Phosphorylation of cardiac junctional and free sarcoplasmic reticulum by PKC alpha, PKC beta, PKA and the Ca2+/calmodulin-dependent protein kinase.
Phosphorylation of cardiac junctional and free sarcoplasmic reticulum (SR) by protein kinase C (PKC) isoforms alpha and beta 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 PKC alpha and PKC beta 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, PKC alpha, PKC beta 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 microM Ca2+ whereas the Ca2+/calmodulin dependent kinase required concentrations in excess of 5 microM Ca2+. In the in vitro system employed in these studies, the concentrations of either PKC alpha 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, PKC alpha and PKC beta do not show differences in selectivity towards these substrates.
Allen
B G
BG
Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Katz
S
S
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004285:Dogs; D007527:Isoenzymes / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D011494:Protein Kinases / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D013379:Substrate Specificity; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D007527:Isoenzymes; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D011494:Protein Kinases; D000099211:Phospholamban