J. Biochem. 109, 163-170 (1991)
Regulation of the Cardiac Ryanodine Receptor by Protein Kinase-Dependent Phosphorylation1 Toshiyuki Takasago, Toshiaki Imagawa, Ken-ichi Furukawa, Tarou Ogurusu, and Munekazu Shigekawa Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565
The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [ r - 31 P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PRC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [ 3 H]ryanodine binding in cardiac microsomes, whereas the level of CaMdependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 2 2 ± 5 , 1 7 ± 4 , and 15±9% (average±SD, n=4-5), respectively. In contrast, incubation of microsomes with 5 fiM CaM alone and 5 fM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38±14 and 53± 15% (average±SD, n=6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residues) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoamnity labeling of microsomes with photoreactive 126I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmlc reticulum Ca2+ release channel.
The contraction of cardiac muscle cells depends on the cytosolic level of Ca2+ ions, whose availability is determined by their entry from the extracellular space as well as their release and uptake by the sarcoplasmic reticulum. The regulatory mechanisms for Ca2+ entry through the L-type Ca2+ channel in the sarcolemma and Ca2+ uptake by means of the sarcoplasmic reticulum Ca2+ pump have been extensively studied (for reviews see Refs. 1 to 3). It has been demonstrated that protein phosphorylation plays an important role in the regulation of these activities in cardiac muscle cells. In contrast, it is still unclear how the release of Ca2+ from the sarcoplasmic reticulum is regulated in these cells. Sarcoplasmic reticulum Ca2+ release channel proteins have recently been isolated from cardiac and skeletal muscles as ryanodine receptors (for a review see Ref. 4). Flux measurements using 45Ca2+ and single-channel record1 This study was supported in part by a Grant-in-Aid (for 1989) from the Japan Heart Foundation and by a Research Grant for Cardiovascular Diseases (62A-1) from the Ministry of Health and Welfare of Japan. Abbreviations: PKA, catalytic subunit of cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PKC, protein kinase C; CaM, calmodulin; Ry-4-actigel A, actigel A conjugated with a monoclonal antibody (Ry-4) against the cardiac ryanodine receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CBB, Coomassie Brilliant Blue; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonic acid; PC, phosphatidylcholine; EGTA, [ethylenebis(oiyethylenenitrilo)]-tetraacetic acid.
Vol. 109, No. 1, 1991
ings using a planar lipid bilayer have yielded results showing that Ca2+ release channel activity is stimulated by micromolar levels of Ca2+, and millimolar levels of adenine nucleotides and caffeine, and inhibited by millimolar levels of Mg*+ and micromolar levels of ruthenium red (4). Calmodulin (CaM) was shown to inhibit the Ca2+ release channel activity in both the cardiac and skeletal sarcoplasmic reticulum (5, 6). Several investigators have also suggested that CaM-dependent phosphorylation of membrane proteins such as a 60-kDa protein is involved in inhibition of Ca2+ release from the sarcoplasmic reticulum of skeletal muscle (7, 8). In the previous paper (9), we demonstrated that the catalytic subunit of cAMP-dependent protein kinase (PKA) phosphorylates the cardiac ryanodine receptor and that this phosphorylation results in enhanced binding of [3H]ryanodine to the receptor. Based on these findings we suggested that PKA-dependent phosphorylation of the sarcoplasmic reticulum Ca2+ release channel may be important for the positive inotropic effect of /?-adrenergic stimulation on cardiac contraction. In this study, to further investigate the mechanism for regulation of the Ca2+ release channel, we phosphorylated the cardiac ryanodine receptor with PKA, cGMP-dependent protein kinase (PKG), protein kinase C (PKC), and endogenous calmodulin (CaM)-dependent protein kinase, and compared the effects of these phosphorylations on [3H] ryanodine binding to the cardiac ryanodine receptor. [ 3 H]163
Downloaded from https://academic.oup.com/jb/article-abstract/109/1/163/774630 by Lancaster University user on 14 January 2019
