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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Proc. Natl. Acad. Sci. USA 92 (1995) 5367
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-
1 2
0, .o
B1 2 3 4
kDa
97 -o
69-...
34 46
34
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).
Biochemistry: Timchenko et at
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5370 Biochemistry: Timchenko et at
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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