From the
The principal (
L-type, or dihydropyridine-sensitive, calcium channels mediate a
voltage-sensitive increase in cytosolic calcium in response to
depolarization in virtually all excitable tissues(1) . They are
the major route for voltage-gated calcium entry in heart and smooth
muscle (2) and are also thought to serve as the voltage sensor that
initiates excitation-contraction coupling in skeletal muscle directly
without calcium influx from the extracellular
solution(3, 4) . The L-type calcium channel purified
from skeletal muscle transverse tubules (5) is a complex of five
subunits(6, 7, 8, 9) . The principal
The
The cDNA encoding the
Antibodies directed against synthetic calcium channel
Synthetic calcium channel peptides were
synthesized using the solid phase method of Merrifield(37) . The
identity of the synthetic peptides was confirmed by amino acid analysis
or mass spectrometry. The synthetic peptide (CP38) used to identify
tryptic phosphopeptide 6 corresponds to residues 1752-1769 of the
calcium channel
For
immunoprecipitation of calcium channels from myotube cultures,
individual dishes were washed on ice two times with 1 ml of bRIA (50
mM Tris-HCl (pH 7.5), 0.5 mM MgCl
The fusion proteins
CaFSk
Purified fusion proteins were
phosphorylated as described above in 50 mM Tris, pH 7.4, 5
mM MgCl
We thank Dr. Steven B. Ellis, Dr. Arnold Schwartz, and
Dr. Michael M. Harpold for providing the clone pSkmCaCh
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1) subunit of purified skeletal muscle
dihydropyridine-sensitive (L-type) calcium channels is present in
full-length (212 kDa) and COOH-terminal truncated (190 kDa) forms,
which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. Immunoprecipitation of the calcium channel from
rabbit muscle myotubes in primary cell culture followed by
phosphorylation with cA-PK, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, and two-dimensional phosphopeptide mapping revealed
comparable phosphorylation of three COOH-terminal phosphopeptides found
in the purified full-length
1 subunit. Stimulation of muscle
myotubes with a permeant cAMP analogue, 8-(4-chlorophenylthio)
adenosine 3&cjs1227;,5&cjs1227;-cyclic monophosphate, prior to
immunoprecipitation of
1 results in a 60-80% reduction of
cA-PK catalyzed ``back'' phosphorylation of each of these
sites in vitro in calcium channels purified from the cells,
indicating that these sites are phosphorylated in vivo in
response to increased intracellular cAMP. Serine 687, the most rapidly
phosphorylated site in the truncated 190-kDa
1 subunit, was
observed as a minor phosphopeptide whose level of phosphorylation was
not significantly affected by stimulation of endogenous cA-PK in the
myotubes. The COOH-terminal sites, designated tryptic phosphopeptides
4, 5, and 6, were identified as serine 1757 (phosphopeptides 4 and 6)
and 1854 (phosphopeptide 5) by a combination of protease cleavage,
phosphorylation of synthetic peptides and fusion proteins, specific
immunoprecipitation, and phosphopeptide mapping. Phosphorylation of
serines 1757 and 1854 in the COOH-terminal region of the 212-kDa
1
subunit in intact skeletal muscle cells may play a pivotal role in the
regulation of calcium channel function by cA-PK.
1 subunit has receptor sites for calcium antagonist drugs (6, 10, 11, 12) and multiple
transmembrane segments (6, 13) and alone can form a
functional voltage-sensitive ion
channel(14, 15, 16) . The
1 subunit is
isolated in association with a
subunit (54 kDa), a glycosylated
subunit (30 kDa), and a glycosylated disulfide-linked dimer of
2 (143 kDa) and
(27 kDa) subunits that are encoded by the
same
gene(5, 6, 7, 8, 9, 17, 18, 19) .
1 and
subunits of the skeletal muscle L-type channel
are substrates in vitro for many protein kinases including
cA-PK,
(
)protein kinase C, casein kinase II, and
a multifunctional Ca
/calmodulin-dependent protein
kinase(6, 20, 21, 22) . Purified and
reconstituted calcium channels are activated by phosphorylation of
their
1 and
subunits by
cA-PK(23, 24, 25) , and it is likely that this
phosphorylation represents the mechanism by which L-type calcium
channels are activated by
-adrenergic stimulation in the heart and
skeletal muscle(2, 26, 27, 28) . More
recently, studies of calcium channels in skeletal muscle cells in
culture (29, 30) and cardiac L-type calcium channel
1 subunits expressed in Chinese hamster ovary cells (31) indicate that
1 subunits of calcium channels may be
phosphorylated by cA-PK in intact cells (29) and that
voltage-dependent phosphorylation of
1 subunits by cA-PK can
modulate channel activity in response to repetitive depolarizing
stimuli(30, 31) .
1
subunit predicts a mature protein of 212 kDa (13), while the purified
protein has an apparent molecular mass of 155-175 kDa in standard
SDS-PAGE(5, 6, 7, 8, 9) . The
skeletal muscle calcium channel
1 subunit exists in two
forms(32, 33) , one truncated at the COOH-terminal tail.