Received for publication, August 22, 1990
164
T. Takasago et al.
Ryanodine binding in cardiac microsomes increased upon phosphorylation by PKA, PKG, and PKC, but decreased upon phosphorylation by CaM-dependent kinase. We present evidence suggesting that the phosphorylation of different serine residues in the cardiac ryanodine receptor is involved in up- or down-regulation of its channel activity. EXPERIMENTAL PROCEDURES
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/109/1/163/774630 by Lancaster University user on 14 January 2019
Preparation of Microsomes and Purification of the Cardiac Ryanodine Receptor—Cardiac microsomes were prepared from canine ventricle tissue as described previously (9). Skeletal muscle microsomes were prepared by a similar method from canine superficial digitalis flexor [type I (slow)] or extensor carpi radialis [type II (fast)] muscle fibers. The ryanodine receptor was purified from canine cardiac microsomes as described in detail in the previous paper (10). All of the samples were quickly frozen in liquid N2 and stored at — 70'C until use. Sources of PKA, PKG, and PKC—PKA from bovine heart was purchased from Sigma. PKG was purified from bovine lung by the method of Lincoln (11). PKC was purified from rat brain by the method of Kikkawa et al. (12). Preparation of Monoclonal Antibody Ry-4 against the Cardiac Ryanodine Receptor and of an Immunoaffinity Adsorbent—The monoclonal antibody, Ry-4, against the cardiac ryanodine receptor was prepared and purified as described in the previous paper (20). The purified Ry-4 was coupled to actigel A beads (2 mg of protein/ml beads) according to the instructions of the manufacturer (Sterogene Biochemicals) to obtain an immunoaffinity adsorbent (Ry-4-actigel A). Phosphorylation of Microsomes and the Purified Ryanodine Receptor—Microsomes (1-2 mg/ml) and the purified cardiac ryanodine receptor (0.1-0.3 mg/ml) were phosphorylated with 50 fiM or 1 mM [y-"P]ATP (Amersham) for 0-15 min at room temperature in the phosphorylation buffer (8mM MgCl2, 10 mM EGTA, and 50 mM piperazine-iV,iV'-bi8(2-ethanesulfonic acid)/Tris, pH 6.8). For the experiments with PKA or PKG, the phosphorylation buffer additionally contained PKA or PKG plus 100//M cGMP. For the experiments with PKC or CaM, on the other hand, the phosphorylation buffer additionally contained 10 mM CaCl, (final free Ca2+ concentration, 80 fxM) and PKC plus 300 nM 12-o-tetradecanoylphorbol-13-acetate or 5 fiM CaM. The amount of each protein kinase added was 1 unit per fig of microsomal protein or 5 units per fig of the purified ryanodine receptor. The reactions were terminated by the addition of an SDS-stop solution comprising 1% SDS, 5% y?-mercaptoethanol, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0. The samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (13) on a 6% gel. The phosphorylated protein bands were identified by autoradiography after the gels had been stained with Coomassie Brilliant Blue (CBB) and dried. The amount of " P incorporated into the cardiac ryanodine receptor was determined by radioactivity counting of the gel slice containing the phosphorylated ryanodine receptor and is expressed as pmol/mg of microsomal protein. [3H]Ryanodine Binding Assay—Cardiac microsomes (2 mg/ml) were phosphorylated with 1 mM ATP in the pres-
ence or absence of PKA, PKG, PKC or CaM, as described above, or treated with 5 //M CaM and 80 //M free Ca2+ in the absence of ATP. The microsomes (75//I) were then passed through a 1-ml Sephadex G-50 column preequilibrated with the binding buffer comprising 150 mM KC1, 2 mM dithiothreitol, 1 mM EGTA, and 50 mM Tris/ HEPES, pH 7.4, in order to replace the phosphorylation buffer with the binding buffer. Microsomes, which were free from ATP contamination, were recovered between 300 and 600//I of the eluate. [3H]Ryanodine binding to the microsomes was measured as described in the previous paper (9), except that the concentration of KC1 used was 150 mM. Specific binding of [3H]ryanodine was calculated by subtracting the value in the absence of Ca2+ from that in the presence of 10 //M free Ca2+. Two-Dimensional Peptide Mapping and Phosphoamino Acid Analysis—Cardiac microsomes (1 mg/ml) were phosphorylated with 50 fM [y- 31 P]ATP for 5 min in the presence of PKA, PKG, PKC, or CaM under the conditions described above. After the