Proc. Nadl. Acad. Sci. USA Vol. 88, pp. 9508-9512, November 1991 Biochemistry Autophosphorylation in vitro of recombinant 42-kilodalton mitogen-activated protein kinase on tyrosine (mitogen-activated protein kinase/cell cycle/peptide mapping/recombinant expression) JIE WU*, ANTHONY J. ROSSOMANDOt, JENG-HORNG HERt, ROBERT DEL VECCHIOt, MICHAEL J. WEBERt, AND THOMAS W. STURGILL*t Departments of tMicrobiology, and *Internal Medicine and Pharmacology, Health Sciences Center, University of Virginia, Charlottesville, VA 22908 Communicated by Oscar L. Miller, Jr., July 29, 1991 (receivedfor review June 21, 1991) ABSTRACT Mitogen-activated protein kinase (MAP ki- lation sites are locatedjust upstream ofthe Ala-Pro-Glu motif nase) is a serine/threonine protein kinase that becomes enzy- in a region where activating autophosphorylations occur in matically activated and phosphorylated on tyrosine and thre- many other kinases (5, 6, 14). onine following treatment of quiescent cells with a variety of To investigate the mechanism(s) for p42maPk activation, we stimulatory agonists. Phosphorylation on both tyrosine and have cloned a full-length cDNA encoding p42maPk (6) and threonine is necessary to maintain full activity, and these two expressed the enzyme in Escherichia coli in soluble form. We regulatory phosphorylations occur close to each other, sepa- now report that recombinant p42maPk is basally active and rated by a single glutamate. To study the mechanisms by which autophosphorylates on tyrosine. This autophosphorylation MAP kinase becomes phosphorylated and activated, we have can occur on the site of regulatory phosphorylation and is cloned a full-length cDNA encoding MAP kinase and have accompanied by a partial enzymatic activation of p42maPk. expressed the enzyme in Escherichia coli as a soluble nonfusion Thus, upstream components regulating MAP kinase activity protein. We find that the enzyme displays a basal, intramo- may conceivably do so by enhancing the rate of autophos- lecular autophosphorylation on tyrosine-185 that is accompa- phorylation on tyrosine. nied by activation of the enzyme's kinase activity towards an exogenous substrate. The tyrosine-phosphorylated protein dis- plays a small fraction of the activity seen with the fully MATERIALS AND METHODS activated, doubly phosphorylated enzyme isolated from mam- Materials. The bacterial expression vector pETila and the malian cells but is activated 10- to 20-fold relative to the E. coli strains HB101 and BL21(DE3) (15) were purchased unphosphorylated enzyme. These findings raise the possibility from Novagen (Madison, WI). Isopropyl f3-D-thiogalactopy- that regulation of MAP kinase activity in response to agonist ranoside (IPTG) and enzymes used in DNA manipulations stimulation could occur in part through the enhancement of were obtained from GIBCO/BRL. The sources for chroma- autophosphorylation on tyrosine. tography media and reagents for culturing E. coli were Pharmacia-LKB and Difco, respectively. Oligonucleotides Mitogen-activated protein kinase (MAP kinase) was orig- were purchased from Operon Technologies (Alameda, CA), inally identified as a serine/threonine protein kinase that and used without further purification. A synthetic peptide became phosphorylated on tyrosine and threonine and en- (Val-Ala-Asp-Pro-Asp-His-Asp-His-Thr-Gly-Phe-Leu-Thr- zymatically activated following insulin-stimulation of 3T3-L1 Glu-Tyr-Val-Ala-Thr-Arg) corresponding to the tryptic pep- cells (1, 2). This enzyme was subsequently found to be tide containing the regulatory phosphorylation sites of identical or closely related to pp42, a protein that becomes p42mapk was purchased from Synthecell (Rockville, MD). tyrosine-phosphorylated in cells treated with various mito- Antibody 1798, which recognizes p42maPk, was raised against gens (3). pp42/MAP kinase recently has been shown to be the carboxyl-terminal 10 amino acids of the predicted ERKi encoded by a member ofa gene family (4-8). Accordingly, we sequence (5) by the MAPS procedure (16). now refer to it as p2mapk to distinguish it from other members Construction of Plasmids for Expression of Unfused p42Pk ofthe family, while still retaining recognizable features ofthe in E. coli. Plasmid pETila was digested with Nde I, and the earlier nomenclature. In addition to being phosphorylated recessive 3' end was filled in with Klenow fragment. The and activated during the Go -* G, transition, p42maPk and/or DNA was then cut with BamHI, generating a 5639-base-pair members ofthis family become phosphorylated and activated (bp) fragment, which was isolated by gel electrophoresis. The during M phase in Xenopus oocytes and in various differen- blunt end ofthe purified fragment starts at the first nucleotide