The major form has an apparent molecular mass of 155-175 kDa
under standard conditions of SDS-PAGE or approximately 190 kDa
(
1
) by Ferguson plot analysis (33) and is
truncated near residue 1700(33) . Anti-peptide antibodies
directed against the COOH terminus of the predicted amino acid sequence
of the
1 subunit detect only a small fraction of full-length
protein (<5%) with an apparent molecular mass of 212 kDa
(
1
)(32, 33) . The most rapidly
phosphorylated site in the 190-kDa form of the
1 subunit is serine
687(34) . However, the predicted amino acid sequence of the
212-kDa form of the
1 subunit contains three potential
phosphorylation sites that are absent from the 190-kDa form, suggesting
a possible mechanism for differential regulation of the two
forms(32, 33, 35) . Studies of phosphorylation
of calcium channel
1 subunits in skeletal muscle cells in culture
indicate that phosphopeptides unique to the 212-kDa form are
phosphorylated in intact cells(29) . We have previously
identified the major site of in vitro phosphorylation of the
full-length
1 subunit immunoprecipitated from purified calcium
channel preparations as serine 1854(35) . This residue was
rapidly and specifically phosphorylated by cA-PK, at least 100-fold
more rapidly than serine 687 in
1
. In this study, we
utilized fetal rabbit skeletal muscle myotubes to examine the
phosphorylation of the full-length
1 subunit by cA-PK and to
identify the predominant residues in the full-length
1 subunit
that are phosphorylated in vitro and in intact cells.
Materials
Materials were obtained from
the following sources: protein A-Sepharose, Triton X-100,
phenylmethanesulfonyl fluoride, benzamidine, pepstatin A, 8-cpt-cAMP,
okadaic acid, penicillin G, streptomycin sulfate, DNase I, ara-C, and
crude trypsin II from Sigma; endoproteinase Lys-C, leupeptin, and
aprotinin from Boehringer Mannheim; calpain inhibitors I and II and
Microcystin LR from Calbiochem; purified rabbit IgG from Zymed;
thin-layer cellulose plates from Kodak; TPCK-treated trypsin from
Worthington Biochemicals; [-
P]ATP from
DuPont NEN; Dulbecco's modified Eagle's medium from Life
Technologies, Inc.; horse and newborn calf serum from Hazleton;
digitonin from Gallard-Schlesinger Inc. (Carle Place, NY). The
catalytic subunit of cA-PK was purified according to Kaczmarek et
al.(36) . Calcium channels were purified from skeletal
muscle microsomes as previously described(5, 6) .
1 subunit
peptides (CP) were prepared as previously
described(32, 33) . Peptide CP1 corresponds to residues
1856-1873 of the
1 subunit plus NH
-terminal
lysine and tyrosine, peptide CP10 corresponds to residues
1738-1752 plus NH
-terminal lysine and tyrosine,
peptide CP15 corresponds to residues 1382-1399 plus
NH
-terminal lysine and tyrosine, and peptide CP20
corresponds to residues 1692-1707 plus NH
-terminal
lysine and tyrosine.
1 subunit.
Two-dimensional Phosphopeptide
Analysis
Phosphopeptide maps were generated by a
modification of the method of Huttner and Greengard (38) as
previously described(35) .
Phosphorylation, Purification, and Proteolytic
Digestion of Synthetic Peptide CP38
For identification of
tryptic phosphopeptide 6 using CP38, the method reported by Murphy et al.(39) was used. Briefly, purified CP38 (50 nmol)
was phosphorylated by incubation at 37 °C in 50 mM
Tris-HCl (pH 7.5), 6 mM MgCl, 1 mM dithiothreitol, 5 mM ATP, 0.25 µM [
-
P]ATP (3000 Ci/mmol) for 16 h in the
presence of 2-10 nM purified catalytic subunit of cA-PK.
Phosphorylation reactions were terminated by addition of 0.05 ml of 30%
acetic acid and rotated for 1 h in the presence of 0.05 ml of settled
Dowex 1-X8 in 30% acetic acid to remove excess
[
-
P]ATP. Dowex was removed by brief
microcentrifugation. Phosphorylated peptide was then purified by
reversed phase high pressure liquid chromatography on an 8
100-mm Waters Delta-Pak C-18 column (15 µM, 300 Å).
Peptide was eluted with a 0-60% B gradient (1%/min) where A
consisted of 0.1% trifluoroacetic acid. Purified phosphorylated CP38
(10,000-20,000 cpm) was digested for 16 h at 37 °C in 0.5 ml
of 25 mM ammonium bicarbonate (pH 8.9) in the presence of
10-20 µg of TPCK-treated trypsin. Supernatants were
lyophilized, resuspended 1 ml of H
O, and relyophilized.
Digested peptide was then resuspended in 10 µl of 1% pyridine, 10%
acetic acid (pH 3.5) containing a trace amount of phenol red and
subjected to two-dimensional phosphopeptide mapping analysis as
described above.
Muscle Cell Culture
Rabbit myotube
cultures were prepared by a modification of the method of Schaffner and
Daniels(40, 41) . Minced muscle from the forelimbs of
day 29 New Zealand White rabbit (R& Rabbitry, Stanwood, WA)
fetuses was dissociated for 20 min at 37 °C in 0.2% trypsin and
0.1% DNase in Dulbecco's modified Eagle's medium.