reaction had been stopped by adding the SDS-stop solution, the sample was applied to a small Sephadex G-50 column pre-equilibrated with the SDS-stop solution to remove [y- 32 P]ATP. The recovered 32 P-labeled microsomes were then subjected to SDSPAGE. The phosphorylated ryanodine receptor in gel slices was digested with trypsin, treated with L-l-tosylamide-2phenylethyl chloromethyl ketone (Worthington) and then subjected to two-dimensional peptide mapping analysis according to the procedure described previously (14). The tryptic peptides were incubated at 110'C for 2 h with 6 N HC1. Acid hydrolysates were analyzed on cellulose thinlayer plates (Kodak) by electrophoresis at pH 1.9 or 3.5, as described (15). Photoaffinity Labeling of Cardiac Microsomes with Photoreactive [l25I]CaM and Immunoprecipitation of the 12S I-Labeled Protein with a Monoclonal Antibody against the Cardiac Ryanodine Receptor—Calmodulin (Amano) was labeled with [I25I]Denny-Jaffe reagent (Du Pont-New England Nuclear), a heterobifunctional crosslinking agent which can be cleaved and photoactivated, as described previously (26). Cardiac microsomes or the purified ryanodine receptor were incubated for 10 min at room temperature in the dark with 1 fxM Denny-Jaffe-labeled CaM in 150 mM KC1, 50 mM Tris/4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.4, 1 mM EGTA, and 0 or 1.01 mM CaCl2 (final free Ca2+ concentration, 10 /*M). Half of each sample was photolyzed for 5 min on ice using a Mineralight lamp (model UVGL-15, UVP, San Gabriel, U.S.A.) as a light source. Cleavage of crosslinking was achieved by 2 h incubation with 10 mM sodium dithionite at room temperature. For the immunoprecipitation experiments, samples were solubilized with 1% CHAPS and the cardiac ryanodine receptor was immunoprecipitated with Ry-4-actigel A beads as described in the previous paper (9). The '"I-labeled proteins were detected by autoradiography following SDS-PAGE. Protein Determination—The protein concentration was measured by the method of Bradford (17) with bovine serum albumin as a standard. Expression of Results—Data are presented as averages ± SD. The results were evaluated by means of Student's t test for paired values.
165
Phosphorylation of the Cardiac Ryanodine Receptor
RESULTS
Protein Kinase-Dependent Phosphorylation of the Ryanodine Receptor—We examined the phosphorylation of canine cardiac microsomes by various protein kinases in the presence of 50^M [y- 32 P]ATP (Fig. 1). For comparative purposes, we also examined the protein kinase-dependent phosphorylation of microsomes isolated from canine fast (extensor carpi radialis) and slow (superficial digitalis flexor) skeletal muscles. The phosphoproteins were identified by autoradiography following SDS-PAGE on a 6% gel. In the cardiac microsomes, PKA heavily phosphorylated a high- molecular- weight protein whose mobility corresponded to that of the cardiac ryanodine receptor. In both the fast and slow skeletal muscle microsomes, however, the major phosphoprotein band was that of a 87-kDa protein and the skeletal ryanodine receptor was not phosphorylated significantly by PKA. These results confirmed our previous data (9). The cardiac ryanodine receptor in microsomes as well as the purified preparation, but not the fast and slow skeletal muscle ryanodine receptors in microsomes, was also phosphorylated significantly in the presence of added CaM. CaM also induced the phosphorylation of a 50-kDa protein in the purified cardiac ryanodine receptor preparation. This 50kDa protein must be a minor component, because we could not detect it in a CBB-stained gel (Fig. 1A). Unlike PKA and CaM, PKG induced significant phosphorylation of both the cardiac and skeletal muscle ryanodine receptors (Fig. IB). PKC, on the other hand, phosphorylated the cardiac
A. CBB stain
B. Autoradiogram Mr(x10-3) PKA
M
- 205 ~ Fig. 1. Phosphorylation of the purified cardiac ryanodine receptor, and microsomes isolated from cardiac and skeletal muscles. The purified cardiac ryanodine receptor (1.0 vg, lanes 1 and 5), and microsomes isolated from cardiac (10 ^g, lanes 2 and 6), superficial digitalis flexor (10 fig, lanes 3 and 7) and extensor carpi radialis (10 fig, lanes 4 and 8) muscle fibers were phosphorylated with 50//M [y-"P]ATP for 3 nun in the absence (lanes 1 to 4) or presence (lanes 5 to 8) of protein kinase or CaM, as described under "EXPERIMENTAL PROCEDURES." Samples were subjected to SDS-PAGE: A, CBB stain; B, autoradiogram. Arrows and numbers represent the positions and molecular weights (Mr x 10"*) of molecular weight standards, respectively. Single and double arrowheads indicate the positions of cardiac and skeletal muscle ryanodine receptors, respectively. Vol. 109, No. 1, 1991
