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• W1963646187 abstract "Multifunctional Ca2+-calmodulin-dependent protein kinase (CaMKII) is a Ser/Thr protein kinase uniformly distributed within the sarcoplasmic reticulum (SR) of skeletal muscle. In fast twitch muscle, no specific substrates of CaMKII have yet been identified in nonjunctional SR. Previous electron microscopy data showed that glycogen particles containing glycogen synthase (GS) associate with SR at the I band level. Furthermore, recent evidence implicates CaMKII in regulation of glucose and glycogen metabolism. Here, we demonstrate that the glycogen- and protein phosphatase 1-targeting subunit, also known as GM, selectively localizes to the SR membranes of rabbit skeletal muscle and that GM and GS co-localize at the level of the I band. We further show that GM, GS, and PP1c assemble in a structural complex that selectively localizes to nonjunctional SR and that GM is phosphorylated by SR-bound CaMKII and dephosphorylated by PP1c. On the other hand, no evidence for a structural interaction between GM and CaMKII was obtained. Using His-tagged GM recombinant fragments and site-directed mutagenesis, we demonstrate that the target of CaMKII is Ser48. Taken together, these data suggest that SR-bound CaMKII participates in the regulation of GS activity through changes in the phosphorylation state of GM. Based on these findings, we propose that SR-bound CaMKII participates in the regulation of glycogen metabolism, under physiological conditions involving repetitive raises elevations of [Ca2+]i. Multifunctional Ca2+-calmodulin-dependent protein kinase (CaMKII) is a Ser/Thr protein kinase uniformly distributed within the sarcoplasmic reticulum (SR) of skeletal muscle. In fast twitch muscle, no specific substrates of CaMKII have yet been identified in nonjunctional SR. Previous electron microscopy data showed that glycogen particles containing glycogen synthase (GS) associate with SR at the I band level. Furthermore, recent evidence implicates CaMKII in regulation of glucose and glycogen metabolism. Here, we demonstrate that the glycogen- and protein phosphatase 1-targeting subunit, also known as GM, selectively localizes to the SR membranes of rabbit skeletal muscle and that GM and GS co-localize at the level of the I band. We further show that GM, GS, and PP1c assemble in a structural complex that selectively localizes to nonjunctional SR and that GM is phosphorylated by SR-bound CaMKII and dephosphorylated by PP1c. On the other hand, no evidence for a structural interaction between GM and CaMKII was obtained. Using His-tagged GM recombinant fragments and site-directed mutagenesis, we demonstrate that the target of CaMKII is Ser48. Taken together, these data suggest that SR-bound CaMKII participates in the regulation of GS activity through changes in the phosphorylation state of GM. Based on these findings, we propose that SR-bound CaMKII participates in the regulation of glycogen metabolism, under physiological conditions involving repetitive raises elevations of [Ca2+]i. In skeletal muscle, a regulatory subunit called GM (also known as G, RGl, R3, and PP1R3A) (1Herzig S. Neuman J. Physiol. Rev. 2000; 80: 173-210Crossref PubMed Scopus (238) Google Scholar, 2Cohen P.T.W. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar) is responsible for the targeting of glycogen and PP1c (protein phosphatase 1 catalytic subunit) to the sarcoplasmic reticulum (SR) 1The abbreviations used are: SR, sarcoplasmic reticulum; CaMKII, Ca2+-calmodulin protein kinase; GS, glycogen synthase; JFM, junctional face membrane of sarcoplasmic reticulum; PKA, cAMP-dependent protein kinase; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase; aa, amino acid(s); Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TRITC, tetramethylrhodamine isothiocyanate. (3Stralfors P. Hiraga A. Cohen P. Eur. J. Biochem. 1985; 149: 295-303Crossref PubMed Scopus (200) Google Scholar, 4Hubbard M.J. Cohen P. Eur. J. Biochem. 1989; 186: 701-709Crossref PubMed Scopus (101) Google Scholar, 5Hubbard M.J. Cohen P. Eur. J. Biochem. 1989; 186: 711-716Crossref PubMed Scopus (87) Google Scholar, 6MacDougall L.K. Jones L.R. Cohen P. Eur. J. Biochem. 1991; 196: 725-734Crossref PubMed Scopus (186) Google Scholar). GM is a striated muscle-specific protein expressed at higher levels in skeletal than in cardiac muscle (7Tang P.M. Bondor J.A. Swiderek K.M. De-Paoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar, 8Lannér C. Suzuki Y. Bi C. Zhang H. Cooper L.D. Bowker-Kinley M.M. DePaoli-Roach A.A. Arch. Biochem. Biophys. 2001; 388: 135-145Crossref PubMed Scopus (15) Google Scholar). GM (glycogen- and PP1c-targeting subunit) belongs to a family of mammalian glycogen- and PP1c-binding proteins (PPP1R6, PTG, and GL) (9Newgard C.B. Brady M.J. O'Doherty R.M. Saltiel A.R. Diabetes. 2000; 49: 1967-1977Crossref PubMed Scopus (150) Google Scholar). GM rabbit skeletal muscle cDNA sequence (7Tang P.M. Bondor J.A. Swiderek K.M. De-Paoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar) predicts a protein of 1109 amino acids with a calculated Mr of 124,257. However, on SDS-PAGE, GM displays an apparent Mr of ∼160,000 (7Tang P.M. Bondor J.A. Swiderek K.M. De-Paoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar, 10MacKintosh C. Campbell D.G. Hiraga A. Cohen P. FEBS Lett. 1988; 234: 189-194Crossref PubMed Scopus (32) Google Scholar), likely because of its acidic pI. Binding sites for PP1c (aa 64–69; see Ref. 11Zhao S. Lee E.Y.C. J. Biol. Chem. 1997; 272: 28368-28372Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and glycogen (aa 150–159 in the case of rabbit GM; see Ref. 12Wu J. Kleiner U. Brautigan D.L. Biochemistry. 1996; 35: 13858-13864Crossref PubMed Scopus (30) Google Scholar) have been identified in the NH2-terminal domain of GM. On the other hand, GM is characterized by the unique presence, at the COOH terminus, of a stretch of hydrophobic residues (aa 1063–1097; see Ref. 7Tang P.M. Bondor J.A. Swiderek K.M. De-Paoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar). It has been proposed that this amino acid sequence is indicative of a transmembrane helix that mediates its binding to SR membranes (7Tang P.M. Bondor J.A. Swiderek K.M. De-Paoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar, 8Lannér C. Suzuki Y. Bi C. Zhang H. Cooper L.D. Bowker-Kinley M.M. DePaoli-Roach A.A. Arch. Biochem. Biophys. 2001; 388: 135-145Crossref PubMed Scopus (15) Google Scholar). Interestingly, this transmembrane region appears to be involved in the interaction of GM with the SERCA2-regulatory protein phospholamban (13Berrebi-Bertrand I. Souchet M. Camelin J.-C. Laville M.-P. Calmels T. Bril A. FEBS Lett. 1998; 439: 224-230Crossref PubMed Scopus (22) Google Scholar). A key role for GM phosphorylation in regulation of glycogen synthase (GS) is suggested by a number of observations: (i) cAMP-dependent protein kinase (PKA) phosphorylates GM on Ser48 and Ser67 both in vitro (3Stralfors P. Hiraga A. Cohen P. Eur. J. Biochem. 1985; 149: 295-303Crossref PubMed Scopus (200) Google Scholar, 4Hubbard M.J. Cohen P. Eur. J. Biochem. 1989; 186: 701-709Crossref PubMed Scopus (101) Google Scholar, 5Hubbard M.J. Cohen P. Eur. J. Biochem. 1989; 186: 711-716Crossref PubMed Scopus (87) Google Scholar) and in vivo in response to adrenaline catecholamines (10MacKintosh C. Campbell D.G. Hiraga A. Cohen P. FEBS Lett. 1988; 234: 189-194Crossref PubMed Scopus (32) Google Scholar, 14Walker K.S. Watt P.W. Cohen P. FEBS Lett. 2000; 466: 121-124Crossref PubMed Scopus (47) Google Scholar). Notably, both phosphorylation sites are conserved between rabbit and human GM (7Tang P.M. Bondor J.A. Swiderek K.M. De-Paoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar, 8Lannér C. Suzuki Y. Bi C. Zhang H. Cooper L.D. Bowker-Kinley M.M. DePaoli-Roach A.A. Arch. Biochem. Biophys. 2001; 388: 135-145Crossref PubMed Scopus (15) Google Scholar). Ser67 lies within the PP1c-binding motif of GM (11Zhao S. Lee E.Y.C. J. Biol. Chem. 1997; 272: 28368-28372Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and PKA-dependent phosphorylation triggers dissociation of PP1c from GM, thereby inactivating its phosphatase activity (4Hubbard M.J. Cohen P. Eur. J. Biochem. 1989; 186: 701-709Crossref PubMed Scopus (101) Google Scholar, 5Hubbard M.J. Cohen P. Eur. J. Biochem. 1989; 186: 711-716Crossref PubMed Scopus (87) Google Scholar). (ii) GM is also phosphorylated by GSK3 at Ser40 and Ser44 (15Dent P. Campbell D.G. Hubbard M.J. Cohen P. FEBS Lett. 1989; 248: 67-72Crossref PubMed Scopus (63) Google Scholar). (iii) GM is possibly phosphorylated by insulin-dependent protein kinase at Ser48 (14Walker K.S. Watt P.W. Cohen P. FEBS Lett. 2000; 466: 121-124Crossref PubMed Scopus (47) Google Scholar, 16Dent P. Lavoinne A. Nakielny S. Caudwell F.B. Watt P. Cohen P. Nature. 1990; 348: 302-308Crossref PubMed Scopus (404) Google Scholar). Experiments using GM null mice (17Aschenbach W.G. Suzuki Y. Breeden K. Prats C. Hirshmaan M.F. Dufresne S.D. Sakamoto K. Vilardo P.G. Steele M. Kim J.-H. Jing S.-L. Goodyear L.J. DePaoli-Roach A. J. Biol. Chem. 2001; 276: 39959-39967Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 18Suzuki Y. Lanner C. Kim J.-H. Vilardo P.G. Zhang H. Yang J. Cooper L.D. Steele M. Kennedy A. Bock C.B. Scrimgeour A. Lawrence J.C. DePaoli-Roach A. Mol. Cell. Biol. 2001; 21: 2683-2694Crossref PubMed Scopus (129) Google Scholar) further support the possibility that GM may be involved in regulation of GS activity. Recently, using COS7 cells and C2C12 myotubes transfected with GM, Liu and Brautigan (19Liu J. Brautigan D.L. J. Biol. Chem. 2000; 275: 26074-26081Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) found that transfected GM co-immunoprecipitated with endogenous GS. The interaction between GM and GS occurs at specific sites of GM, localized between aa 77–118 and 219–240 (19Liu J. Brautigan D.L. J. Biol. Chem. 2000; 275: 26074-26081Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). However, thus far, there is no experimental evidence supporting a direct, structural association between GM and GS in native SR membranes. Here, we provide clear evidence that GM associates with nonjunctional SR membranes, where it forms a complex with GS and PP1c. In addition, we demonstrate that GM is phosphodephosphorylated by SR-bound CaMKII and PP1c. Using His-tagged GM recombinant protein corresponding to the aa 40–338 sequence, we also demonstrate that endogenous, as well as exogenous CaMKII are able to phosphorylate serine residue(s) in the NH2-terminal region of GM. The finding that CaMKII is unable to phosphorylate both a truncated GM (aa 69–338) lacking Ser48 and Ser67 and a point-mutated form in which Ser48 is replaced by Ala identifies this residue as a CaMKII target in GM. We thus suggest that Ca2+-calmodulin activation of SR-bound CaMKII might play a pivotal role in the regulation of glycogen metabolism during physical exercise through phosphorylation of GM. Materials—Molecular mass standards were purchased from BDH Laboratories (Poole, UK; molecular