Non-dissociated tissue was pelleted by centrifugation at 1000 rpm for
10 s; the supernatant was decanted, adjusted to 10% newborn calf serum
to inhibit trypsin, and saved; the pelleted tissue was then subjected
to two additional 20-min incubations with enzyme. The cell suspensions
were pooled and passed through a Nitex filter (pore size, 70
µm) to remove large pieces of tissue. Cells were
pelleted by centrifugation for 10 min at 1200 rpm and plated in 100-mm
gelatin-coated plastic tissue culture dishes at a density of 4
10
cells/ml in 80% Dulbecco's modified Eagle's
medium, 15% horse serum (heated at 56 °C for 45 min prior to use),
5% newborn calf serum, 10 µg/ml streptomycin, and 30 µg/ml
penicillin G. After cells reached confluence (4-5 days), fresh
medium was added including 10 µM cytosine arabinoside to
inhibit fibroblast proliferation. Cytosine arabinoside was removed
after 2-3 days, and the fused myotube cultures were fed once more
prior to use at 10-12 days.
Immunoprecipitation and Phosphorylation of Calcium
Channels
All buffers and solutions contained protease
inhibitors as above (without calpain inhibitors). Standard
phosphorylation and immunoprecipitation of purified calcium channels
was performed as previously described(35) .
, 0.2 M NaCl, 10 mM EDTA, 20 mM sodium
pyrophosphate, 100 mM sodium fluoride, and 1.0 mg/ml bovine
serum albumin). 300 µl of bRIA including 1% Triton X-100 were added
to each dish, the myotubes were scraped, pooled, lysed, and homogenized
with 50 strokes in a Dounce tissue homogenizer, and the homogenate was
centrifuged at 11,000 rpm for 15 min in a SS-34 rotor. Supernatants
were rotated on ice for 3-4 h with a 1:30 dilution of the
appropriate antibody and then incubated with protein A-Sepharose (PAS)
beads for 1 h. The antigen/antibody/PAS conjugates were then pelleted,
washed once with bRIA including 1% Triton X-100, washed twice with
phosphorylation buffer (50 mM Tris-HCl, pH 7.5, 5 mM magnesium acetate, 1 mM EGTA) including 0.1% Triton
X-100, and incubated for 10 min at 37 °C in 50 µl of
phosphorylation buffer including 0.25 µM [
-
P]ATP and 10 nM catalytic
subunit of cA-PK. Phosphorylation reactions were terminated by addition
of 20 mM EDTA. The PAS pellets were washed twice with bRIA (1%
Triton X-100), and 100 µl of 50 mM Tris-HCl, 1% SDS buffer
were added. After boiling for 2 min, the samples were centrifuged, and
400 µl of bRIA including 2% Triton X-100 (6-fold Triton/SDS ratio)
were added to the supernatants. Once again, appropriate antibody was
added to samples at a 1:30 dilution for a second immunoprecipitation at
4 °C for 16 h. After a second PAS precipitation step, samples were
washed three times with 1% Triton X-100 bRIA and once with
H
O; the PAS pellets were then released into 50 µl of
Laemmli SDS sample buffer.
Construction and Expression of Recombinant
Glutathione S-Transferase Fusion Proteins
The fusion
protein CaFSk1-wt was generated from pSkmCaCh
1.8 (18) using the polymerase chain reaction. A 336-base pair
fragment containing amino acid residues 1722-1834 of the cardiac
1 subunit was amplified by polymerase chain reaction and cloned
into the pGEX-3X expression vector (Pharmacia Biotech Inc.) to obtain
in-frame recombinant proteins containing glutathione S-transferase. EcoRI and BamHI sites were
included at the ends of the amino- and carboxyl-terminal
oligonucleotides, respectively, to facilitate cloning. All constructs
were verified by DNA sequencing and transformed into a
protease-deficient strain BL26 of Escherichia coli (Novagen).
Overnight cultures grown in 20 ml of YT media supplemented with 100
mg/ml ampicillin (YT-Amp) were used to innoculate 500 ml of YT-Amp
containing 0.4% glucose and incubated at 37 °C for 3 h with
shaking. Fusion protein synthesis was induced by the addition of 2
mM isopropyl-
-D-thiogalactopyranoside, and the
cells were cultured for an additional 2.5 h before harvesting by
centrifugation at 5000
g for 10 min. Cell pellets were
resuspended in 10 ml of phosphate-buffered saline containing 0.2 mM 4-(aminoethyl)benzenesulfonyl fluoride, 2 µM pepstatin A, 2 mg/ml aprotinin, 2 µM leupeptin, 0.2
mM benzamidine, 0.5 mM EDTA, and 0.5 mM EGTA, and the bacteria were lysed by mild sonication. The mixture
was adjusted to 1% Triton X-100, incubated 15 min on ice, and
centrifuged at 10,000
g for 10 min. The supernatants
were stored at -80 °C. The glutathione S-transferase
fusion protein was purified from cell lysates by affinity
chromatography on glutathione-Sepharose 4B according to the
manufacturer's instructions (Pharmacia).
1-S1771/2A, and CaFSk
1-S1757A are identical to
CaFSk
1-wt, except that the serine residues corresponding to amino
acids 1771, 1772, and 1757 of the skeletal
1 subunit were mutated
to alanine residues using polymerase chain reaction
mutagenesis(42) .
, 1 mM EGTA, 10 mM dithiothreitol, 0.1% Triton X-100 with 0.15 µM [
-
P]ATP (3000 Ci/mmol). The reaction,
initiated by the addition of 1 µg of catalytic subunit of cA-PK,
was carried out at 20 °C for 1 min and stopped by heating in
SDS-PAGE sample buffer at 65 °C for 5 min.