1 2 3 4 5 6 7 8
Mr(x10-3) PKG
- 205 -
1 2 3 4 5 6 7 8 CaM
Mr(x10-3)
4* 1
2 3 4 5 6 7 8
1
2 3 4 5 6 7 8
Downloaded from https://academic.oup.com/jb/article-abstract/109/1/163/774630 by Lancaster University user on 14 January 2019
ryanodine receptor only slightly. Table I summarizes the results of measurement of the amount of "P- incorporation into the ryanodine receptors in cardiac and skeletal muscle microsome8 under the conditions in Fig. 1. It should be pointed out that the " P incorporation was certainly underestimated in the presence of CaM or PKC. CaM-dependent kinase and PKC require Ca2+ for their activation. The inclusion of 80 ^M free Ca2+ in the phosphorylation buffer would have caused the consumption of a sizable fraction of 50^M [y- 32 P]ATP, because Ca1+ would have activated other ATP-hydrolyzing enzymes, such as the Ca l+ pump. In addition, the incubation time (3 min) was not optimal for the PKC-dependent phosphorylation (cf. Fig. 2). Figure 2 shows the time courses for phosphorylation of the ryanodine receptor in cardiac microsomes by various protein kinases with a high concentration (1 mM) of [ y3I P] ATP. In the presence of PKA, PKG, or CaM, phosphorylation of the cardiac ryanodine receptor proceeded at a similar rapid rate. On the other hand, PKC induced relatively slow phosphorylation, although the final level of "P-incorporation was similar to those with PKA and PKG. Under the conditions in Fig. 2, the amounts of " P incorporated into the cardiac ryanodine receptor at 10 min were 6.0±0.4 (n=3), 5.5±1.0 (n=3), 4.7±1.1 (n=3), and 20.7±3.5 (n=3) pmol/mg of microsomal protein in the presence of PKA, PKG, PKC, and CaM, respectively. These values obtained with PKA, PKG, and PKC were comparable to the maximum level of [3H] ryanodine binding in our cardiac microsomes (3 to 6 pmol/mg) (10). In contrast, the level of CaM-dependent 32P incorporation was about 4 times greater.
186
T. Takasago et al.
TABLE I. Protein kinase-dependent phosphorylation of cardiac and skeletal muscle ryanodine receptors. Microsomes isolated from cardiac, slow (superficial digitalis flexor), and fast (extensor carpi radialis) skeletal muscles were phosphorylated with 50 //M [y-"P]ATP for 3 min in the presence of PKA, PKG, PKC, or CaM, as described under "EXPERIMENTAL PROCEDURES." Samples (10 Mg) were subjected to SDS-PAGE. The amount of "P incorporated into the ryanodine receptor was also determined as descnbed under "EXPERIMENTAL PROCEDURES." Values are presented as averages±SD for three independent experiments. Amount of "P incorporated into ryanodine receptor (pmol/mg microsomal protem) PKA PKG PKC CaM Cardiac microsomes 6.3±1 .1 4.8±1.7 0.7±0.3 3.5±13 Slow skeletal 0.5±0 .4 4.4±0.4 0.5±0 2 0.3±0.3 muscle microsomes Fast skeletal 0.6±0 .4 7 1±0.3 0.3±0.0 0.1±0 1 muscle microsomes
5
10 Time (mm)
0.01 versus the control value). When 1 mM ATP was present together with CaM, ryanodine binding decreased by 53±15% (n=6, p
Regulation of the Cardiac Ryanodine Receptor by Protein Kinase-Dependent Phosphorylation1 Toshiyuki Takasago, Toshiaki Imagawa, Ken-ichi Furukawa, Tarou Ogurusu, and Munekazu Shigekawa Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565
The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [ r - 31 P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PRC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [ 3 H]ryanodine binding in cardiac microsomes, whereas the level of CaMdependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 2 2 ± 5 , 1 7 ± 4 , and 15±9% (average±SD, n=4-5), respectively. In contrast, incubation of microsomes with 5 fiM CaM alone and 5 fM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38±14 and 53± 15% (average±SD, n=6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residues) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoamnity labeling of microsomes with photoreactive 126I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmlc reticulum Ca2+ release channel.
