C. Mundinaweilenmann et al., IMMUNODETECTION OF PHOSPHORYLATION SITES GIVES NEW INSIGHTS INTO THE MECHANISMS UNDERLYING PHOSPHOLAMBAN PHOSPHORYLATION IN THE INTACT HEART, The Journal of biological chemistry, 271(52), 1996, pp. 33561-33567
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.