P-Labeled
fusion proteins were analyzed by SDS-PAGE on 8.5% porous polyacrylamide
gels (43) or on 10-20% Tricine gradient gels (NOVEX, San
Diego, CA) and located by autoradiography.
SDS-PAGE
PAS-bound immunoprecipitates
were boiled and subjected to SDS-PAGE according to the method of
Laemmli (44) using 6.5% acrylamide, 0.17% bisacrylamide.
Autoradiography was performed on wet gels. To quantify P
incorporation, gel slices corresponding to proteins of interest were
excised and counted by Cerenkov spectrometry. To detect endoproteinase
Lys-C calcium channel fragments, the modification of the Laemmli method
reported by Schagger and von Jagow (45) was used. This
Tricine/SDS-PAGE method is useful for the separation of proteins in the
range from 1 to 100 kDa.
Immunoprecipitation of Phosphorylated 212-kDa
Solubilization and immunoprecipitation of calcium
channels from primary cultures of rabbit skeletal muscle myotubes,
followed by cA-PK phosphorylation, a second immunoprecipitation,
SDS-PAGE, and autoradiography as described under ``Experimental
Procedures'' revealed a single band at approximately 200 kDa (Fig. 1A, lane 2), while immunoprecipitation
with non-immune IgG did not reveal any immunoreactive protein bands (Fig. 1A, lane 1). A less prominent band was
detected just above the 200-kDa protein that represents nonspecific
immunoprecipitation as determined by prolonged exposure of the
non-immune IgG lane. To identify the 200-kDa protein band, we compared
its mobility on SDS-PAGE with the 212-kDa form of the purified skeletal
muscle 1 Subunit from Fetal Rabbit Skeletal Muscle Myotube Calcium
Channels
1 subunit recognized by anti-CP20 (Fig. 1B, lane 1) and the mixture of 190- and 212-kDa forms recognized
by anti-CP15 (Fig. 1B, lane 2). Anti-CP20 and
anti-CP1, which are directed to the COOH segment of the
1 subunit
that is present only in the 212-kDa form, both immunoprecipitated
1 subunits from muscle cells with an apparent molecular mass
comparable to
1
from purified channel in the double
immunoprecipitation protocol (Fig. 1B, lanes 3 and 5). Non-immune IgG again was ineffective in
immunoprecipitating
1 subunits (Fig. 1B, lane
6). Anti-CP15, which recognizes both full-length and truncated
forms of the
1 subunit, also primarily immunoprecipitated the
212-kDa form (lane 4). The apparent molecular masses of the
1 subunits immunoprecipitated from primary cultures of skeletal
muscle myotubes by these three different antibodies (Fig. 1B, lanes 3-5) are greater than
that of the 190-kDa form (lane 2) but comparable to that of
the 212-kDa form (lane 1). These results show that the
full-length
1 subunit (
1
) is the major form
expressed in these fetal rabbit muscle cell cultures.
Figure 1:
Immunoprecipitation of 1 subunits
from fetal rabbit skeletal muscle. A, calcium channel was
immunoprecipitated from digitonin-solubilized fetal muscle myotubes as
described under ``Experimental Procedures'' with anti-peptide
antibody CP20 (lane 2) or non-immune rabbit IgG control (lane 1) and analyzed by SDS-PAGE (6.5% acrylamide) and
autoradiography. B, calcium channel purified (PCh) by
wheat germ agglutinin-Sepharose chromatography and sucrose density
gradient sedimentation was phosphorylated with cA-PK,
immunoprecipitated as described under ``Experimental
Procedures'' with anti-peptide antibodies CP20 (lane 1)
or CP15 (lane 2), and analyzed by SDS-PAGE (5 pmol of
channel/lane) as above. Calcium channel was immunoprecipitated and
phosphorylated from Triton X-100 solubilized fetal muscle myotubes (MT) as described under ``Experimental Procedures''
with anti-peptide antibodies CP20 (lane 3), CP15 (lane
4), CP1 (lane 5), or non-immune rabbit IgG control (lane 6). All immunoprecipitations from fetal muscle myotubes
in panelsA and B represent double
immunoprecipitation protocols. Second immunoprecipitations were
performed by releasing PAS pellets by boiling for 2 min and incubating
supernatants with the same antibody, as indicated, overnight at 4
°C. The second PAS precipitations (1 h) were released into SDS
sample buffer and analyzed as above.
Phosphopeptides of
Two-dimensional tryptic phosphopeptide maps
of the single 1
from Cultured
Rabbit Muscle Cells
1 band immunoprecipitated from the muscle cell
cultures confirmed the identity of this form as
1
.
Phosphopeptide maps of the 190-kDa protein isolated from purified
calcium channels revealed a distinctive pattern of a single predominant
phosphopeptide (Fig. 2A,phosphopeptide2) and a phosphopeptide of intermediate intensity (phosphopeptide1, Ref. 35). Each of these
phosphopeptides was previously sequenced (35) and found to
derive from alternate trypsin cleavage of the sequence rich in basic
residues surrounding serine 687, the most rapidly phosphorylated
residue in
1
. In contrast, the three major
COOH-terminal phosphopeptides (numbers4-6) are
preferentially phosphorylated in
1
immunoprecipitated from purified channel with anti-CP20 (Fig. 2B). Virtually identical phosphopeptide maps
containing predominantly phosphopeptides 4-6 were obtained after
immunoprecipitation of the
1 subunit from cultured skeletal muscle
myotubes with anti-CP20 (Fig. 2C) and with anti-CP1 or
anti-CP15 (data not shown). These data show that skeletal muscle
calcium channels containing full-length
1, when isolated from
either purified preparations or directly from primary cell culture, are
preferentially phosphorylated on three sites that are unique to the
1
.