The contraction of cardiac muscle cells depends on the cytosolic level of Ca2+ ions, whose availability is determined by their entry from the extracellular space as well as their release and uptake by the sarcoplasmic reticulum. The regulatory mechanisms for Ca2+ entry through the L-type Ca2+ channel in the sarcolemma and Ca2+ uptake by means of the sarcoplasmic reticulum Ca2+ pump have been extensively studied (for reviews see Refs. 1 to 3). It has been demonstrated that protein phosphorylation plays an important role in the regulation of these activities in cardiac muscle cells. In contrast, it is still unclear how the release of Ca2+ from the sarcoplasmic reticulum is regulated in these cells. Sarcoplasmic reticulum Ca2+ release channel proteins have recently been isolated from cardiac and skeletal muscles as ryanodine receptors (for a review see Ref. 4). Flux measurements using 45Ca2+ and single-channel record1 This study was supported in part by a Grant-in-Aid (for 1989) from the Japan Heart Foundation and by a Research Grant for Cardiovascular Diseases (62A-1) from the Ministry of Health and Welfare of Japan. Abbreviations: PKA, catalytic subunit of cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PKC, protein kinase C; CaM, calmodulin; Ry-4-actigel A, actigel A conjugated with a monoclonal antibody (Ry-4) against the cardiac ryanodine receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CBB, Coomassie Brilliant Blue; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonic acid; PC, phosphatidylcholine; EGTA, [ethylenebis(oiyethylenenitrilo)]-tetraacetic acid.
Vol. 109, No. 1, 1991
ings using a planar lipid bilayer have yielded results showing that Ca2+ release channel activity is stimulated by micromolar levels of Ca2+, and millimolar levels of adenine nucleotides and caffeine, and inhibited by millimolar levels of Mg*+ and micromolar levels of ruthenium red (4). Calmodulin (CaM) was shown to inhibit the Ca2+ release channel activity in both the cardiac and skeletal sarcoplasmic reticulum (5, 6). Several investigators have also suggested that CaM-dependent phosphorylation of membrane proteins such as a 60-kDa protein is involved in inhibition of Ca2+ release from the sarcoplasmic reticulum of skeletal muscle (7, 8). In the previous paper (9), we demonstrated that the catalytic subunit of cAMP-dependent protein kinase (PKA) phosphorylates the cardiac ryanodine receptor and that this phosphorylation results in enhanced binding of [3H]ryanodine to the receptor. Based on these findings we suggested that PKA-dependent phosphorylation of the sarcoplasmic reticulum Ca2+ release channel may be important for the positive inotropic effect of /?-adrenergic stimulation on cardiac contraction. In this study, to further investigate the mechanism for regulation of the Ca2+ release channel, we phosphorylated the cardiac ryanodine receptor with PKA, cGMP-dependent protein kinase (PKG), protein kinase C (PKC), and endogenous calmodulin (CaM)-dependent protein kinase, and compared the effects of these phosphorylations on [3H] ryanodine binding to the cardiac ryanodine receptor. [ 3 H]163
Downloaded from https://academic.oup.com/jb/article-abstract/109/1/163/774630 by Lancaster University user on 14 January 2019
