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Phosphorylation of cardiac junctional and free sarcoplasmic reticulum by PKCα, PKCβ, PKA and the Ca2+/calmodulin-dependent protein kinase

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Abstract

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.

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Abbreviations

Ca2+ :

free calcium

CaM kinase:

Ca2+/calmodulin-dependent protein kinase

DTT:

dithiothreitol

EDTA:

ethylenediaminetetraacetic acid

EGTA:

ethylene glycol bis(b-aminoethylether)-N,N,N′,N′-tetraacetic acid

FSR:

free sarcoplasmic reticulum

JSR:

junctional sarcoplasmic reticulum

PKC:

protein kinase C

PS:

phosphatidylserine

SDS:

sodium dodecyl sulfate

SAG:

1-stearoyl-2-arachidonylglycerol

TPCK:

L-1-tosylamido-2-phenylethyl chloromethyl ketone

Tris/HCI:

tris(hydroxymethyl)aminomethane hydrochloride

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Author notes

  1. Bruce G. Allen

    Present address: Smooth Muscle Research Group and Department of Medical Biochemistry, University of Calgary, 3330 Hospital Drive NW, T2N 4N1, Calgary, Alberta, Canada

Authors and Affiliations

  1. Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, V6T 1Z3, Vancouver, BC, Canada

    Bruce G. Allen & Sidney Katz

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This work was supported by a grant (to S.K.) from the Heart and Stroke Foundation of B.C. and Yukon. The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked ‘advertisement’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Studentship form the Heart and Stroke Foundation of Canada.

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Allen, B.G., Katz, S. Phosphorylation of cardiac junctional and free sarcoplasmic reticulum by PKCα, PKCβ, PKA and the Ca2+/calmodulin-dependent protein kinase. Mol Cell Biochem 155, 91–103 (1996). https://doi.org/10.1007/BF00229306

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