weight range 200,000–43,000 or 77,000–12,000). [γ-33P]ATP (3000–6000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Exogenous Ca2+-calmodulin-dependent protein kinase (500,000 units/ml) was purchased from New England Biolabs. Uridine-diphospho-d-[6-3H]glucose, NH4 salt (14.1 Ci/mmol) was purchased from Amersham Biosciences. Hog brain calmodulin was purchased from Roche Applied Science. CaMKII inhibitor KN-93 was purchased from Calbiochem (San Diego, CA). Protein kinase A from bovine heart (1–2 units/μg of protein kinase), dl-propranolol, protein A-Sepharose, and glucose assay kit were purchased from Sigma-Aldrich. Okadaic acid was purchased from Calbiochem-Novabiochem (Bad Soden, Germany). All other chemicals were analytical grade and were purchased from Sigma-Aldrich. Animals and Muscles—New Zealand male adult rabbits were lawfully acquired and properly housed, fed, and taken care of in the Animal Colony of the Department of Experimental Biomedical Sciences of the University of Padova in compliance with Italian Law (Decreto Legge, September 27, 1992, no. 116). The adductor magnus was used as a representative fast twitch muscle (20Damiani E. Sacchetto R. Margreth A. Biochem. Biophys. Res. Commun. 2000; 279: 181-189Crossref PubMed Scopus (12) Google Scholar). Generation and Purification of His6-GM Fusion Proteins—Total cellular RNA extraction from rabbit adductor and cDNA synthesis were performed as described (21Damiani E. Sacchetto R. Salviati L. Margreth A. Biochem. Biophys. Res. Commun. 2003; 302: 73-83Crossref PubMed Scopus (9) Google Scholar). GM cDNA was amplified to generate two forms of GM: GM-(40–338) and GM-(69–338). We used an identical cloning strategy with the same reverse primer and a different forward primer. The primers were designed based on the rabbit GM sequence (7Tang P.M. Bondor J.A. Swiderek K.M. De-Paoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar) (GenBank™ accession number M65109) and are: 40 Forward (5′-CTC TTC ATA TGG CAC ATC ACC ATC ACC ATC ACA TTG ATG ACG ACG ACA AGT CCC CTC AAC CGA GAC GAG-3′, containing the NdeI restriction enzyme and a His6 encoding sequences); 96 Forward (5′-CGA CAT ATG CAC CAC CAC CAC CAC CAC GTC GAC AAC TTT GGA TTC AAT-3′, containing the NdeI restriction enzyme and a His6 encoding sequences); and 338 Reverse (5′-CTT CCT GAA TTC CTA TTC AGA AGC AGC ACA TCT AG-3′, containing the EcoRI restriction enzyme sequence). The first set of primers amplifies the region that encodes for a 299-aa fragment protein (NH2-terminal residues from aa 40 to 338). The second, amplifies the region from aa 69 to 338. PCR conditions were as follow: 94 °C for 3 min, 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 2.5 min for 5 cycles followed by 30 cycles of 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 2.5 min. The extension step was performed using the Pfu DNA polymerase by Promega. The PCR products, purified using a Qiagen kit, were digested with NdeI and EcoRI enzymes and cloned into the pET 21a+ vector (Novagen). The amplified mutated vector was used for heat-pulsing transformation of Novablue cells. Vector extraction was performed using a QIAprep Miniprep kit (Qiagen) After confirmation of the sequences, the plasmid was heat pulse transferred into the Escherichia coli strain BL21 (DE). Cultures were grown at 37 °C in a medium containing ampicillin. When cultures reached an A550 of 0.5 units, the His-tagged fusion protein fragments were induced with 0.5 mm isopropyl β-d-thiogalactopyranoside for3hat 37 °C. His-tagged protein fragments were purified from bacterial lysate by nickel-nitrilotriacetic acid affinity chromatography (Qiagen) according to the manufacturer's instructions. Purified