Received for publication, August 22, 1990
164
T. Takasago et al.
Ryanodine binding in cardiac microsomes increased upon phosphorylation by PKA, PKG, and PKC, but decreased upon phosphorylation by CaM-dependent kinase. We present evidence suggesting that the phosphorylation of different serine residues in the cardiac ryanodine receptor is involved in up- or down-regulation of its channel activity. EXPERIMENTAL PROCEDURES
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/109/1/163/774630 by Lancaster University user on 14 January 2019
Preparation of Microsomes and Purification of the Cardiac Ryanodine Receptor—Cardiac microsomes were prepared from canine ventricle tissue as described previously (9). Skeletal muscle microsomes were prepared by a similar method from canine superficial digitalis flexor [type I (slow)] or extensor carpi radialis [type II (fast)] muscle fibers. The ryanodine receptor was purified from canine cardiac microsomes as described in detail in the previous paper (10). All of the samples were quickly frozen in liquid N2 and stored at — 70'C until use. Sources of PKA, PKG, and PKC—PKA from bovine heart was purchased from Sigma. PKG was purified from bovine lung by the method of Lincoln (11). PKC was purified from rat brain by the method of Kikkawa et al. (12). Preparation of Monoclonal Antibody Ry-4 against the Cardiac Ryanodine Receptor and of an Immunoaffinity Adsorbent—The monoclonal antibody, Ry-4, against the cardiac ryanodine receptor was prepared and purified as described in the previous paper (20). The purified Ry-4 was coupled to actigel A beads (2 mg of protein/ml beads) according to the instructions of the manufacturer (Sterogene Biochemicals) to obtain an immunoaffinity adsorbent (Ry-4-actigel A). Phosphorylation of Microsomes and the Purified Ryanodine Receptor—Microsomes (1-2 mg/ml) and the purified cardiac ryanodine receptor (0.1-0.3 mg/ml) were phosphorylated with 50 fiM or 1 mM [y-"P]ATP (Amersham) for 0-15 min at room temperature in the phosphorylation buffer (8mM MgCl2, 10 mM EGTA, and 50 mM piperazine-iV,iV'-bi8(2-ethanesulfonic acid)/Tris, pH 6.8). For the experiments with PKA or PKG, the phosphorylation buffer additionally contained PKA or PKG plus 100//M cGMP. For the experiments with PKC or CaM, on the other hand, the phosphorylation buffer additionally contained 10 mM CaCl, (final free Ca2+ concentration, 80 fxM) and PKC plus 300 nM 12-o-tetradecanoylphorbol-13-acetate or 5 fiM CaM. The amount of each protein kinase added was 1 unit per fig of microsomal protein or 5 units per fig of the purified ryanodine receptor. The reactions were terminated by the addition of an SDS-stop solution comprising 1% SDS, 5% y?-mercaptoethanol, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0. The samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (13) on a 6% gel. The phosphorylated protein bands were identified by autoradiography after the gels had been stained with Coomassie Brilliant Blue (CBB) and dried. The amount of " P incorporated into the cardiac ryanodine receptor was determined by radioactivity counting of the gel slice containing the phosphorylated ryanodine receptor and is expressed as pmol/mg of microsomal protein. [3H]Ryanodine Binding Assay—Cardiac microsomes (2 mg/ml) were phosphorylated with 1 mM ATP in the pres-
ence or absence of PKA, PKG, PKC or CaM, as described above, or treated with 5 //M CaM and 80 //M free Ca2+ in the absence of ATP. The microsomes (75//I) were then passed through a 1-ml Sephadex G-50 column preequilibrated with the binding buffer comprising 150 mM KC1, 2 mM dithiothreitol, 1 mM EGTA, and 50 mM Tris/ HEPES, pH 7.4, in order to replace the phosphorylation buffer with the binding buffer. Microsomes, which were free from ATP contamination, were recovered between 300 and 600//I of the eluate. [3H]Ryanodine binding to the microsomes was measured as described in the previous paper (9), except that the concentration of KC1 used was 150 mM. Specific binding of [3H]ryanodine was