Translation of Ser16 and Thr17 phosphorylation of phospholamban into Ca 2+-pump stimulation.
Stimulation of cardiac sarcoplasmic reticulum Ca 2+-pump activity is achieved by phosphorylation of the oligomeric protein phospholamban at either Ser16 or Thr17. The altered mobility of phosphorylated forms of pentameric phospholamban has been utilized to demonstrate that the mechanisms of phosphorylation of the two sites differ. Phosphorylation of Ser16 by the AMP-dependent protein kinase proceeds via a random mechanism [Li, Wang and Colyer (1990) Biochemistry 29, 4535-4540], whereas phosphorylation of Thr17 by calmodulin-dependent protein kinase is shown here to proceed via a co-operative mechanism. This co-operative reaction mechanism was unaffected by the phosphorylation status of Ser16. These two mechanisms of phosphorylation generate very different phosphoprotein profiles depending on whether the Ser16 or Thr17 residue is phosphorylated. The translation of these patterns of phosphorylation into Ca 2+-pump function was reviewed using a fluorimetric Ca 2+-indicator dye, fluo-3, to measure Ca2+ uptake by cardiac sarcoplasmic reticulum vesicles. The rate of Ca2+ accumulation, which parallels Ca 2+-pump activity, was stimulated in proportion with the stoichiometry of phospholamban phosphorylation, irrespective of whether phosphorylation was on Ser16 or Thr17.
Jackson
W A
WA
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Colyer
J
J
D000814:Aniline Compounds; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004285:Dogs; D005456:Fluorescent Dyes; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012694:Serine; D013737:Testis / Q000378:metabolism; D013912:Threonine; D014966:Xanthenes; D000099211:Phospholamban
D000814:Aniline Compounds; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D002147:Calmodulin; D017868:Cyclic AMP-Dependent Protein Kinases; D005456:Fluorescent Dyes; D012694:Serine; D013912:Threonine; D014966:Xanthenes; D000099211:Phospholamban; C059715:Fluo-3