Proc. Natl. Acad. Sci. USAVol. 92, pp. 5366-5370, June 1995Biochemistry

Full-length myotonin protein kinase (72 kDa) displays serinekinase activityL. TIMCHENKO*, W. NASTAINCZYKt, T. SCHNEIDERt, B. PATEL*, F. HOFMANNt, AND C. T. CASKEY*t§*Department of Molecular and Human Genetics and tHoward Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030; andtInstitut fur Pharmakologie and Toxikologie der Technischen Universitat, Munich, Federal Republic of Germany

Contributed by C. T. Caskey, January 31, 1995

ABSTRACT We describe the full-length (72 kDa) myoto-nin protein kinase (Mt-PK) and demonstrate its kinaseactivity. The 72-kDa protein corresponds to the translationproduct from the first in-frame AUG codon. This protein wasfound in the cytoplasmic fraction, whereas the previouslyreported 55-kDa protein was observed in nuclear extracts.Only the 72-kDa protein was phosphorylated by [32P]phos-phate in normal human fibroblasts. To investigate the puta-tive kinase activity of Mt-PK, a construct containing thefull-length open reading frame of Mt-PK was expressed inbacterial cells. The recombinant Mt-PK autophosphorylates aSer residue and phosphorylates the synthetic peptide Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg, which contains a Ser residue in thephosphorylation site. We examined phosphorylation of thevoltage-dependent Ca2+ release channel, or dihydropyridinereceptor (DHPR), by recombinant Mt-PK. We observed thatthe f3 subunit ofDHPR was phosphorylated in vitro by Mt-PK.A a8-subunit DHPR peptide containing some of the Ser resi-dues predicted to be phosphorylated was synthesized andfound to be a substrate for Mt-PK in vitro. We conclude thatthe 72-kDa Mt-PK has a protein kinase activity specific for Serresidues.

Myotonic dystrophy (DM) is an autosomal dominant multi-system disease that is characterized by muscle weakness,atrophy, and myotonia (1). The molecular basis of DM isthought to be an amplified trinucleotide (CTG)n repeat lo-cated in the 3' untranslated region of the myotonin proteinkinase (Mt-PK) gene. The predicted amino acid sequence ofMt-PK shows a high degree of homology to Ser/Thr kinases(2-4). A number of polyclonal anti-peptide antibodies to thepredicted protein sequence of Mt-PK were developed, allrecognizing a 52- to 55-kDa protein in muscle extracts (5-7).One antiserum also recognized a major 42-kDa protein inbrain (6). We observed a reduced level of 55-kDa protein in theskeletal muscle of adult-onset DM patients (5), associated withtrinucleotide amplification. Contradicting results regardingthe steady-state levels of Mt-PK mRNA have been reported.We found reduced levels of Mt-PK mRNA (5), in agreementwith data observed by others (8-11), whereas Sabourin et aL(12) have described increased expression of the mutant Mt-PKmRNA in DM patients (12). To our knowledge, no data onprotein expression in the DM patients with increased orunchanged steady-state levels of Mt-PK mRNA have beenreported. The size of the primary translation product of Mt-PKmRNA has also been unclear. The expected size of thetranslation product from the longest open reading frame is-72 kDa, but analysis of the Kozak sequence around AUGcodons within the Mt-PK mRNA raises the possibility ofalternatively initiated translation products. Translation initi-ating from alternative AUG codons could yield several dif-ferent Mt-PK isoforms, including a 55-kDa protein. Further-

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more, multiple splice forms of Mt-PK mRNA have beendescribed (5, 13), predicting a diversity in the range of proteinisoforms. To our knowledge, no data were reported regardingthe Mt-PK activity in normal and DM cells. Study of the54-kDa protein, precipitated from muscle cells extract, re-vealed Tyr phosphorylation (14). In another study (15), re-combinant Mt-PK, expressed from constructs containing onlyputative kinase domain or the kinase and a-helical coiled-coildomain, was reported to have autophosphorylated the recom-binant truncated Mt-PK, with specificity for Thr and Serresidues. Our failed attempts to find kinase activity in the55-kDa protein from normal muscle prompted a search for aMt-PK with kinase activity.