calculated by subtracting the value in the absence of Ca2+ from that in the presence of 10 //M free Ca2+. Two-Dimensional Peptide Mapping and Phosphoamino Acid Analysis—Cardiac microsomes (1 mg/ml) were phosphorylated with 50 fM [y- 31 P]ATP for 5 min in the presence of PKA, PKG, PKC, or CaM under the conditions described above. After the reaction had been stopped by adding the SDS-stop solution, the sample was applied to a small Sephadex G-50 column pre-equilibrated with the SDS-stop solution to remove [y- 32 P]ATP. The recovered 32 P-labeled microsomes were then subjected to SDSPAGE. The phosphorylated ryanodine receptor in gel slices was digested with trypsin, treated with L-l-tosylamide-2phenylethyl chloromethyl ketone (Worthington) and then subjected to two-dimensional peptide mapping analysis according to the procedure described previously (14). The tryptic peptides were incubated at 110'C for 2 h with 6 N HC1. Acid hydrolysates were analyzed on cellulose thinlayer plates (Kodak) by electrophoresis at pH 1.9 or 3.5, as described (15). Photoaffinity Labeling of Cardiac Microsomes with Photoreactive [l25I]CaM and Immunoprecipitation of the 12S I-Labeled Protein with a Monoclonal Antibody against the Cardiac Ryanodine Receptor—Calmodulin (Amano) was labeled with [I25I]Denny-Jaffe reagent (Du Pont-New England Nuclear), a heterobifunctional crosslinking agent which can be cleaved and photoactivated, as described previously (26). Cardiac microsomes or the purified ryanodine receptor were incubated for 10 min at room temperature in the dark with 1 fxM Denny-Jaffe-labeled CaM in 150 mM KC1, 50 mM Tris/4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.4, 1 mM EGTA, and 0 or 1.01 mM CaCl2 (final free Ca2+ concentration, 10 /*M). Half of each sample was photolyzed for 5 min on ice using a Mineralight lamp (model UVGL-15, UVP, San Gabriel, U.S.A.) as a light source. Cleavage of crosslinking was achieved by 2 h incubation with 10 mM sodium dithionite at room temperature. For the immunoprecipitation experiments, samples were solubilized with 1% CHAPS and the cardiac ryanodine receptor was immunoprecipitated with Ry-4-actigel A beads as described in the previous paper (9). The '"I-labeled proteins were detected by autoradiography following SDS-PAGE. Protein Determination—The protein concentration was measured by the method of Bradford (17) with bovine serum albumin as a standard. Expression of Results—Data are presented as averages ± SD. The results were evaluated by means of Student's t test for paired values.
165
Phosphorylation of the Cardiac Ryanodine Receptor
RESULTS
Protein Kinase-Dependent Phosphorylation of the Ryanodine Receptor—We examined the phosphorylation of canine cardiac microsomes by various protein kinases in the presence of 50^M [y- 32 P]ATP (Fig. 1). For comparative purposes, we also examined the protein kinase-dependent phosphorylation of microsomes isolated from canine fast (extensor carpi radialis) and slow (superficial digitalis flexor) skeletal muscles. The phosphoproteins were identified by autoradiography following SDS-PAGE on a 6% gel. In the cardiac microsomes, PKA heavily phosphorylated a high- molecular- weight protein whose mobility corresponded to that of the cardiac ryanodine receptor. In both the fast and slow skeletal muscle microsomes, however, the major phosphoprotein band was that of a 87-kDa protein and the skeletal ryanodine receptor was not phosphorylated significantly by PKA. These results confirmed our previous data (9). The cardiac ryanodine receptor in microsomes as well as the purified preparation, but not the fast and slow skeletal muscle ryanodine receptors in microsomes, was also phosphorylated significantly in the presence of added CaM. CaM also induced the phosphorylation of a 50-kDa protein in the purified cardiac ryanodine receptor preparation. This 50kDa protein must be a minor component, because we could not detect it in a CBB-stained gel (Fig. 1A). Unlike PKA and CaM, PKG induced significant phosphorylation of both the cardiac and skeletal muscle ryanodine receptors (Fig. IB). PKC, on the other hand, phosphorylated the cardiac
