IMMUNODETECTION OF PHOSPHORYLATION SITES GIVES NEW INSIGHTS INTO THE MECHANISMS UNDERLYING PHOSPHOLAMBAN PHOSPHORYLATION IN THE INTACT HEART

Phosphorylation site-specific antibodies, quantification of P-32 incor poration into phospholamban, and simultaneous measurements of mechanic al activity were used in Langendorff-perfused rat hearts to provide fu rther insights into the underlying mechanisms of phospholamban phospho rylation. Immunological detection of phospholamban phosphorylation sit es showed that the isoproterenol concentration-dependent increase in p hospholamban phosphorylation was due to increases in phosphorylation o f both Ser(16) and Thr(17) residues. When isoproterenol concentration was increased at extremely low Ca2+ supply to the myocardium, phosphor ylation of Thr(17) was virtually absent. Under these conditions, P-32 incorporation into phospholamban, due to Ser(16), decreased by 50%, Ch anges in Ca2+ supply to the myocardium either at constant beta-adrener gic stimulation or in the presence of okadaic acid, a phosphatase inhi bitor, exclusively modified Thr(17) phosphorylation. Changes in phosph olamban phosphorylation due to either Ser(16) and/or Thr(17) were para lleled by changes in myocardial relaxation. The results indicate that cAMP-(Ser(16)) and Ca2+-calmodulin (Thr(17))-dependent pathways of pho spholamban phosphorylation can occur independently of each other. Howe ver, in the absence of beta-adrenergic stimulation, phosphorylation of Thr(17) could only be detected after simultaneous activation of Ca2+- calmodulin-dependent protein kinase and inactivation of phosphatase. I t is suggested that under physiological conditions, this requisite is only filled by cAMP-dependent mechanisms.