In this paper, we describe a full-length Mt-PK of 72 kDa anddetermine the specificity of kinase activity of recombinantMt-PK with synthetic substrates. In addition, we investigatedthe phosphorylation of a protein that may be a candidateligand for Mt-PK in muscle. Since Ca2+ conductance abnor-malities may contribute to hyperexcitability of the sarcoplas-mic membrane in DM, we initially focused on the voltage-gating L-type Ca2+ channel, or dihydropyridine receptor(DHPR), as a potential substrate for Mt-PK. Here we describethe phosphorylation of the ,B subunit of DHPR in vitro byrecombinant Mt-PK.

METHODSProduction of Mt-PK Antibodies. Mt-PK amino acid se-

quence was analyzed to predict hydrophilicity, surface prob-ability, secondary structure, and antigenicity. One peptide(Pro-Gly-Thr-Gly-Ser-Tyr-Gly-Pro-Glu-Cys-Asp-Trp) fromthe kinase domain was chosen as a potential antigen. Thispeptide was synthesized and used for antibody production inrabbits by Research Genetics (Huntsville, AL). The immuno-globulin fraction of antisera against Pro-Gly-Thr-Gly-Ser-Tyr-Gly-Pro-Glu-Cys-Asp-Trp (antiserum 8391) was purified onprotein A-agarose and used for immunoblot analysis.Two additional Mt-PK-specific antibodies were used, one

(antibody 10033) was raised against a truncated Mt-PK pro-duced with the prokaryotic expression vector pRSET (Invitro-gen) and the other (antibody 254) was raised against syntheticMt-PK peptide (5).

Generation of Construct Containing the Mt-PK CodingRegion. A SfaNI-HindIII fragment containing the Mt-PKcoding region was cloned into the Pvu II and HindIII sites ofpRSETc (Invitrogen). The Mt-PK insert (2307-bp fragmentencoding residues 546-2853) was cloned downstream with thesequence that encodes an N-terminal fusion peptide. ThisN-terminal sequence encodes, from N-terminal to C-terminalends, an AUG translation initiation codon, a tract of 6 Hisresidues that function as a metal binding domain, and a

Abbreviations: IPTG, isopropyl ,B-D-thiogalactoside; Mt-PK, myoto-nin protein kinase; DM, myotonic dystrophy.§To whom reprint requests should be addressed at: Department ofMolecular and Human Genetics, Baylor College of Medicine, OneBaylor Plaza, Houston, TX 77030.

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transcript-stabilizing sequence from gene 10 of phage T7. Theresulting construct was called pRMK.

Generation of Mt-PK Fusion Protein from the pRSETcVector. XLI Blue cells were transformed with pRMK plasmidDNA and expression of fusion protein was induced with M13phage helper and 2 mM isopropyl 13-D-thiogalactopyranoside(IPTG). Cells were collected by centrifugation at 4000 x g for20 min at 4°C and resuspended in 50 mM sodium phosphate,pH 7.0/500 mM NaCl/leupeptin (0.5 ,ug/ml)/pepstatin (20,ug/ml). Cells were frozen overnight at -20°C and then lysedby sonication. The lysate was centrifuged at 8000 x g for 20 minat 4°C and fusion proteins were isolated from the supernatantby affinity chromatography with Ni2+ resin (Qiagen, Chats-worth, CA), by the manufacturer's protocol.Immunoblot Analysis. Human fibroblast cell lines were

grown in lx minimum essential medium supplemented withglutamine (0.29 mg/ml) and penicillin/streptomycin (1000units/ml and 100 ,ug/ml, respectively). Cells were washedtwice with PBS. One milliliter of RIPA buffer (50 mMNaCl/1% Nonidet P-40/0.5% sodium deoxycholate/0.1%SDS/50 mM Tris HCl, pH 7.5) was added to a 100-mm dish,which was incubated on ice for 30 min. Cells and debris werescraped and centrifuged for 10 min at 10,000 x g and 4°C.For assessment of protein phosphorylation, human fibro-

blasts were grown as above in medium without phosphate butwith the addition of [32P]phosphate (0.25 mCi/100-mM dish;1 Ci = 37 GBq). Cells were grown with radioactivity for 6 h andprotein extracts were made with RIPA buffer. For Mt-PKimmunoprecipitation, 100 ,A of the protein extract was dilutedwith 150 mM NaCl/50 mM Tris-HCl, pH 7.6/leupeptin (0.5,ug/ml)/pepstatin (20 ,ug/ml) and 15 ,ul of the protein A-agarose and 10 ,ul of antibody 10033 were added. Afterovernight incubation at 4°C, immunoprecipitate was collectedby centrifugation at 4000 x g and washed five times with 1.0ml of 150 mM NaCl/50 mM Tris HCl/leupeptin (0.5 jig/ml)/pepstatin (20 ,ug/ml). Labeled Mt-PK was eluted with 20 ,1 of100 mM glycine (pH 3.0), neutralized with 1.0 M Tris-HCl (pH8.0), and loaded onto an 8% polyacrylamide gel.