A. CBB stain
B. Autoradiogram Mr(x10-3) PKA
M
- 205 ~ Fig. 1. Phosphorylation of the purified cardiac ryanodine receptor, and microsomes isolated from cardiac and skeletal muscles. The purified cardiac ryanodine receptor (1.0 vg, lanes 1 and 5), and microsomes isolated from cardiac (10 ^g, lanes 2 and 6), superficial digitalis flexor (10 fig, lanes 3 and 7) and extensor carpi radialis (10 fig, lanes 4 and 8) muscle fibers were phosphorylated with 50//M [y-"P]ATP for 3 nun in the absence (lanes 1 to 4) or presence (lanes 5 to 8) of protein kinase or CaM, as described under "EXPERIMENTAL PROCEDURES." Samples were subjected to SDS-PAGE: A, CBB stain; B, autoradiogram. Arrows and numbers represent the positions and molecular weights (Mr x 10"*) of molecular weight standards, respectively. Single and double arrowheads indicate the positions of cardiac and skeletal muscle ryanodine receptors, respectively. Vol. 109, No. 1, 1991
1 2 3 4 5 6 7 8
Mr(x10-3) PKG
- 205 -
1 2 3 4 5 6 7 8 CaM
Mr(x10-3)
4* 1
2 3 4 5 6 7 8
1
2 3 4 5 6 7 8
Downloaded from https://academic.oup.com/jb/article-abstract/109/1/163/774630 by Lancaster University user on 14 January 2019
ryanodine receptor only slightly. Table I summarizes the results of measurement of the amount of "P- incorporation into the ryanodine receptors in cardiac and skeletal muscle microsome8 under the conditions in Fig. 1. It should be pointed out that the " P incorporation was certainly underestimated in the presence of CaM or PKC. CaM-dependent kinase and PKC require Ca2+ for their activation. The inclusion of 80 ^M free Ca2+ in the phosphorylation buffer would have caused the consumption of a sizable fraction of 50^M [y- 32 P]ATP, because Ca1+ would have activated other ATP-hydrolyzing enzymes, such as the Ca l+ pump. In addition, the incubation time (3 min) was not optimal for the PKC-dependent phosphorylation (cf. Fig. 2). Figure 2 shows the time courses for phosphorylation of the ryanodine receptor in cardiac microsomes by various protein kinases with a high concentration (1 mM) of [ y3I P] ATP. In the presence of PKA, PKG, or CaM, phosphorylation of the cardiac ryanodine receptor proceeded at a similar rapid rate. On the other hand, PKC induced relatively slow phosphorylation, although the final level of "P-incorporation was similar to those with PKA and PKG. Under the conditions in Fig. 2, the amounts of " P incorporated into the cardiac ryanodine receptor at 10 min were 6.0±0.4 (n=3), 5.5±1.0 (n=3), 4.7±1.1 (n=3), and 20.7±3.5 (n=3) pmol/mg of microsomal protein in the presence of PKA, PKG, PKC, and CaM, respectively. These values obtained with PKA, PKG, and PKC were comparable to the maximum level of [3H] ryanodine binding in our cardiac microsomes (3 to 6 pmol/mg) (10). In contrast, the level of CaM-dependent 32P incorporation was about 4 times greater.
186
T. Takasago et al.
TABLE I. Protein kinase-dependent phosphorylation of cardiac and skeletal muscle ryanodine receptors. Microsomes isolated from cardiac, slow (superficial digitalis flexor), and fast (extensor carpi radialis) skeletal muscles were phosphorylated with 50 //M [y-"P]ATP for 3 min in the presence of PKA, PKG, PKC, or CaM, as described under "EXPERIMENTAL PROCEDURES." Samples (10 Mg) were subjected to SDS-PAGE. The amount of "P incorporated into the ryanodine receptor was also determined as descnbed under "EXPERIMENTAL PROCEDURES." Values are presented as averages±SD for three independent experiments. Amount of "P incorporated into ryanodine receptor (pmol/mg microsomal protem) PKA PKG PKC CaM Cardiac microsomes 6.3±1 .1 4.8±1.7 0.7±0.3 3.5±13 Slow skeletal 0.5±0 .4 4.4±0.4 0.5±0 2 0.3±0.3 muscle microsomes Fast skeletal 0.6±0 .4 7 1±0.3 0.3±0.0 0.1±0 1 muscle microsomes
5
10 Time (mm)
0.01 versus the control value). When 1 mM ATP was present together with CaM, ryanodine binding decreased by 53±15% (n=6, p