protein was quantified by densitometry of Coomassie Blue-stained gels, with reference to a calibration curve of bovine serum albumin (0.5–10 μg). Site-directed Mutagenesis—Alanine substitution in GM-(40–338) was generated by introducing a mutation with the use of the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's specifications. The mutagenic primers were designed complementary to opposite strands of the vector and containing two bases mismatched (bold letters) in correspondence of serine 48 to obtain alanine. They were as follow: sense primer 5′-CCG AGC AGA CGA GGT GCG GAA TCT TCT GAA GAG GTC-3′; and antisense primer 5′-GAC CTC TTC AGA AGA TTC CGC ACC TCG TCT GCT CGG-3′. PCR conditions were as follows: 1 cycle of 95 °C for 30 s and 16 cycles of 95 °C for 30 s, 65 °C for 1 min, and 72 °C for 7 min. The amplified mutated vector was used for heat-pulsing transformation of Novablue cells. Vector extraction was performed using a QIAprep Miniprep kit (Qiagen). Mutation was confirmed by DNA sequencing of the region containing the mutation. The cloning strategy of the mutated GM-(40–338)-S48A was identical of that described under “Purification of His-GM Fusion Proteins. Mutated His tag GM-(40–338)-S48A was purified from bacterial lysate by nickel-nitrilotriacetic acid affinity chromatography (Qiagen) according to the manufacturer's instructions. Preparative Procedures—To avoid β-adrenergic stimulation and to maximize association of glycogen to SR, rabbits were anesthetized with Zoletil 100 (10 mg/kg per vena) and injected intravenously with propranolol (3 mg/kg), as described by Walker et al. (14Walker K.S. Watt P.W. Cohen P. FEBS Lett. 2000; 466: 121-124Crossref PubMed Scopus (47) Google Scholar). After 10 min, the animals were killed by intravenous injection of 1.5 ml of Tanax (Intervet International B.V., Boxmee, The Netherlands) in accordance with procedure approved by the Animal Committee of the University of Padova. Bilateral adductor muscles (a representative fast twitch muscle) were homogenized in 0.3 m sucrose (20% weight/volume), 5 mm imidazole, pH 7.0, 1 mm EGTA, 1 μg/ml leupeptin, and 100 μm phenylmethylsulfonyl fluoride. Muscle microsomes enriched in content of nonjunctional SR were isolated by the method of Chu et al. (22Chu A. Saito A. Fleischer S. Arch. Biochem. Biophys. 1987; 258: 13-23Crossref PubMed Scopus (18) Google Scholar) by centrifuging muscles homogenates at 7700 × g for 10 min. The post-myofibrillar supernatant was recentrifuged for 20 min at 15,000 × g. The final post-mitochondrial supernatant was centrifuged at 150,000 × g for 90 min to obtain soluble cytoplasm and a microsomal fraction. SR membranes were further purified by isopycnic sucrose density centrifugation (23Saito A. Seiler S. Chu A. Fleischer S. J. Cell Biol. 1984; 99: 875-885Crossref PubMed Scopus (420) Google Scholar) to yield four distinct fractions, labeled F1–F4 from top to bottom of gradients. The Ca2+ pump membrane and the junctional face membrane (JFM) were dissociated by incubating F4 membranes with 0.25% detergent Chaps at low Ca2+ and centrifuging, as described (24Damiani E. Tobaldin G. Bortoloso E. Margreth A. Cell Calcium. 1997; 22: 129-150Crossref PubMed Scopus (23) Google Scholar). Ca2+ pump membrane corresponded to the solubilized material. Crude JFM was further purified by the method of Sacchetto et al. (25Sacchetto R. Turcato F. Damiani E. Margreth A. J. Musc. Res. Cell Motil. 