Nuclear extracts and cytoplasm were prepared with a rapidprocedure. Fibroblasts were scraped, washed with PBS, andsuspended in 150 Al of buffer A [10 mM Tris HCl, pH 7.6/1.5mM MgCl2/10 mM KCl/0.5 mM dithiothreitol/leupeptin (0.5,ug/ml)/pepstatin (20 ,ug/ml)]. After a 15-min incubation onice, cells were homogenized by pulling them through a 23-gauge needle (six to eight strokes), and the sample wascentrifuged for 5 min at 10,000 rpm in a microcentrifuge at 4°C.Supernatant (cytoplasm) was collected and stored at -80°C.Nuclei (pellet) were resuspended in 20-50 Al of buffer B [20mM Tris HCl, pH 7.6/20% (wt/vol) sucrose/0.420 M NaCl/1.5 mM MgCl2/0.2 mM EDTA/0.5 mM dithiothreitol] andincubated on ice for 30 min. The nuclei were pelleted andsupernatant (nuclear extract) was removed, dialyzed againstbuffer C [20 mM Tris HCl, pH 7.6/20% (vol/vol) glycerol/20mM KCl/1.5 mM MgCl2/0.2 mM EDTA], and used immedi-ately.

Protein concentration was determined with Bradford re-agent by the Bio-Rad protocol. Twenty to 50 ,g of the proteinwas subjected to SDS/PAGE in 8% polyacrylamide gels by themethod of Laemmli (16). Proteins were transferred onto anitrocellulose membrane and incubated with affinity-purifiedantibodies 10033 (1:10,000 dilution), 254 (1:10,000 dilution), or8391 (1:8000 dilution) for 1 h at room temperature. Therecombinant fusion Mt-PK was detected with monoclonalantibodies to the gene 10 leader peptide (Novagen) (1:15,000dilution). The reaction was visualized by using a chemilumi-nescence kit (ECL detection kit, Amersham).

Determination of Mt-PK Kinase Activity. Enzyme assayswere performed at room temperature for 5 min in 50 ,ul ofassay buffer (50 mM Mops, pH 7.2/150 mM KCl/10 mMMgCl2/0.001 mM microsystin), 100 ,uM [y-32P]ATP, and 300

,M peptide substrate (Sigma) or 2-5 ,ug DHPR. The reactionwas stopped by dilution with electrophoretic loading buffer(0.015 M Tris-HCl, pH 6.8/2.5% glycerol/0.5% SDS/1.25%2-mercaptoethanol/0.0125% bromphenol blue). Phosphory-lated proteins were separated by SDS/PAGE on 10% poly-acrylamide gels and visualized by autoradiography. Whenpeptide was used as a substrate, the reaction was stopped withice-cold 75 mM H3PO4, and phosphorylated peptides wereseparated from incorporated [,y-32P]ATP on SpinZyme basicseparation units (Pierce) by the Pierce protocol.Immune Complex Protein Kinase Procedure. Frozen biop-

sies of human skeletal muscle were homogenized in 10 vol of50 mM Tris HCl, pH 7.6/1 mM EDTA/4 mM dithiothreitol/leupeptin (0.5 ,ug/ml)/pepstatin (20 gg/ml). The homogenatewas centrifuged at 10,000 rpm in a microcentrifuge at 4°C for5 min and supernatant was collected. The supernatants wereincubated with 10 ,pl of monoclonal antibodies against the f3subunit and 15 ,lI of protein A-agarose as described above. Theantigen-antibody complexes were washed five times with 150mM NaCl/50 mM Tris HCl, pH 7.6/leupeptin (0.5 ,g/ml)/pepstatin (20 ,ug/ml) and twice with kinase assay buffer (seeabove). The immune pellet was suspended in 20 ,ul of proteinkinase buffer containing 50 ,ul of ['y-32P]ATP and recombinantMt-PK (50 gg). After incubation for S min at room temper-ature, immunoprecipitate was collected by centrifugation at2500 rpm in a microcentrifuge for 15 min at 4°C. The pellet waswashed four times with 1.0 ml of assay buffer, resuspended in20 ,ul of electrophoresis sample buffer, and analyzed byelectrophoresis.

