caa a22 4500 477119948 CHVBK 20180405111633.0 cr unu---uuuuu 170330e19960201xx s 000 0 eng 10.1007/BF00229306 doi (NATIONALLICENCE)springer-10.1007/BF00229306 Phosphorylation of cardiac junctional and free sarcoplasmic reticulum by PKCα, PKCβ, PKA and the Ca2+/calmodulin-dependent protein kinase [Elektronische Daten] [Bruce Allen, Sidney Katz] Phosphorylation of cardiac junctional and free sarcoplasmic reticulum (SR) by protein kinase C (PKC) isoforms α and β was investigated. Both SR and PKC were isolated from canine heart. Junctional and free SR vesicles were prepared by calcium-phosphate-loading. The substrate specificities of PKCα and PKCβ were found to be similar in both SR fractions. A high molecular weight junctionally-associated protein was phosphorylated by PKA, PKC and an endogenous Ca2+/calmodulin-dependent protein kinase activity: the highest levels of phosphate incorporation being catalysed by the latter kinase. In addition to this high molecular weight junctionally-associated protein, PKC induced phosphorylation of 45, 96 kDa and several proteins of greater than 200 kDa in junctional SR. A protein of 96 kDa was phosphorylated by both isoforms in junctional and free SR. The major substrate for PKA, PKCα, PKCβ and the Ca2+/calmodulin-dependent protein kinase, in both junctional and free SR, was phospholamban. Although the phosphorylation of phospholamban by PKC was activated by Ca2+, a component of this activity appeared to be independent of Ca2+. PKC-mediated phosphorylation of phospholamban was fully activated by 1 μM Ca2+ whereas the Ca2+/calmodulin dependent kinase required concentrations in excess of 5 μM Ca2+. In the in vitro system employed in these studies, the concentrations of either PKCα or the catalytic subunit of PKA required to phosphorylate phospholamban were found to be similar. In addition, in the presence of a 15 kDa sarcolemmal-associated protein, which becomes phosphorylated upon activation of PKC in vivo, phosphorylation of phospholamban by PKC was unaffected. These results demonstrate that, although substrates for both subtypes are found in both junctional and free SR, PKCα and PKCβ do not show differences in selectivity towards these substrates. Kluwer Academic Publishers, 1996 cardiac nationallicence sarcoplasmic reticulum nationallicence protein kinase C nationallicence isoenzymes nationallicence phosphorylation nationallicence Ca2+ : free calcium nationallicence CaM kinase : Ca2+/calmodulin-dependent protein kinase nationallicence DTT : dithiothreitol nationallicence EDTA : ethylenediaminetetraacetic acid nationallicence EGTA : ethylene glycol bis(b-aminoethylether)-N,N,N′,N′-tetraacetic acid nationallicence FSR : free sarcoplasmic reticulum nationallicence JSR : junctional sarcoplasmic reticulum nationallicence PKC : protein kinase C nationallicence PS : phosphatidylserine nationallicence SDS : sodium dodecyl sulfate nationallicence SAG : 1-stearoyl-2-arachidonylglycerol nationallicence TPCK : L-1-tosylamido-2-phenylethyl chloromethyl ketone nationallicence Tris/HCI : tris(hydroxymethyl)aminomethane hydrochloride nationallicence Allen Bruce Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, V6T 1Z3, Vancouver, BC, Canada aut Katz Sidney Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, V6T 1Z3, Vancouver, BC, Canada aut Molecular and Cellular Biochemistry Kluwer Academic Publishers 155/2(1996-02-01), 91-103 0300-8177 155:2<91 1996 155 11010 https://doi.org/10.1007/BF00229306 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00229306 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Allen Bruce Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, V6T 1Z3, Vancouver, BC, Canada aut NATIONALLICENCE 700 1- Katz Sidney Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, V6T 1Z3, Vancouver, BC, Canada aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Kluwer Academic Publishers 155/2(1996-02-01), 91-103 0300-8177 155:2<91 1996 155 11010 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer