP phosphotransferase activity of the Ala-71-GST- posed to 200 nM PMA for 5 min (P-MKK) as described in Materials Erkl was stimulated 16-fold by the MAP kinase kinase. and Methods. Approximately 1 ,ug of GSoT-Erkl (K71) and GST- The MAP kinase activation results presented here are Erkl in which Lys-71 that is required for catalytic activity is comparable to those reported by others using partially converted to Ala (A71) bound to agarose was incubated with the purified preparations of MAP kinase kinase (1, 79). How- MAP kinase kinase preparations and [y-321P]ATP for 30 min (A). In ever, Robbins et al. (57) have described 500-fold and greater some experiments (B), the MBP phosphotiransferase activty of the activations of bacterially expressed histidine-tagged p42maPk MAP kinases was subsequently quantitatecd and by MAP of the agarose beads. The reactions were tterminatedafter extensiveupon washingaddition p44erkl highly purified kinase kinase from rabbit of SDS-polyacrylamide gel electrophoresis sample buffer. Following skeletal muscle. This difference from our results likely SDS-polyacrylamide gel electrophoresis, autoradiography for 15 reflects the lower concentrations of MAP kinase that were min was performed on the dried gels. The positions of the prestained used in that study. Apparently, higher concentrations of standards bovine serum albumin (80 kDa) and ovalbumin (50 kDa) MAP kinase can lead to inhibition of its activation by MAP correspond to the top and bottom, respeictively, of panel A. The kinase kinase. It is possible that this might reflect the positions of the prestained standards soybean trypsin inhibitor (28 phosphorylation and inactivation of MAP kinase kinase by kDa) and lysozyme (19 kDa) correspond to the top and bottom, MAP kinase, as has been implied from studies with yeast respectively, of panel B. The counts per second (cps) of 32p p40ft3 and p56ste7 (20). Indeed, vertebrate MAP kinase incorporated in the excised GST-Erkl (A);and MBP (B) bands were kinase features an ideal consensus sequence (13) at its C quantitated by liquid scintillation counting and are provided at the bottom of each lane. In panel C, the rcrsuats are presented tfr terminus for phosphorylation by MAP kinase (3, 17, 33, 68, phosphoamino acid analysis of the excised band that is shown in 78). Further studies are warranted to establish the role of this lane 3 of panel A of GST-Erkl (K71) after incubation with P-MKK. putative MAP kinase phosphorylation site in MAP kinase Migrations of the phosphoamino acid stanclards are indicated to the kinase. right of panel C. ACKNOWLEDGMENTS were also tested. Despite the ability of these two kinases to D.L.C. and G.M. contributed equally to this study. strongly phosphorylate angiotensin I]I, they were approxi- We thank the following associates at the Biomedical Research kk Centre for the valuable reagents and services that they contributed mately twofold-less efficient than p56' in the phosphoryla- to this study: Allen Delaney for the computer data base searches, tion of GST-Erkl, and they also failecJ to stimulate its MBP Jasbinder Sanghera for the purified p44mP , Ian Clark-Lewis for the phosphotransferase activity (data not shown). synthesis of peptides, Michael Williams for the injection of MAP Activation of GST-Erkl by a MAP kiinase kinase from sheep kinase peptides into rabbits, Harry Paddon and Faye Chow for platelets. Several recent studies hasve identified a MAP affinity purification of the MAP kinase antibodies, and Scott Pownall kinase activator from PMA-treated jmammalian cells that and John Babcook for the synthesis of the oligonucleotides. We are phosphorylates both regulatory threonline and tyrosine phos- also grateful to D. Brautigan, F. Hall, N. Tonks, and J. Wang for the sites This MAkP kinase kinase enzymes that they provided. phorylation (1, 59, 69). does D.L.C. was the recipient of a Roman Matthew Babicki Student- not bind to DEAE resins at neutral pfWI, and it appears to be ship Award and a Medical Research Council of Canada (M.R.C.C.) highly specific for MAP kinases. To:investigate the activa- Studentship. G.M. was supported by a studentship award from the tion of GST-Erkl by this kinase, we p.artially purified it from Conseil Regional de Bretagne (France). S.L.P. is a M.R.C.C. sheep platelets that had been pretrealted for 5 min with 200 Scientist awardee. F.J. and K.W.H. were recipients of a Canadian nM PMA. Incubation of GST-Erkl wiith the activated plate- Arthritis Society Associateship and a studentship, respectively. let MAP kinase kinase for 30 min resulted in a threefold This work was supported by operating grants from the M.R.C. of increase in the phosphorylation of the GST-Erkl compared Canada and the National Institute of Canada (to S.L.P.). with autophosphorylation of the MAT kinase in its absence (Fig. 7A, lanes 1 and 3). Phosphoaimino acid analysis of REFERENCES GST-Erkl after incubation with the MAP kinase kinase 1. Alessandrini, A., C. M. Crews, and R. L. Erikson. 1992. Phorbol revealed enhanced phosphorylation on ester stimulates a protein-tyrosine/threonine kinase that phos- predominantly ty- phorylates and activates the Erk-1 gene product. Proc. Natl. rosine, with threonine and serine phos;phorylation evident to Acad. Sci. USA 89:8200-8204. a much lesser degree (Fig. 7C). MAP Ikinase kinase similarly 2. Anderson, N. 1992. MAP kinases-ubiquitous signal transduc- prepared from untreated platelets waLs relatively ineffective ers and potentially important components of the cell cycling for enhancement of GST-Erkl phosphorylation (Fig. 7A, machinery in eukaryotes. Cell. Sign. 4:239-246. lane 2). The phosphorylation of GST'-Erkl by MAP kinase 3. Ashworth, A., S. Nakielny, P. Cohen, and C. Marshall. 1992. VOL. 13, 1993 CHARACTERIZATION OF HMAN p4e1*l 4689

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