Figure 2:
Phosphopeptide map of calcium channel
1 subunits from rabbit muscle cells. A, purified calcium
channels were phosphorylated and immunoprecipitated by anti-CP15, which
recognizes both full-length and truncated forms, as described under
``Experimental Procedures'' and in the legend to Fig. 1.
Phosphopeptides were generated from immunoprecipitated, phosphorylated
1 subunits as described under ``Experimental
Procedures'' and were first separated by electrophoresis (negative
on left) on the horizontal dimension and then thin-layer
chromatography on the vertical dimension. The plates were dried, and
autoradiography was performed. B, a two-dimensional tryptic
phosphopeptide map of purified calcium channel
1 subunit
immunoprecipitated by anti-CP20. C, calcium channels were
immunoprecipitated from muscle myotubes by anti-CP20 and analyzed by
SDS-PAGE. A two-dimensional phosphopeptide map of the phosphorylated
1 subunit was generated as described under ``Experimental
Procedures.'' Arrows point to consistently observed
phosphopeptides. Dotted circles represent COOH-terminal
phosphopeptides absent from
1
. The origin of
migration is denoted by
``o.''
Endogenous Phosphorylation of
To determine whether
the phosphopeptides generated from the 1
in
Stimulated Rabbit Skeletal Muscle Myotubes
1 subunit are
phosphorylated in intact cells, we compared the amount of
P incorporated into the
1 subunit immunoprecipitated
from cells that had been incubated with or without 8-cpt-cAMP to
increase intracellular cAMP levels and activate cA-PK. Calcium channel
1 subunits were substantially phosphorylated in vitro after immunoprecipitation from untreated cells (Fig. 3, lane2). In contrast, after treatment of the intact
cells with 8-cpt-cAMP, incorporation of
P into the
1
subunit in vitro was diminished 60-70% from that
observed in untreated control cells (Fig. 3, lane3). Fig. 3, lane1, represents an
IgG control, demonstrating the specificity of the immunoprecipitation
method. These results indicate that substantial in vivo phosphorylation of the
1 subunit induced by treatment with
8-cpt-cAMP caused a corresponding reduction in back phosphorylation in vitro.
Figure 3:
Stimulation of fetal rabbit myotubes with
8-cpt-cAMP. Individual dishes of fetal muscle myotubes were incubated
at 37 °C for 10 min with or without 100 µM 8-cpt-cAMP.
Dishes were washed twice with ice-cold bRIA, and myotubes were lysed
and prepared for immunoprecipitation as described under
``Experimental Procedures'' with the following exceptions:
digitonin (2% for cell lysis, concentrations at all other steps
followed ``Experimental Procedures'') replaced Triton X-100
in all buffers and solutions; beginning with the lysis buffer, all
solutions contained okadaic acid (0.1 µM, final
concentration) and microcystin LR (2 µM, final
concentration) to inhibit endogenous phosphatase activity.
Immunoprecipitations for each treatment group utilized anti-CP20 or
control nonimmune IgG, and phosphorylation and SDS-PAGE were performed
as described under ``Experimental Procedures.'' Lane
1, non-immune rabbit IgG; lane 2, anti-CP20; lane3, anti-CP20 after treatment with
8-cpt-cAMP.
Phosphorylation of Phosphopeptides 4, 5, and 6 in
Intact Cells
To assess the effect of stimulation with
8-cpt-cAMP on P incorporation into specific
phosphopeptides, we generated tryptic phosphopeptide maps of
1
subunits isolated from treated or untreated cells. Fig. 4A represents the typical pattern of phosphorylation of COOH-terminal
sites in
1
immunoprecipitated from untreated
myotubes. Diminished back phosphorylation of each of the three
COOH-terminal phosphopeptides was observed in the map from
8-cpt-cAMP-treated cells (Fig. 4B, phosphopeptides4, 5, and 6). The magnitude of this
diminishment was 70% for phosphopeptide 4, 60% for phosphopeptide 5,
and 80% for phosphopeptide 6. The mapping experiments also indicate
that serine 687 (Fig. 4, phosphopeptide2) is
not a substrate for in vivo phosphorylation of
1
in response to elevation of cAMP as no significant change in the
low level of
P incorporation into this site was observed.
This result serves as a useful internal control for the effect of
8-cpt-cAMP treatment on phosphorylation of phosphopeptides 4, 5, and 6.
Thus, treatment with 8-cpt-cAMP caused a substantial and specific
increase in endogenous phosphorylation of each of the three
phosphopeptides that are unique to the full-length form of the
1
subunit.
Figure 4:
Phosphorylation of individual
phosphopeptides in intact cells. A, calcium channels were
immunoprecipitated from fetal rabbit myotubes incubated under control
conditions with anti-CP20, and the phosphopeptides were mapped as
described under ``Experimental Procedures.'' B,
companion cell cultures were treated with 8-cpt-cAMP; the calcium
channels were isolated by immunoprecipitation with anti-CP20, and
phosphopeptide maps were generated as described under
``Experimental Procedures.'' Arrows point to
consistently observed phosphopeptides. The origin of migration is
denoted by ``o.''