tiated, nonmitogenic cells following treatment with stimula- ofthe ATG start codon ofthe phage T7 4510 gene. The p42maPk tory agonists (9-11). Therefore, we suspect that this enzyme cDNA was excised from plasmid pA4 (6) by sequential plays a fundamental role in some process common to a treatment with Bgl I, mung bean nuclease, and BamHI. The number of regulatory events (for review, see ref. 11). -1.25-kilobase (kb) 42mapk cDNA fragment was ligated to Phosphorylation of$2maPk on both tyrosine and threonine the above 5639-bp fragment of pETila and transformed into is required for it to display full enzymatic activity (12). The E. coliHB1O1. Plasmids in which the second nucleotide ofthe sites of regulatory phosphorylation were identified by mass p42mIPk cDNA were ligated directly to the first nucleotide of spectrometry and found to be on a single peptide separated the phage T7 )10 gene on pETlia reconstitute a translational by only one amino acid, glutamic acid, in a sequence (Thr- start codon ATG and thus an unfused 42mapk coding se- Glu-Tyr-Val-Ala-Thr-Arg) that is absolutely conserved in the related protein kinases ERKi (extracellular signal-related Abbreviations: MAP kinase, mitogen-activated protein kinase; protein kinase 1), KSS1, and FUS3 (13). These phosphory- p42maPk, 42-kDa MAP kinase; IPTG, isopropyl f-D-thiogalactopy- ranoside; MBP, myelin basic protein; ERK1, extracellular signal- related protein kinase 1. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Box 419, De- payment. This article must therefore be hereby marked "advertisement" partment of Internal Medicine, University of Virginia Health Sci- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ences Center, Charlottesville, VA 22908. 9508 Downloaded by guest on September 26, 2021 Biochemistry: Wu et al. Proc. Natl. Acad. Sci. USA 88 (1991) 9509 quence under the control of T7 transcriptional and transla- BL21 MK K52R tional control signals. One such plasmid, pET-MK, was - + - + - + IPTG isolated and transformed into expression host E. coli BL21(DE3). The kinase-defective Lys-52 -- Arg mutant was generated using the Promega "Altered Sites" in vitro muta- 45 kDa - genesis kit. _ Expression of p42IPk in E. coli. A 10-ml overnight culture 40 ~p42'P42 mapk ofE. coli BL21(DE3)[pET-MK] was inoculated into 1 liter of Luria-Bertoni medium (10% tryptone/5% yeast extract/10%o NaCI) containing 0.1 mg of ampicillin per ml. The bacteria were grown at 370C with vigorous shaking to late logarithmic phase (ODwo = 0.8). IPTG was then added to a final con- 45 kDa - centration of 0.4 mM, and the cells were induced for 4-5 hr - p42 mapk before harvesting by centrifugation. For labeling of the phosphorylated p42maPk synthesized in E. coli, 0.2 mCi (1 Ci = 37 GBq) of 32p, per ml was added to the bacteria prior to the IPTG induction. The bacterial pellets were frozen at FIG. 1. Expression and tyrosine phosphorylation of p42mapk in -700C. bacteria. Wild-type p42mapk (lanes MK) and the kinase-defective Purification ofp421)Pk Produced in E. colU. Bacterial pellets Lys-52 -- Arg mutant (lanes K52R) were expressed in E. coli by were thawed, resuspended in 25 mM Tris-HCl, pH 7.5 induction with IPTG for 4 hr. The bacterial lysates were separated by (40C)/40 mM p-nitrophenyl phosphate/2 mM EGTA/2 mM SDS/PAGE, transferred to nitrocellulose, and blotted with either EDTA/1 mM dithiothreitol/l mM phenylmethylsulfonyl flu- anti-p42IaPk antibody 1798 (Upper) or anti-phosphotyrosine anti- oride and lysed by three passages through a chilled French body (Lower). pressure cell. The cell extracts were centrifuged at 12,000 x g for 20 min at 40C. p42maPk in the supernatant was then an autophosphorylation reaction. To test this hypothesis, we purified sequentially by batch absorption to phenyl- generated and expressed a kinase-defective mutant of Sepharose/Mono Q chromatography and phenyl-Superose p421Pk, with a substitution of arginine for lysine at position chromatography as described (13). 52 (in the ATP binding site) (6). Although IPTG also induced Protein Kinase Assay. Autophosphorylation of recombi- the appearance of this mutant protein (Fig. 1 Upper), the nant p42 Pk was performed by incubation in "autokinase protein did not become detectably phosphorylated on tyro- buffer" [25 mM Hepes/10 mM MgCl2/2 mM MnCl2/1 mM sine (Fig. 1 Lower). Thus, the appearance ofphosphotyrosine dithiothreitol, pH 7.5/50 ,uM [y-32P]ATP (5000 cpm/pmol)] at on bacterially expressed p42maPk requires a kinase-active 30°C. The amounts of protein and the time of autophosphor- enzyme, indicating that this tyrosine phosphorylation is a ylation are specified in the figure legends. The reaction was consequence of autophosphorylation. stopped by adding SDS electrophoresis