Frequency-encoding Thr(17) phospholamban phosphorylation is independent ofSer(16) phosphorylation in cardiac myocytes

Both Ser(16) and Thr(17) Of phospholamban (PLB) are phosphorylated, respect ively, by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), PLB phosphorylation relieves cardiac sarcoplas mic reticulum Ca2+ pump from inhibition by PLB. Previous studies have sugge sted that phosphorylation of Ser(16) by PKA is a prerequisite for Thr(17) p hosphorylation by CaMKII and is essential to the relaxant effect of beta-ad renergic stimulation. To determine the role of Thr(17) PLB phosphorylation, we investigated the dual-site phosphorylation of PLB in isolated adult rat cardiac myocytes in response to beta(1)-adrenergic stimulation or electric al field stimulation (0.1-3 Hz) or both. A beta(1)-adrenergic agonist, nore pinephrine (10(-9)-10(-6) M), in the presence of an alpha(1)-adrenergic ant agonist, prazosin (10(-6) hr), selectively increases the PKA-dependent phos - phorylation of PLB at Ser(16) in quiescent myocytes, In contrast, electri cal pacing induces an opposite phosphorylation pattern, selectively enhanci ng the CaMKII-mediated Thr(17) PLB phosphorylation in a frequency-dependent manner. When combined, electric stimulation (2 Hz) and beta(1)-adrenergic stimulation lead to dual phosphorylation of PLB and exert a synergistic eff ect on phosphorylation of Thr(17) but not Ser(16) Frequency-dependent Thr(1 7) phosphorylation is closely correlated with a decrease in 50% relaxation time (t(50)) of cell contraction, which is independent of, but additive to, the relaxant effect of Ser(16) phosphorylation, resulting in hastened cont ractile relaxation at high stimulation frequencies. Thus, we conclude that in intact cardiac myocytes, phosphorylation of PLB at Thr(17) occurs in the absence of prior Ser(16) phosphorylation, and that frequencydependent Thr( 17) PLB phosphorylation may provide an intrinsic mechanism for cardiac myoc ytes to adapt to a sudden change of heart rate.