1999; 20: 403-415Crossref PubMed Scopus (28) Google Scholar). The membrane fractions were resuspended in 0.3 m sucrose, 5 mm imidazole, pH 7.4, 100 μm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, divided in aliquots, and stored at -80 °C, until used. Protein concentration was determined by the method of Lowry et al. (26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 265: 10118-10124Google Scholar), using bovine serum albumin as a standard. Measurement of Glycogen—Glycogen content of membrane fractions was measured under the general conditions described by Gomez-Lechon et al. (27Gomez-Lechon M. Ponsoda X. Castell J.V. Anal. Biochem. 1996; 236: 296-301Crossref PubMed Scopus (58) Google Scholar) and Lees et al. (28Lees S.J. Franks P.D. Spangenburg E.E. Williams J.H. J. Appl. Physiol. 2001; 91: 1638-1644Crossref PubMed Scopus (46) Google Scholar). Membranes resuspended (0.5–1 mg/ml) in 0.2 m sodium-acetate buffer, pH 4.8, were incubated at 40 °C with 1750 milliunits/100 μl of amyloglucosidase. At the end of 2 h of incubation, distilled water was added to the assay mixture up to final volume of 1 ml and neutralized with 5.5 μl of 1 n NaOH. Glucose liberated after enzymatic hydrolysis of glycogen was determined by a colorimetric method, using a glucose assay kit (Sigma). A blank of the reaction was performed by incubating SR membranes without amyloglucosidase. A calibration curve was constructed with known amounts of glycogen. Assay of Glycogen Synthase Activity—GS activity was assayed at 37 °C, by the method of Thomas et al. (29Thomas J.A. Schlender K.K. Larner J. Anal. Biochem. 1968; 25: 486-499Crossref PubMed Scopus (949) Google Scholar), measuring the incorporation of [3H]glucose from UDP[3H]glucose into glycogen. Membranes (final protein concentration, 0.4 mg/ml) were incubated in duplicate in a standard assay mixture (final volume, 50 μl), containing 50 mm Tris-HCl, pH 7.8, 10 mm EDTA, 50 mm NaF, 15 mg/ml glycogen, 2 μm okadaic acid, 10 mm UDP[3H]glucose (specific activity, 0.15 μCi/μmol), and 10 mm glucose 6-phosphate. At the end of 2 min of incubation, the assay mixture was spotted on Whatman C glass filters. The filters were washed twice for 30 min with ice-cold 70% ethanol (10 ml/filter) and dried. [3H]Glucose incorporated into glycogen was measured by liquid scintillation. Background radioactivity was measured in the absence of the enzyme. Zero time controls were prepared by mixing membranes and test mixture on ice and immediately spotting the assay mixture on filters. Phosphorylation Assays—CaMKII phosphorylation assay: Phosphorylation of SR proteins and of GM-(40–338) by endogenous CaMKII was carried out, as described previously (21Damiani E. Sacchetto R. Salviati L. Margreth A. Biochem. Biophys. Res. Commun. 2003; 302: 73-83Crossref PubMed Scopus (9) Google Scholar), using a standard medium containing 100 μm free Ca2+ and 1 μm CaM, unless otherwise stated. Incubation was at 30 °C for 30 min. Phosphorylation by exogenous CaMKII of His-tagged GM recombinant fragments was carried out using a commercially available CaMKII (New England Biolabs). Prior to substrate phosphorylation, CaMKII was activated by incubation with ATP/Mg2+ in the presence of CaCl2 and calmodulin as indicated by the manufacturer. Activated CaMKII enzyme was then added to the assay mixture (250 units/50 μl) containing substrate proteins. Composition of phosphorylation assay medium was identical to that used for endogenous phosphorylation. Incubation was at 30 °C for 30 min. PKA Phosphorylation Assay—Phosphorylation by exogenous PKA of SR membranes and of His-tagged GM fusion proteins was carried out at 30 °C as described by Damiani et al. (30Damiani E. Sacchetto R. Margreth A. Biochim. Biophys. Acta. 2000; 1464: 231-241Crossref PubMed Scopus (33) Google Scholar), except that the incubation time was 30 min. Incubation medium contained 5 μm cAMP (final concentration) and 2.6 μm PKA (5–10 units/50 μl). All of the phosphorylation reactions were started by the addition of 50 μm [γ-33P]ATP (specific radioactivity, 0.10 Ci/mmol) and were terminated by adding SDS-solubilizing buffer to samples. 