©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sites of Selective cAMP-dependent Phosphorylation of the L-type Calcium Channel 1 Subunit from Intact Rabbit Skeletal Muscle Myotubes (*)

Eric I. Rotman , Brian J. Murphy , William A. Catterall

From the (1)Department of Pharmacology, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The principal (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.


INTRODUCTION

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 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) .

The 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) .

The cDNA encoding the 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.


EXPERIMENTAL PROCEDURES

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) .

Antibodies directed against synthetic calcium channel 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.

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 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 HO, 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) .

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, 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 HO; 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 pSkmCaCh1.8 (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).

The fusion proteins CaFSk1-S1771/2A, and CaFSk1-S1757A are identical to CaFSk1-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) .

Purified fusion proteins were phosphorylated as described above in 50 mM Tris, pH 7.4, 5 mM MgCl, 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.


RESULTS

Immunoprecipitation of Phosphorylated 212-kDa 1 Subunit from Fetal Rabbit Skeletal Muscle Myotube Calcium Channels

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 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 1 from Cultured Rabbit Muscle Cells

Two-dimensional tryptic phosphopeptide maps of the single 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 1 in Stimulated Rabbit Skeletal Muscle Myotubes

To determine whether the phosphopeptides generated from the 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, CaFSk1, 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 CaFSk1 (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 CaFSk1 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 CaFSk1-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 CaFSk1-wt fusion protein. B, phosphopeptide A from CaFSk1 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 CaFSk1 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 (CaFSk1 S1757A and CaFSk1 S1771A/S1772A) were prepared, corresponding in sequence to CaFSk1 but with the designated serines mutated to alanine residues. These fusion proteins were expressed and purified as described under ``Experimental Procedures.'' When CaFSk1 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 CaFSk1-wt and the 13-kDa endoproteinase Lys-C fragment (data not shown). When an identical amount of purified CaFSk1 S1757A was phosphorylated by cA-PK and analyzed, no P-labeled phosphoproteins were detected (Fig. 7, right). These results show that CaFSk1 S1771/72A is a substrate for phosphorylation by cA-PK while CaFSk1 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 CaFSk1 S1757A fusion proteins. Equivalent amounts (2 µg) of purified full-length CaFSk1 S1771A/S1772A and CaFSk1 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.




DISCUSSION

Size Forms of the Rabbit Skeletal Muscle Calcium Channel 1 Subunit

In earlier work from our laboratory(35) , two forms of the 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 1 in Rabbit Myotubes

In these experiments, we found that three phosphopeptides are nearly equally represented in two-dimensional tryptic maps from fetal muscle myotube 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 1

Three phosphopeptides in 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 1 Subunits of Calcium Channels in Rat Skeletal Muscle Cells

In previous studies, we found that rat skeletal muscle cells contain long (200 kDa) and short (160 kDa) forms of the 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grant NS22625 and a research grant from the Muscular Dystrophy Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: cA-PK, cAMP-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PAS, protein A-Sepharose; CP, synthetic calcium channel 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.


ACKNOWLEDGEMENTS

We thank Dr. Steven B. Ellis, Dr. Arnold Schwartz, and Dr. Michael M. Harpold for providing the clone pSkmCaCh1.8.


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