RESULTSImmunologic Characterization and Cellular Localization of

the Full-Length Mt-PK (72 kDa). To search for the full-lengthMt-PK, a number of different antibodies were tested. It hasbeen reported that antibodies against bacterial fusion protein10033 and anti-peptide antibody 254 recognized a 55-kDaprotein in human skeletal muscle biopsies (5). In addition,antibodies to the peptide Pro-Gly-Thr-Gly-Ser-Tyr-Gly-Pro-Glu-Cys-Asp-Trp from the Mt-PK kinase domain were devel-oped (antibody 8391). All antibodies recognized the full-lengthrecombinant Mt-PK expressed in pRSET expression vector inbacterial cells (Fig. 1). In these studies, Mt-PK was purified asa fusion protein from Escherichia coli cells transfected withpRMK, which contains the full-length Mt-PK cDNA codingregion in the pRSET expression vector. The molecular size ofthe Mt-PK fusion protein from pRMK is -75 kDa, whichcorresponds to the full-length Mt-PK (72 kDa) plus the fusionportion (3 kDa) (Fig. 1A, lane 1). The recombinant protein

AkDa97 -o69 -o46 -o-

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FIG. 1. Electrophoretic and immunoanalysis of the purified Mt-PKfusion protein after expression of pRMK in bacterial cells. (A)Coomassie brilliant blue R staining of the purified Mt-PK fusionprotein (lane 1) or control protein that was purified under the sameconditions as Mt-PK but without induction by IPTG (lane 2). (B)Products of pRMK were analyzed by electrophoresis, transferred tonitrocellulose, and probed with antibodies against the leader peptideof gene 10 (lane 1), with antibodies against Mt-PK 10033 (lane 3), orwith anti-peptide antibodies 8391 (lane 4). Control protein waspurified under the same conditions as Mt-PK but without induction byIPTG (lane 2).

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1 2 3

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FIG. 2. Immunoblot analysis of protein extract from human fibro-blasts with antibodies 10033 (lane 1), 254 (lane 2), and 8391 (lane 3).Positions of the 72-kDa and 55-kDa Mt-PK proteins are shown.

reacts with monoclonal antibodies to the gene 10 leaderpeptide (Fig. 1B, lane 1), antibody 10033 (Fig. 1B, lane 3), andanti-peptide antibodies 8391 (Fig. 1B, lane 4) and 254 (5).Since Western blot analysis shows no immunoreactivity inuninduced bacterial extract (Fig. 1B, lane 2), we conclude thatall three antibodies recognize the same fusion protein. Wethen determined the immunoreactive proteins recognizedfrom human cultured cells. Western blot analysis of proteinextracts from human fibroblast cell lines with antibody 10033detected the major 72-kDa protein and a minor 55-kDa protein(Fig. 2, lane 1). Antibody 254 recognized only the 55-kDaisoform from the same protein extracts (Fig. 2, lane 2) andantibody 8391 recognized both 72-kDa and 55-kDa proteins inthe total protein extracts from human fibroblast cell line (Fig.2, lane 3). Our results demonstrated that two independentlydeveloped antibodies to Mt-PK (10033 and 8391) interact witha 72-kDa protein, the predicted full length of Mt-PK. Based onthis observation, we suggest that the 72-kDa protein is afull-length translational product of Mt-PK mRNA. The lack ofantibody 254 recognition of the 72-kDa protein may reflect aprotected epitope in the 72-kDa protein or immunocrossre-activity.The cellular localization (cytoplasmic and/or nuclear) of the

putative immunoreactive Mt-PK proteins was determinedfrom fibroblast cell lines. By using antibody 8391, we detectedthe 72-kDa protein in the cytoplasm and the 55-kDa protein inthe nuclear extract (Fig. 3).The Full-Length (72 kDa) Mt-PK Phosphorylation and

