T-as consensus sequence motif for

Vol. 269, No. 47, Issue of November 25, pp. 29855-29859, 1994 Printed in U.S.A.

THEJOURNAL OF BIOLCGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Identification of -R-X-(X)-S/T-X3-S/Tas Consensus Sequence Motif for Autophosphorylation-dependent Protein Kinase* (Received for publication, May 3, 1994, and in revised form, July 13, 1994)

Shiaw-Der YangS, Tze-Jen Huang, and ThomasR Soderling From the Institute of Biomedical Sciences, National Tsing Hua University, Hsinchu, 30043 Taiwan, the Institute of Basic Medicine, Chang Gung Medical College, Tao-Yuan, Taiwan, Republic of China, and the Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232

protein (Yang et al., 1987a) possibly involved in the regulation To identify consensus sequence motif for a new family of protein kinase termedautophosphorylation-depend- of diverse cell functions and may representa newly described, ent protein serindthreonine kinase (auto-kinase), we previouslyundiscovered multisubstrate/multifunctional prohave tested several synthetic peptides.The well estab- tein kinase(Yang et al.,1987a, 1987b; Guoet al.,1993; Guoand lished protein serinelthreonine kinases such as CAMP- Damuni, 1993). dependentproteinkinase, Ca2+lcalmodulin-dependent In an attempt to identifyspecific consensus sequence motif protein kinase (CaM-kinase), and protein kinase C were for this new family of protein serinekhreonine kinase, we have synfound to be inactive toward phosphorylation of demonstrated a unique phosphorylation site with peptide setide-3 (RPFWASVPPSPSLSRHA), which turned out to be quence of -AARTTHYGS- frommyelinbasic protein, which an excellent substrate only for auto-kinase, indicating could be specifically recognized by this new kinase but notby that syntide-3 is a specific substrate for auto-kinase. any other well established protein serinehhreonine kinases Modification of syntide-3 to become RPRPASVPPSfl' did By contrast, auto- such as CAMP-dependent proteinkinase (Kishimoto et al., not affect the activity of auto-kinase. kinase became rather or almost inactive when the pep- 1985), Ca'+/phospholipid-dependentprotein kinase (Kishimoto tide was modified to become RPRPASVPPNGIFI"R/DI et al., 19851, Ca2+/calmodulin-dependentprotein kinase (Shoji et al., E/Y,indicating that amino acid number 10 in syntide-3is et al., 19871, mitogen-activated protein kinase (Erickson crucial to the sequence motif recognized by auto-kinase. 19901, and protein kinase FMglycogen synthase kinase-3 (Yu and Yang, 1994). Similarly, we also identified a unique peptide Phosphorylation of myelin basic protein (MBP) by autokinase revealed that auto-kinase predominantly phos- fragment with sequence of RPRPASWPSPSLSRHA derived particular site with RT- from the site3 of glycogen synthase (syntide-3),which could be phorylates MBP on one T(p)HYGSas the phosphorylation site sequence, which specifically phosphorylated only by autophosphorylation-decould not be phosphorylated by any other reported MBP pendent protein serinekhreonine kinase but not by any other kinases including CAMP-dependent protein kinase, well established protein serinehhreonine kinases tested. ModiCaM-kinase,proteinkinase C, mitogen-activated pro- fications of syntide-3 further revealed that RPRPASWPS/T tein kinase, and kinase FNGSK-3. Taken together, the but notRPRPASWPMG/F/K/lUDIEN are uniquesequence moresults provide initial evidence that -kg-X-(X)-Serfl'hr- tifs specific for auto-kinase. When taken together with all of X,-Ser/Thr- may representa unique consensus sequence these results, a unique consensus sequence motif specifically motif specifically recognized by autophosphorylation- recognized by this new family of protein serinekhreonine kidependentproteinkinase,anewfamilyofmultinase is therefore proposed in this report. substratelmultifunctional protein serinelthreonine kinase. EXPERIMENTALPROCEDURES