Identification of a COOH-terminal Peptide Containing
Tryptic Phosphopeptides 4 and 6
The three major tryptic
phosphopeptides generated from cA-PK phosphorylation of the intact
1 subunit are likely to be located between residues 1685 and 1873
in the intracellular COOH-terminal domain of the skeletal muscle L-type
calcium channel since they are phosphorylated in the full-length form
of the
1 subunit but not in the truncated
form(32, 33, 35) . Analysis of the predicted
amino acid sequence of the
1 subunit indicates that there are
three potential sites in this region with the preferred consensus
sequence RRX(S/T) (46) corresponding to Ser-1757,
Ser-1772, and Ser-1854. Consistent with this prediction, previous work
showed that phosphopeptide 5 is derived from phosphorylation in
vitro of the site at Ser-1854(35) . Inspection of the amino
acid sequence in this region shows that Ser-1757 and Ser-1772 would be
contained within a single 13-kDa peptide fragment containing peptide
CP10 after complete digestion by endoproteinase Lys-C, which cleaves at
the COOH-terminal side of lysine residues. To identify tryptic
phosphopeptides 4 and 6 within this predicted COOH-terminal fragment,
we utilized endoproteinase Lys-C to digest purified calcium channel in
detergent solution followed by immunoprecipitation by anti-CP10, in
vitro phosphorylation with cA-PK, and Tricine/SDS-PAGE. These
procedures isolated a phosphoprotein fragment of approximately 13 kDa
from the COOH-terminal domain (Fig. 5A) that should
include the cA-PK consensus sites containing Ser-1757 and Ser-1772.
This 13-kDa fragment was excised from the gel, re-electrophoresed on a
standard 6.5% SDS-PAGE gel (data not shown), and then subjected to
two-dimensional tryptic phosphopeptide mapping. PanelsB and C in Fig. 5compare the phosphopeptide maps
from the intact
1 subunit and the 13-kDa Lys-C peptide. Evidently,
both tryptic phosphopeptides 4 and 6 derive from the 13-kDa Lys-C
peptide fragment that is specifically immunoprecipitated with
anti-CP10. As Ser-1757 and Ser-1772 are exact consensus sequences for
phosphorylation by cA-PK (46) and the 13-kDa Lys-C fragment
contains no other sequence having the minimal requirements for a cA-PK
consensus site(46) , these results show that phosphorylation of
one or both of these two sites produces phosphopeptides 4 and 6.
Figure 5:
Identification of a COOH-terminal peptide
containing phosphopeptides 4 and 6. A, purified calcium
channel (20 pmol) was diluted 1:10 with 25 mM ammonium
bicarbonate (pH 8.3) and subjected to digestion in solution with
endoproteinase Lys-C (20 µg) at 37 °C for 16 h. The solution
was adjusted to 1% Triton X-100 and incubated with a 1:20 dilution of
anti-CP10 at 4 °C for 3 h. The antigen/antibody complex was
precipitated with PAS, washed, phosphorylated by cA-PK, and
precipitated with anti-CP10 and PAS; the final PAS precipitate was
prepared for SDS-PAGE as described under ``Experimental
Procedures.'' Tricine/16.5% acrylamide SDS-PAGE as reported by
Schagger and von Jagow (45) followed by autoradiography was used to
detect a 13-kDa immunoprecipitated Lys-C fragment. B, purified
channel (5 pmol) was phosphorylated with cA-PK for 30 min and
immunoprecipitated with anti-CP10 followed by SDS-PAGE, trypsin
digestion of 1
, and two-dimensional phosphopeptide
mapping as described under ``Experimental Procedures.'' C, the immunoprecipitated Lys-C fragment from panelA was excised from the tricine gel, re-electrophoresed on
standard 6.5% acrylamide SDS-PAGE, and prepared for two-dimensional
tryptic phosphopeptide mapping as described under ``Experimental
Procedures.''
Phosphorylation and Two-dimensional Tryptic Peptide
Mapping of Calcium Channel Fusion Protein
Phosphopeptides 4
and 6 in the rabbit skeletal muscle 1 subunit were identified
using a glutathione S-transferase-linked fusion protein,
CaFSk
1, corresponding exactly to the predicted 13-kDa
endoproteinase Lys-C fragment of the
1 subunit (residues
1721-1834 of the
1 sequence) (13). This fusion protein was
purified by chromatography on glutathione-Sepharose, phosphorylated in
the presence of cA-PK and [
-
P]ATP, and
analyzed by SDS-PAGE as described under ``Experimental
Procedures.'' The fusion protein was a substrate for cA-PK (data
not shown). The two-dimensional tryptic phosphopeptide map of
P-labeled CaFSk
1 (Fig. 6A) was
identical to that obtained from the 13-kDa endoproteinase Lys-C
fragment immunoprecipitated from purified
1 subunit (Fig. 5C), with the two phosphopeptides corresponding in
migration position to phosphopeptides 4 and 6 from intact skeletal
muscle
1 subunit (Fig. 5B). The two phosphopeptides
derived from CaFSk
1 were designated A and B. To confirm that these
phosphopeptides were identical, respectively, to phosphopeptides 4 and
6 from skeletal muscle
1 subunit, mixing experiments were
performed (Fig. 6, panelsB and C). In
each case, only a single spot was revealed after two-dimensional
mapping of the mixed phosphopeptides.
Figure 6:
Identification of calcium channel
phosphopeptides by tryptic mapping of CaFSk1 fusion protein.