Kinase Activity. The nucleotide sequence of Mt-PK cDNAshows a high identity with Ser/Thr kinases. However, infor-mation about Mt-PK activity is very limited (14, 15). Weinvestigated whether Mt-PK proteins are phosphorylated invivo. Human fibroblasts cell line were, therefore, grown with[32P]phosphate and labeled proteins were isolated. Mt-PK wasimmunoprecipitated with antibody 10033 and analyzed byPAGE on 8% gels. Antibody 10033 interacted with both72-kDa and 55-kDa isoforms (Fig. 4A), but only the 72-kDaisoform was phosphorylated by [32P]phosphate in vivo (Fig.4B).To demonstrate protein kinase activity of the full-length

Mt-PK in vitro, recombinant fusion Mt-PK was purified from

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FIG. 3. Intracellular localization of Mt-PKs. One hundred micro-grams of total protein extract from human fibroblasts (lane 1), nuclearextract (lane 2), or cytoplasm (lane 3) was separated by electrophore-sis, transferred to nitrocellulose, and probed with antibody 8391.

FIG. 4. Phosphorylation of the 72-kDa Mt-PK in vivo. Immuno-detection of Mt-PK by Western blot analysis with antibody 10033 (A).Human fibroblasts were grown with [32P]phosphate and Mt-PK wasprecipitated with antibody 10033 (B). Immunoprecipitates were re-solved by SDS/PAGE in 8% gels and exposed to film for 4 days.

bacterial cells transfected with pRMK and analyzed as de-scribed above (Fig. 1A, lane 1). We first attempted to deter-mine whether Mt-PK demonstrates autophosphorylation, afeature of many other kinases. As a control we used a proteinfraction that was isolated from uninduced cells by the sameprocedure as Mt-PK purification (Fig. 1A, lane 2). As shownin Fig. SA, lane 2, the recombinant full-length Mt-PK hasstrong autophosphorylation but the control protein fractiondoes not (Fig. 5A, lane 1). The results of the thin layerchromatography of the radioactive residues from the phos-phorylated Mt-PK indicated significant radioactivity in thephosphoserine residue position and minor radioactivity in thephosphothreonine position (data not shown). We then testedwhether Mt-PK is a member of an established kinase family byusing synthetic peptides, including Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly (a peptide for Tyr kinase), Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg (a substrate for cAMP-dependent protein kinase), Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys (syntide 2, a substrate forCa2+/calmodulin-dependent kinase II and for Ca2+/phospholipid-dependent protein kinase), Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys (a substrate for protein ki-nase C), and Leu-Arg-Arg-Ala-Hse-Leu-Gly (a peptide con-taining homoserine, Hse). The recombinant Mt-PKsignificantly phosphorylated the peptide Gly-Arg-Gly-Leu-

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FIG. 5. Determination of kinase activity of Mt-PK in vitro. (A)Mt-PK autophosphorylation. Fusion protein (50 ng) was phosphory-lated and loaded onto a 10% SDS/polyacrylamide gel (lane 2).Phosphorylation of the control protein, which was purified under thesame condition as Mt-PK but without induction by IPTG (lane 1). Theautoradiogram was prepared after a 4-h exposure. (B) Phosphoryla-tion of synthetic peptides by Mt-PK. Each peptide at 1 jig [Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly (Pep. 1), Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg (Pep. 2), Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys (Pep. 3), Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys (Pep. 4), and Leu-Arg-Arg-Ala-Hse-Leu-Gly (Pep. 5, where Hse is homoserine)] was incubated withrecombinant Mt-PK. Incorporated radioactivity was determined bythe Pierce protocol.

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FIG. 6. (A) Phosphorylation ofDHPR by Mt-PK in vitro. One hundred nanograms ofDHPR was phosphorylated with 100 ng of Mt-PK, purifiedafter expression of pRMK (lane 2). Lane 1 is a control with no Mt-PK. The membrane from A was probed with monoclonal antibodies againstthe ,3 subunit of DHPR (lane 3). The 1B subunit of DHPR was precipitated from human muscle extract and the immunoprecipitate was incubatedwith fusion Mt-PK in kinase buffer. Immunoprecipitate from the kinase reaction mixture was then analyzed by electrophoresis and exposed to x-rayfilm overnight (lane 5). Lanes 4 and 6 are controls where antibodies to the subunit were phosphorylated without protein extract (lane 4) or withoutprotein extract or Mt-PK (lane 6). (B) Phosphorylation of the synthetic peptide from the subunit of DHPR by recombinant Mt-PK.

