Preservation of the in vivo phosphorylation status of phospholamban in the heart: evidence for a site-specific difference in the dephosphorylation of phospholamban.
The phosphorylation status of the cardiac sarcoplasmic reticular (SR) protein phospholamban determines the activity of the SR Ca(2+)-ATPase. In order to predict SR Ca2+ transport in vivo, it is vital that techniques used to measure the phosphorylation status of phospholamban adequately clamp the endogenous kinases and phosphatases which modify phosphorylation during sample preparation. A recent study (Boateng, S., Seymour, A-M., Dunn, M., Yacoub, M., and Boheler, K. (1997) Biochem. Biophys. Res. Comm. 239, 701-705) has suggested that phosphatase inhibitors must be present in quenching media to prevent almost total dephosphorylation of phospholamban. We addressed this issue by assessing the effect of both kinase and phosphatase inhibition on site-specific phosphorylation of phospholamban in ferret ventricular muscle and isolated rat ventricular myocytes quenched with Laemmli sample buffer. Under these clearly defined quenching conditions in isolated myocytes, we demonstrated that the phosphorylation status of phospholamban was low under basal conditions, and high following exposure to the beta-agonist isoprenaline. The only significant effect of inhibitor inclusion in the quench solution was in isolated myocyte preparations where phosphatase inhibition increased phosphorylation at Ser16 by about a third. The differential effect of phosphatase inclusion on phosphorylation at Ser16 and Thr17 may indicate that different enzymes are involved in dephosphorylation of the two sites.
School of Biochemistry and Molecular Biology, University of Leeds, United Kingdom. s.c.calaghan@leeds.ac.uk
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Calaghan
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White
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Colyer
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005289:Ferrets; D006352:Heart Ventricles; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D010749:Phosphoprotein Phosphatases; D011494:Protein Kinases; D012969:Sodium Fluoride; D019311:Staurosporine; D000099211:Phospholamban