33P-Labeled protein were detected by autoradiography (Hyperfilm Amersham) or by a Bio-Rad model GS-250 Molecular Imager. Bound radioactivity was quantified by using a β-particle sensitive screen and a model GS-250 Molecular Imager (Bio-Rad). One unit of exogenous CaMKII and PKA is defined as the amount of enzyme that will transfer 1 pmol of phosphate to specific substrate in 1 min at 30 °C. Protein Dephosphorylation—Isolated SR membranes were incubated at 30 °C with 50 μm [γ-33P]ATP in assay medium for phosphorylation by endogenous CaMKII. After 30 min of incubation, CaMKII was inhibited by 2 μm staurosporin. As reported in detail in the legends to Fig. 5, protein dephosphorylation by endogenous PP1c was carried out in the same medium for 30 min at 30 °C. Phosphoamino acid Analysis—GM, 33P-phosphorylated by exogenous CaMKII as described above, was blotted to nitrocellulose membrane, localized by autoradiography, and digested by trypsin. The tryptic peptides were subjected to acidic hydrolysis (6 m HClfor4hat110 °C), and the radiolabeled phosphoamino acids were separated by high voltage paper electrophoresis at pH 1.9, as described by Perich et al. (31Perich J.W. Meggio F. Reynolds E.C. Marin O. Pinna L. Biochemistry. 1992; 31: 5893-5897Crossref PubMed Scopus (48) Google Scholar). Preparation of Antisera against GM Fusion Proteins—Chicken polyclonal antibodies against the purified His-tagged GM-(40–338) fusion protein were raised, as previously reported by Damiani et al. (32Damiani E. Betto R. Salvatori S. Volpe P. Salviati G. Margreth A. Biochem. J. 1981; 197: 245-248Crossref PubMed Scopus (41) Google Scholar). Guinea pig polyclonal antibodies were raised against a 11.2-kDa peptide corresponding to aa 754–852 of rabbit GM (7Tang P.M. Bondor J.A. Swiderek K.M. De-Paoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar). The immunogen was expressed and purified by on-column purification through an IMAC column and was then used to raise the antiserum (Harlan Sera-Lab Ltd., Bicester Oxon, UK). Immunoprecipitation—Immunoprecipitation experiments were carried out as described (21Damiani E. Sacchetto R. Salviati L. Margreth A. Biochem. Biophys. Res. Commun. 2003; 302: 73-83Crossref PubMed Scopus (9) Google Scholar). 250 μg of F4 protein, after solubilization, was incubated for 2 h in cold room with the guinea pig anti-GM antibody to (1:1000 dilution). Protein A-Sepharose (40 μl) was added to the mixture, incubated for 1 h, and sedimented using an Eppendorf centrifuge. After washing, the pellets were solubilized with SDS solubilization buffer. Gel Electrophoresis and Immunoblotting—SDS-PAGE (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar) and immunoblotting were carried out, as described (21Damiani E. Sacchetto R. Salviati L. Margreth A. Biochem. Biophys. Res. Commun. 2003; 302: 73-83Crossref PubMed Scopus (9) Google Scholar). Slab gels were stained with Coomassie Blue and then with Stains All. Apparent Mr values were calculated from a graph of relative mobilities versus log Mr of standard proteins. The blots were probed with: (i) mouse monoclonal antibodies to α-actinin (Sigma), RyR1 (sarcoplasmic reticulum Ca2+ release channel, skeletal isoform; BioMol, Plymouth Meeting, PA), GP53 (a SR glycoprotein of 53 kDa) (ABR, Golden, CO), GS (Chemicon International, Temecula, CA), and PP1c (Santa Cruz Biotechnology, CA); (ii) guinea pig polyclonal antibodies to GM; or (iii) chicken polyclonal antibodies to GM. Antibody binding was detected by immunoenzymic staining (21Damiani E. Sacchetto R. Salviati L. Margreth A. Biochem. Biophys. Res. Commun. 2003; 302: 73-83Crossref PubMed Scopus (9) Google Scholar). Densitometry of blotted proteins, after immunostaining, was carried out using a Bio-Rad model GS-670 imaging densitometer. GS content was determined by densitometry of proteins immunostained with mouse monoclonal antibody GS, with reference to a calibration curve of purified GS. GM content was determined by densitometry of blots immunostained with chicken polyclonal antibodies to GM, with reference to a calibration curve obtained with purified GM-(40–338). Immunofluorescence Microscopy—Muscle cryosections were fixed with paraformaldehyde and immunostained by incubating with primary antibodies to GM and GS (dilution 1:100), followed by incubation with the appropriate secondary antibody conjugated with TRITC or fluorescein isothiocyanate (Dako), as described by Sacchetto et al. (34Sacchetto R. Damiani E. Margreth A. J. Muscle Res. Cell Motil. 2002; 22: 545-559Crossref Scopus (14) Google Scholar). The images were captured by a B/W chilled CCD camera (Hamamatsu, Japan), transmitted to an interactive image analysis system equipped with image memory (High Fish Beta, version 2.0) and image processing software (Image Processing, version 3.4, Casti Imaging, Venice, Italy). The images were printed on a CP-D1E printer (Mitsubishi, Japan). Localization of GM to SR—When longitudinal cryosections from rabbit adductor muscle were probed with a guinea pig anti-GM antibody, a cross-striated pattern was observed (Fig. 1A). By comparison with phase contrast microscopy, the fluorescent striations were found to correspond to the I band of the sarcomere. Fig. 1B shows that GM co-localized with GS, which also is resident in the I band (35Nielsen J.N. Derave W. Kristiansens S. Ralston E. Ploug T. Richter E.A. J. Physiol. 2001; 531: 757-769Crossref PubMed Scopus (110) Google Scholar). The overlapping between the two proteins was virtually complete. It was verified that non-specific antibody or secondary alone did not give this fluorescence pattern (not shown). The subcellular localization of GM was further investigated by differential centrifugation of muscle homogenates. The immunoblot analysis shown in Fig. 1C clearly shows that most GM sedimented mostly in the microsomal fraction, which was devoid of contamination by myofibrillar proteins and highly enriched in SR membranes, as demonstrated by the localization of the SR-specific protein markers, RyR1 and GP53. It is noteworthy that GM and GS co-fractionated throughout the entire preparation. A fraction enriched in nonjunctional SR membranes was obtained from muscle microsomes by isopycnic-sucrose density centrifugation (22Chu A. Saito A. Fleischer S. Arch. Biochem. Biophys. 1987; 258: 13-23Crossref PubMed Scopus (18) Google Scholar). This procedure, at variance with that of Saito et al. (23Saito A. Seiler S. Chu A. Fleischer S. J. Cell Biol. 1984; 99: 875-885Crossref PubMed Scopus (420) Google Scholar), which yields a much higher proportion of membranes deriving from the terminal cisternae of junctional SR, yields mainly vesicles derived from nonjunctional SR (22Chu A. Saito A. Fleischer S. Arch. Biochem. Biophys. 1987; 258: 13-23Crossref PubMed Scopus (18) Google Scholar). The nonjunctional SR origin of this fraction was confirmed by [3H]ryanodine binding measurements, carried out at optimal conditions of free Ca2+ (100 μm) and ionic strength (1 m KCl). The specific SR illustrated in Fig. 2A gave a Bmax value of 1.1 pmol/mg protein, which is almost 20-fold lower than that previously reported for pur" @default.
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