Ser¹⁶prevails over Thr¹⁷phospholamban phosphorylation in the β-adrenergic regulation of cardiac relaxation

Abstract

Phospholamban is a critical regulator of sarcoplasmic reticulum Ca2+-ATPase and myocardial contractility. To determine the extent of cross signaling between Ca2+ and cAMP pathways, we have investigated the β-adrenergic-induced phosphorylation of Ser16 and Thr17 of phospholamban in perfused rat hearts using antibodies recognizing phospholamban phosphorylated at either position. Isoproterenol caused the dose-dependent phosphorylation of Ser16 and Thr17 with strikingly different half-maximal values (EC50 = 4.5 ± 1.6 and 28.2 ± 1.4 nmol/l, respectively). The phosphorylation of Ser16 induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine correlated to increased cardiac relaxation ( r = 0.96), whereas phosphorylation of Thr17 did not. Elevation of extracellular Ca2+did not induce phosphorylation at Thr17; only in the presence of a submaximal dose of isoproterenol, phosphorylation at Thr17 increased eightfold without additional effects on relaxation rate. Thr17 phosphorylation was partially affected by ryanodine and was completely abolished in the presence of 1 μmol/l verapamil or nifedipine. The data indicate that 1) phosphorylation of phospholamban at Ser16 by cAMP-dependent protein kinase is the main regulator of β-adrenergic-induced cardiac relaxation definitely preceding Thr17 phosphorylation and 2) the β-adrenergic-mediated phosphorylation of Thr17 by Ca2+-calmodulin-dependent protein kinase required influx of Ca2+through the L-type Ca2+ channel.

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

“…The activity of CaMKII is regulated in response to frequency and amplitude of Ca 2+ transients [38,39]. At low stimulation frequencies CaMKII mediated phosphorylation is small but increases substantially at high frequencies [14,40,41]. Given the large number of effector proteins, specific targeting of CaMKII activity is critical to coordinate the dynamic functional requirements of the cardiomyocyte.…”

Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses

“…Following β-AR activation, and thus production of cAMP by AC, PKA phosphorylates PLN on serine-16 (10) in addition to RyR2 on serine-2808 and serine-2814 (11). PKA-mediated serine-16 phosphorylation (and thus SERCA2a activation) is the major mechanism of the lusitropic and also inotropic (12) effects of β-AR stimulation in regulating cardiac function (13). In failing hearts, in contrast, decreased PLN phosphorylation, and thus a decrease in Ca 2+ uptake by SERCA2a, is a central feature (14,15).…”

Phospholamban knockout increases CaM kinase II activity and intracellular Ca2+wave activity and alters contractile responses of murine gastric antrum

“…In cardiac, skeletal, and smooth muscles, SERCA activity is under rheostat control by the SR membrane protein phospholamban (PLB) to continuously vary the rate of Ca 2ϩ influx into the SR via its phosphorylation levels, from maximal SERCA inhibition of 50% by dephospho-PLB to maximal SERCA activity by phospho-PLB (23,38). Phosphorylation of PLB by cAMP-dependent protein kinase or cGMP-dependent protein kinase at Ser16 or by CaM kinase II at Thr17 removes its inhibitory effects on SERCA, thereby increasing Ca 2ϩ uptake into the SR and contributing to relaxation (8,25,32,43). The role of PLB in the regulation of contractility by SR Ca 2ϩ uptake and release has been investigated through the use of gene knock out and transgenic mouse models (32,38,43).…”