Purified CaFSk
1-wt fusion protein was phosphorylated in the
presence of cA-PK and [
-
P]ATP, analyzed by
SDS-PAGE as described under ``Experimental Procedures,'' and
detected by autoradiography. Purified calcium channel was
phosphorylated with cA-PK, immunoprecipitated with anti-CP20, and
analyzed by SDS-PAGE and autoradiography as described in Fig. 1.
P-Labeled proteins were excised from wet gels and
subjected to tryptic digestion and phosphopeptide mapping as described
under ``Experimental Procedures.'' A, phosphopeptide
map of CaFSk
1-wt fusion protein. B, phosphopeptide A from
CaFSk
1 and phosphopeptide 4 (see Fig. 2, panelB) were excised from cellulose TLC plates, radioactivity
was eluted with water, and the concentrated samples were mixed and
re-analyzed by two-dimensional mapping. C, phosphopeptide B
from CaFSk
1 mixed with phosphopeptide 6 from purified channel and
re-analyzed by two-dimensional electrophoresis. Circle represents the origin, and arrows represent directions of
electrophoresis (-) and chromatography (C).
The CaFSk1 fusion protein
contains two cA-PK consensus sequences surrounding serine residues
corresponding to amino acids 1757 and 1771/72 of the skeletal muscle
1 subunit. Each of these contains two arginine residues for
potential alternate cleavage by trypsin, raising the possibility that
both phosphopeptides 4 and 6 could arise from phosphorylation of a
single serine residue within one of the consensus sequences. To address
this possibility, additional fusion proteins (CaFSk
1 S1757A and
CaFSk
1 S1771A/S1772A) were prepared, corresponding in sequence to
CaFSk
1 but with the designated serines mutated to alanine
residues. These fusion proteins were expressed and purified as
described under ``Experimental Procedures.'' When CaFSk
1
S1771A/S1772A was phosphorylated by cA-PK in the presence of
[
-
P]ATP and analyzed on a 10-20%
tricine gel followed by autoradiography, a single major
P-labeled phosphoprotein of the predicted molecular mass
was detected (Fig. 7, left). A two-dimensional tryptic
phosphopeptide map of this protein proved identical to maps of
CaFSk
1-wt and the 13-kDa endoproteinase Lys-C fragment (data not
shown). When an identical amount of purified CaFSk
1 S1757A was
phosphorylated by cA-PK and analyzed, no
P-labeled
phosphoproteins were detected (Fig. 7, right). These
results show that CaFSk
1 S1771/72A is a substrate for
phosphorylation by cA-PK while CaFSk
1 S1757A is not. We have
performed phosphoamino acid analysis of calcium channel
1 subunit
(data not shown) and have determined that phosphorylation occurs
exclusively on serine. These data, therefore, indicate that threonine
1756 is not a substrate for phosphorylation by cA-PK. These data are
consistent with identification of Ser-1757 as the residue
phosphorylated by cA-PK in tryptic phosphopeptides 4 and 6 in the
1 subunit of the rabbit skeletal muscle calcium channel.
Figure 7:
Phosphorylation and SDS-PAGE of
CaFSk1 S1771/2A and CaFSk
1 S1757A fusion proteins. Equivalent
amounts (2 µg) of purified full-length CaFSk
1 S1771A/S1772A
and CaFSk
1 S1757A fusion proteins were phosphorylated as described
under ``Experimental Procedures'' in the presence of cA-PK
and [
-
P]ATP, analyzed by SDS-PAGE on a
10-20% Tricine gradient gel (NOVEX, San Diego, CA), and located
by autoradiography. Molecular weight markers are represented as M
10
.
Size Forms of the Rabbit Skeletal Muscle Calcium
Channel
In earlier work from our
laboratory(35) , two forms of the 1 Subunit
1 subunit of the skeletal
muscle calcium channel were observed using a phosphorylation and
immunoprecipitation protocol. In purified channel preparations, the
predominant 190-kDa form migrates on standard SDS gels at an apparent
size of approximately 175 kDa, the size most often described in the
literature for the
1 subunit. A less prominent 212-kDa protein,
migrating at an apparent size of 190-200 kDa on SDS gels, was
observed using antibodies directed to the COOH-terminal sequence of the
predicted full-length protein. In the present study, antibodies
directed against amino acid sequences present on either side of the
proposed cleavage site responsible for converting full-length to
truncated
1 (33) immunoprecipitated a predominant 212-kDa
1 subunit from fetal rabbit muscle myotubes. Two-dimensional
tryptic phosphopeptide mapping confirmed the results of the SDS gels,
as
1 subunits immunoprecipitated from the myotubes contained
tryptic phosphopeptides 4, 5, and 6 derived from the COOH terminus of
1
(35) . Only occasional experiments showing
increased phosphorylation of Ser-687 when the
1 subunit is
immunoprecipitated by an antibody directed to the 190-kDa form of the
protein (data not shown) suggested that truncated
1 exists in
these cells. However, we have been unable to distinguish a truncated
form of
1 as a discrete band on one-dimensional SDS gels, and
two-dimensional maps of
1 subunit immunoprecipitated with
190-kDa-directed antibodies closely resemble maps of
1 subunit
immunoprecipitated with antibodies directed to full-length protein. It
is possible that the finding that
1
predominates in
these myotubes is due to the rapid sequence of cell lysis and
immunoprecipitation achieved in our experiments, preventing the
proteolytic activity found in most preparations of calcium channel.