Materials-Most materials were as described in previous reports (Yang et al., 1993; Yu and Yang, 1994). The peptide fragment derived A new family of protein serinekhreonine kinase termed au- from site 3 of glycogen synthasetermedsyntide-3(RPRPASVPPtophosphorylation-dependent protein kinase has been identiSPSLSRHA) and its derivatives and G-peptide were synthesized from by HPLC,' and fied and characterizedfrom mammalian nervous and non-nerv- peptide synthesizer (MilliGenBioresearch) and purified ous tissues (Yang et a l . , 1987a, 198713; Guo et al.,1993;Guo and the amino acid sequences were demonstrated by the amino acid analysis and peptide sequence analysis. Damuni, 1993). This protein kinase exists in an inactive form Purification of Various Protein Kinases and Substrate-Autophosbut can be activated in the presence of MgATP. The activation phorylation-dependent protein serine/threonine kinase was purified to process involves an intramolecular autophosphorylation. The homogeneity from porcine brain basically as described in a previous activated protein kinase becomes capable of phosphorylating report (Yanget al., 1987a). After histone-Sepharose 4B chromatography, several substrates including histones(Yang et al., 1987a), gly- the inactive auto-kinasewas preactivated inthe presence of cold ATP cogen synthase (Yang et al., 1987b), protein phosphatase 2A and repurified by the second histone-Sepharose 4B column and then used in the kinase assays.When analyzedby gel electrophoresis inthe (Guo et al., 1993; Guo and Damuni, 19931, and myelin basic presence of SDS and Coomassie Bluestaining, the purified kinase gave a single major protein band at M , = 36,000. Analysisof the radioactively * This work was supported by Grants NSC 82-0203-B007-029 and autophosphorylated kinaseon the autoradiogram also revealeda single NSC 83-0203-B-007-001 from the National Science Council of Taiwan major phosphorylated protein band at M, = 36,000.Phosphorylase b and by Grant CMRP-263 from Chang Gung Medical College and Memorial Hospitalsof Taiwan, Republicof China. The costsof publication of this article were defrayed in part by the payment of page charges. The abbreviations used are: HPLC, high performance liquid chroThis article must therefore be hereby marked "aduertisernent" in acmatography; MBP, myelin basic protein;auto-kinase, autophosphorylacordance with 18 U.S.C. Section 1734 solely toindicate this fact. tion-dependent protein serinehhreonine kinase; CaM-kinase, Ca2+/calj: To whom correspondence should be addressed: Inst. of Biomedical modulin-dependentproteinkinase;MAP-kinase,mitogen-activated ROC. Fax: proteinkinase; FMGSK-3,protein kinase FNglycogen synthase kiSciences, National Tsing-Hua University, Hsinchu, Taiwan, 886-35-721746. nase-3.

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Consensus Auto-kinase Sequence for Motif

kinase (Cohen, 1973) was purified from rabbit skeletal muscle. The catalytic subunit of cAMP-dependent protein kinase (Kinzel and Kubler, 1976) was purified frombovine heart. Ca'+/phospholipid-dependent protein kinase (Kikkawa et al., 1982),kinase FNglycogen synthase kinase-3 flang, 1986; Yu and Yang, 1993), Ca'+/calmodulin-dependent protein kinase I1 (Bennett et al., 1983), and casein kinase I1 (DePaoli-Roach, 1984) were purified from bovine brain. The substrate myelin basic protein (MBP) was purified from bovine brain following the procedure of Eylar et al. (1969)with some modification.The detailed purification procedures were as described in a previous report (Yu and Yang, 1994). Calmodulin waspurified from bovine brain following the procedure of Dedman and Kaetzel(l983). Standard Phosphorylation of Peptide Substrates by Protein Kinases-Standard phosphorylation of synthetic peptides by protein kinases was performed at 30 "C for various time intervals as indicated in a 0.1-ml reaction mixture containing 20 mM Tris-HC1 at pH 7.0, 20 mM MgCl,, 0.5 mM dithiothreitol, 0.2 m~ [y3'P1ATP (1 pmol, -1,000 counts/min), 1.0 mM peptide substrates, and 1 p pure kinases. 32P incorporation into substrate was determined by spotting 10 pl of the reaction mixture onto Whatman P81 paper, dropping into 75 m~ phosphoric acid, and processing as described by Reimann et al. (1971). Preparation of Immobilized Metal (Fe3+) Affinity columnImmobilized metal affinity column was prepared according to Andersson and Porath (1986).The chelating Sepharose CL-GB column (1.5 x 5 cm) was washed with deionized water and equilibrated with FeCl, solution. The column was further washed with 3 column volumesof 0.1 M acetic acid, NaOH at pH 3.1 and ready for use. Dypsin Digestion and Purification of Dyptic Digests of P2P]MBP Phosphorylated by Auto-kinase-The reaction mixture at a totalvolume of 0.1 ml containing 0.1 mM MBP and 1p pure auto-kinase was incubated at 30 "C for various time points. The 100%trichloroacetic acid at a volume of 25 p1 was next added to stop the phosphorylation reaction. The detailed procedures for trypsin digestion and purification of the tryptic digests by HPLC were as described in previous reports (Yang et al., 1993; Yu and Yang, 1994). PhosphoaminoAcid Analysis-Phosphoamino acidanalysis was performed as described byBoyle et al. (1991). The positions of phosphoamino acids in plates were localized by ninhydrin staining of the phosphorylated amino acid standards. Amino Acid Sequence Analysis and Determination of Phosphorylation Site Sequences-The amino acid sequence analysis of [32Plsyntide-3 and the phosphopeptide isolated from HPLC of the tryptic digests of [32PlMBPwasperformed on MilliGedBioresearch model6600 Sequencer, and the phosphorylation site was determined by sequential manual Edman degradation essentially according to Laursen (1966) and Laursen and Machleidt (1980) and processed as described in a previous report (Yang et al., 1993). AnalyticMethods-Protein concentration was determined by the method of Lowry etal. (1951)using bovine serum albumin as standard. Peptide concentration was determined by amino acid analysis. SDSpolyacrylamide gelelectrophoresiswas performedessentially according to Laemmli (1970).Autoradiography was carried out with an Fuji Rx x-ray film using Kodak X-Omatic cassette with intensifying screens.