Ser-Leu-Ser-Arg, a substrate for cAMP-dependent kinases(Fig. SB). This peptide contains a Ser residue in the phos-phorylation site. Collectively, these results indicate that Mt-PKis a member of the Ser/Thr kinase family.

Phosphorylation of the ,3 Subunit ofDHPR by Mt-PK. Thephosphorylation of DHPR by Mt-PK fusion protein wasstudied in vitro (Fig. 6A). Biochemically purified DHPR (17),kindly provided by S. Hamilton, was used in the reactionmixture with Mt-PK fusion protein. DHPR contains an endo-genous kinase that phosphorylates the major proteins ofDHPR (18) requiring use of a low concentration ofDHPR (60,g/ml). As shown in Fig. 6A, lane 2, Mt-PK does not change thephosphorylation of the high molecular weight protein (at sub-unit) but strongly phosphorylates the 56-kDa protein (( subunit)(19). Confirmation of the 56-kDa protein as the (3 subunit ofDHPR (20) was made by two studies. (i) The subunits ofDHPRwere phosphorylated as described, electrophoresed, and trans-ferred to nitrocellulose. The membrane was then probed withmonoclonal antibodies to the 1B subunit of DHPR. Fig. 6A, lanes2 and 3, shows that the phosphorylated 56-kDa protein reactingwith the antibodies has the same mobility as the 13 subunit ofDHPR. (ii) These antibodies were used to precipitate the 56-kDaprotein from human muscle biopsy material; this protein was thenincubated with recombinant Mt-PK. The recombinant Mt-PKstrongly phosphorylated the ,B subunit within the immunopre-cipitate (Fig. 6A, lane 5). Lanes 4 and 6 are controls whereantibodies to the 3subunit were phosphorylated without proteinextract (lane 4) or without protein extract or Mt-PK (lane 6).These results indicate that the (3 subunit of DHPR can bephosphorylated by Mt-PK in vitro. It is important to note that wehave observed coimmunoprecipitation of Mt-PK and the (3subunit of DHPR during immunoprecipitation of Mt-PK fromhuman cells (unpublished data).The (3 subunit of DHPR contains 19 sites for phosphoryla-

tion by various protein kinases (19) including a specific Leu-Arg-Gln-Ser-Arg-Leu-Ser-Ser-Ser-Lys sequence. This peptidecontains two Ser residues (Ser-179 and Ser-183) predicted tobe phosphorylated and a Ser (Ser-182) phosphorylated bycAMP-dependent protein kinase in vitro (19). RecombinantMt-PK also phosphorylates this peptide (Fig. 6B).

DISCUSSIONAnalysis of Mt-PK cDNA (5) shows that the first in-frameAUG codon is in nt 546. If this AUG codon is the authenticsite of initiation, the size of the translation product should be-72 kDa. Mt-PK cDNA contains four additional in-frameAUG codons within the translated region in nt 870, 1017, 1704,and 1836. The initiation of translation from theseAUG codons

would give alternative protein products with approximatemolecular sizes of 59, 55, 30, and 28 kDa, respectively. Noneof the AUG codons possesses a favorable "Kozak" sequence(21, 22) for translational initiation, so leaky initiation oftranslation could produce alternative isoforms. It is possiblethat the 72-kDa protein is a full-length version of Mt-PK andthat the 52- to 55-kDa proteins are formed because of alter-native initiation of translation. We have investigated thekinetics of 35S-labeling of the 72- and 55-kDa proteins in vivoand found that both proteins had identical kinetics of labeling(unpublished data). Translation of Mt-PK mRNA isolated byselective hybridization in a cell-free system and immunopre-cipitation of translational products indicated biosynthesis ofthe major 72-kDa protein and minor 55-kDa protein (unpub-lished data). Thus, these observations suggest that the 55-kDaprotein probably is an alternative translational product oranother kinase with significant homology to Mt-PK.