Alternatively, the predominance of full-length
1 subunits in these
cells may reflect the immature structure of developing skeletal muscle
myotubes in culture. Calcium channels are concentrated in the junctions
between the transverse tubule and sarcoplasmic reticulum membrane
systems(41, 47) , where they serve primarily as voltage
sensors mediating rapid protein-protein interactions that initiate
release of calcium from the sarcoplasmic reticulum in E-C coupling. The
immature structure of the sarcoplasmic reticulum/transverse tubule
junctions in developing myotubes may be responsible for the presence of
an incompletely processed full-length form of the
1 subunit
compared to the predominant mature form of 190 kDa in adult muscle. Our
results demonstrating preferential phosphorylation of COOH-terminal
sites in
1
indicate that the calcium channels
containing
1
in fetal and adult muscle are likely to
be regulated differently by cA-PK phosphorylation than the truncated
form lacking these sites.
Sites of Phosphorylation of
In these experiments, we found that three
phosphopeptides are nearly equally represented in two-dimensional
tryptic maps from fetal muscle myotube 1
in
Rabbit Myotubes
1 subunits. This result
differs somewhat from our earlier work where a single major and two
minor sites of phosphorylation by cA-PK were observed in
1
in purified calcium channels(35) . The total number and
migration positions of phosphopeptides were identical in each
preparation. The discrepancy in degree of phosphorylation of individual
sites most likely reflects alterations in the endogenous level of
phosphorylation or the conformational accessibility of sites in calcium
channels purified by a vigorous and lengthy procedure from whole
tissue. Calcium channels immunoprecipitated quickly and directly from
functioning myotubes as in these experiments should more accurately
provide insight into the physiological relevance of specific sites and
their magnitude of phosphorylation. Therefore, we conclude that three
distinct phosphopeptides derived from the COOH-terminal between
residues 1685 and 1873 are phosphorylated in intact skeletal muscle
cells in response to increases in cAMP.
Identification and in Vivo Phosphorylation of Sites
in the COOH-terminal of
Three
phosphopeptides in 1
1
are phosphorylated by cA-PK
when the calcium channel is immunoprecipitated from myotubes lysed with
1% Triton X-100 that dissociates channel subunits or with 2% digitonin
where the channel retains functional activity. An earlier study from
our laboratory identified tryptic phosphopeptide 5 (T5) as serine 1854;
time course experiments indicated that this site is phosphorylated in vitro at least 100-fold more rapidly than serine 687 in
1
(35) . In the current study, we found the
two additional phosphopeptides unique to
1
to be
major sites of phosphorylation and identified them as serine 1757 (T4
and T6). As with purified rabbit skeletal muscle calcium channel,
efficient phosphorylation of sites in the NH
-terminal 1685
residues of
1
was not observed. We observed a
significant decrease in cA-PK back phosphorylation of each of the three
observed COOH-terminal phosphopeptides after elevation of levels of
cAMP in intact myotubes. No detectable change in phosphorylation of
serine 687 was observed supporting the hypothesis (35) that the
large COOH-terminal domain precludes phosphorylation of interior sites.
Phosphorylation of the two sites we have identified in the
COOH-terminal region may play a critical role in regulating the ion
conductance activity of the full-length form of the calcium channel.
However, these sites are not likely to be necessary for function of the
1 subunit in ion conductance or excitation-contraction coupling
since a truncated form of the
1 subunit is sufficient for both of
these activities when expressed in myocytes from mdg mice
(48).
Comparison with Phosphorylation of
In previous
studies, we found that rat skeletal muscle cells contain long (200 kDa)
and short (160 kDa) forms of the 1 Subunits of
Calcium Channels in Rat Skeletal Muscle Cells
1 subunit that are differentially
phosphorylated(29) . The short form was phosphorylated on two
tryptic phosphopeptides while the long form was phosphorylated on these
peptides and on three additional phosphopeptides that were specific for
the long form. These sites of phosphorylation of the rat
1 subunit
could not be identified by peptide mapping and mutagenesis procedures
because the
1 subunit of the skeletal muscle calcium channel has
not been cloned or sequenced. Nevertheless, the specific
phosphorylation of three phosphopeptides in the long form of the
1
subunit from rat skeletal muscle cells is consistent with our present
results and suggests that these phosphopeptides may arise from the
serine residues in the rat sequence that correspond to serine 1757 and
serine 1854 that we have identified in these experiments on rabbit
skeletal muscle. In the rat skeletal muscle cells, phosphopeptides 1
and 2 were phosphorylated after stimulation with forskolin. Assuming
that these two phosphopeptides in rat cells correspond to serine 687 in
rabbit cells, it appears that phosphorylation of serine 687 is
detectable in intact rat skeletal muscle cells but not in rabbit cells.
This difference may reflect species differences in the availability of
this site for phosphorylation by cA-PK or in the level of cA-PK
activity in developing myotubes from these two species. In either case,
phosphorylation of the COOH-terminal sites on full-length
1
subunits is preferred in both cell types. The present studies identify
these sites of preferential phosphorylation in intact skeletal muscle
cells as serine 1757 and serine 1854 and suggest that they are
candidates for an important role in regulation of calcium channel
function.
1 subunit peptides; 8-cpt-AMP, 8-(4-chlorophenylthio)
adenosine 3&cjs1227;,5&cjs1227;-cyclic monophosphate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl glycine.
1.8.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.