A.

1.0 0.8

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Kemptide

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40

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120

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Time (min) FIG.1. Phosphorylation of Kemptide, G-peptide,and syntide-3 by cAMF"dependent protein kinase and auto-kinase. 1p autokinase (0) or cAMP-dependentprotein kinase (0)was incubated with 1 mM Kemptide (A) or G-peptide (B) or syntide-3 ( C )at a totalvolume of 0.1 ml at 30 "C for various time intervals as indicated. At each time point, a 10-4 aliquot was subjected to determination of 3zPincorporation into the peptides as described under "Experimental Procedures."

protein kinase that could potently act on syntide-3 among all the serine/threonine kinases tested (Table I), indicating that syntide-3 is a very unique substrate specifically recognizedby auto-kinase. Peptide sequence analysis and sequential manual Edman degradation of [32P]syntide-3phosphorylated by autokinase further revealed that -RPRPAS(p)VPPSPSLSRH- is the phosphorylation site sequence (Fig. 2). To further identify the more detailed specific consensus sequence motif recognized by auto-kinase, we tried to modify syntide-3 into various forms. When syntide-3 (RPRPASVPPSPSLSRHA) was shortened from 17 to 10 amino acids, the activity of auto-kinase toward phosphorylation of this truncated peptide ( ~ y n t i d e - 3with ) ~ ~ sequence of RPRPASVPPS was not significantly changed, indicating that amino acids number 11-17 in syntide-3 are not critical to the consensus sequence RESULTS motif for auto-kinase (Table 11). In sharp contrast, when synIn comparison with the activities of CAMP-dependentprotein tide-3 was deleted from 17 to 9 amino acids, the activity of kinase and auto-kinase toward phosphorylation of several syn- auto-kinase toward phosphorylation of this 9-amino-acid pepthetic peptides, we found that CAMP-dependent protein kinase tide (syntide-3), with sequence of RPRPASVPP was dramatically impaired (Table 11),indicating that amino acid number 10 was very active toward phosphorylation of Kemptide(LRRASLG) and G-peptide (KPGFSPQPSRRGSESSEEV) (the serine residue) in syntide-3 is critical t o the consensus whereas auto-kinase was almost inactive toward phosphoryla- sequence recognized by auto-kinase. When the serine residue tion of these two synthetic peptides when assayed under sim- at the amino acid number 10 in syntide-3 was replaced by ilar conditions (Fig. 1, A and B ) . In sharp contrast, CAMP- threonine, the activity of auto-kinase was unaffected. However, dependent protein kinase wasfound t o be inactive toward when the serine or threonine residue at theamino acid number phosphorylation of syntide-3 (RPRPASVPPSPSLSRHA) 10 in syntide-3 was replaced by tyrosine or negatively charged whereas auto-kinase was very active toward phosphorylation of amino acids such as glutamic acid or aspartic acid, the activity syntide-3 under similar conditions (Fig. 1C).The results sug- of auto-kinase was significantly impaired. A full kinetic analygest that thisnew family of protein kinase may recognize spe- sis of phosphorylation of these peptide substrates by auto-kicific consensus sequence distinctly different from CAMP-de- nase further confirmed this point (see Table 111).In sharp conpendent protein kinase (Fig. 1).When further tested with the trast, when serine or threonine was replaced by glycine, other protein kinases such as Caz+/phospholipid-dependentpro- alanine, or phenylalanine or by positively charged amino acids tein kinase, CaM-kinase, casein kinase-2, phosphorylase b ki- such as arginine or lysine, the activity of auto-kinase was nase, and protein kinase FNglycogen synthase kinase-3 (FA/ greatly impaired as summarized in Table 11, which further GSK-3), we found that auto-kinase turned out to be the only supported the notion that amino acid number 10 in syntide-3 is

Consensus Sequence Motif for Auto-kinase

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TABLE I11 TABLE I Kinetic analysisof phosphorylation of syntide-3 derivatives Relative activities of several protein kinases toward phosphorylation by auto-kinase of syntide-3 The phosphorylation of various concentrations of synthetic peptides The phosphorylation of 1 mM syntide-3 by 1 pM of various protein kinases was carried out as described under “Experimental Procedures.” by 1 p~ pure auto-kinase at 30 “C for 10 min was camed out as described under “Experimental Procedures.” Data were taken from the Activity is expressed as rate of incorporation of phosphate. Protein kinase C was assayed with 0.5 mM CaC1, and 0.5 mg/ml phosphatidyl- average of three independent experiments. protein kinase (Cd-kinase 11)was sequences serine. Ca2+/calmodulin-dependent Peptide Km vm Vm’Km assayed with 0.5 mM CaC1, and 10 m~ calmodulin. Phosphorylase b kinase was assayed with 0.5 mM CaCl, at pH 8.6. Assay time was 5 01M) nmollminlmg min, and