In these studies, we used two antibodies to Mt-PK (anti-bodies 10033 and 8391) and showed the existence of animmunoreactive polypeptide with a molecular size of 72 kDacorresponding to the predicted size of the full-length Mt-PK.Identification of the 72-kDa immunoreactive proteinprompted us to investigate the steady-state levels of theseproteins in muscle cell lines from normal individuals and DMpatients. We observed decreased levels of the major 72-kDaprotein in muscle cells with reduced levels of Mt-PK (unpub-lished data). In addition, we found that only the 72-kDaprotein was phosphorylated in human normal fibroblasts.Kinase activity studies in an in vitro assay show that thefull-length recombinant Mt-PK displays autophosphorylation.Phosphorylation of the 72-kDa Mt-PK in vivo in humanfibroblasts may be a result of autophosphorylation, or thefull-length protein may be a substrate for other kinases. Thesedata demonstrate that the 72-kDa protein is a translationalproduct of Mt-PK mRNA and implicates the kinase in thepathology of DM. The nature of immunoreactive 55-kDaprotein should be investigated.Mt-PK shows very high homology to Ser/Thr kinases (2-4).

We observed Mt-PK activity on a Ser peptide that is also asubstrate for cAMP-dependent kinases. In addition, Mt-PKfusion protein phosphorylated in vitro a Ser-containing peptidefrom DHPR. These data suggest that the full-length Mt-PKcan phosphorylate Ser residues in vitro. The phosphorylationof the Ser-containing substrates by the full-length Mt-PK is inagreement with results of phosphorylation of Thr and Serresidues by truncated Mt-PK (15). One group also found thatthe 54-kDa Mt-PK, precipitated from muscle cell extracts,phosphorylated Tyr (14). It is possible that different immu-noreactive Mt-PKs have different substrate specificities. Be-

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cause the 72-kDa Mt-PK was phosphorylated in vivo, westggest that the kinase activity of the full-length Mt-PK isregulated by phosphorylation. No information is availableabout the substrate(s) molecule for Mt-PK. Since DM is amuscle disease with defects in muscle excitability, ion channelsthat are involved in the regulation of muscle activity would begood candidates for biological substrates for Mt-PK. Severaldisorders are characterized by abnormal muscle membranehyperexcitability. For most of these diseases, specific muta-tions have been found in ion channel genes. In Thompsendisease (23) and recessive generalized myotonia (24), pointmutations in chloride channel genes have been found. Pointmutations were observed in sodium channel genes in paramyo-tonia congenita and hyperkalemic periodic paralysis (25-27).In hypokalemic periodic paralysis, mutations in the al subunitof DHPR were demonstrated (28). In this paper we describethe phosphorylation of the Ca2+ channel, ,B subunit of DHPR,by recombinant Mt-PK in vitro as a possible candidate sub-strate of Mt-PK. The biological role of this phosphorylationshould be investigated in vivo, as should the phosphorylationof other ion channels.

We thank S. L. Hamilton, D. J. Sweatt, L. Birnbaumer, C. Wei,B. J. F. Rossiter, T. Ashizawa, and P. R. Clemens for their interest andconstructive comments and S. Vaishnav for skilled technical assis-tance. This work was supported in part by The Welch Foundation.C.T.C. is an Investigator with the Howard Hughes Medical Institute.

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3. Fu, Y.-H., Pizzuti, A., Fenwick, R. G., Jr., King, J., Rajnarayan,S., Dunne, P. W., Dubel, J., Nasser, G. A., Ashizawa, T., de Jong,P., Wieringa, B., Korneluk, R., Perryman, M. B., Epstein, H. F.& Caskey, C. T. (1992) Science 255, 1256-1258.

4. Brook, J. D., McCurrach, M. E., Harley, H. G., Buckler, A. J.,Church, D., Aburatani, H., Hunter, K., Stanton, V. P., Thirion,J.-P., Hudson, T., Sohn, R., Zemelman, B., Snell, R. G., Rundle,S. A., Crow, S., Davies, J., Shelbourne, P., Buxton, J., Jones, C.,Juvonen, V., Johnson, K., Harper, P. S., Shaw, D. J. & Housman,D. E. (1992) Cell 68, 799-808.

5. Fu, Y.-H., Friedman, D. L., Richards, S., Pearlman, J. A., Gibbs,R. A., Pizzuti, A., Ashizawa, T., Perryman, M. B., Scarlato, G.,Fenwick, R. G., Jr., & Caskey, C. T. (1993) Science 260,235-238.

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