Transgenic Approaches to Define the Functional Role of Dual Site Phospholamban Phosphorylation

Abstract

Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca 2؉ -ATPase activity and myocardial contractility. Phosphorylation of phospholamban occurs on both Ser 16 and Thr 17 during isoproterenol stimulation. To determine the physiological significance of dual site phospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser 16 3 Ala mutant phospholamban in the cardiac compartment of the phospholamban knockout mice. Transgenic lines with similar levels of mutant or wildtype phospholamban were studied in parallel. Langendorff perfusion indicated that the basal hyperdynamic cardiac function of the knockout mouse was reversed to the same extent by reinsertion of either wild-type or mutant phospholamban. However, isoproterenol stimulation was associated with much lower responses in the contractile parameters of mutant phospholamban compared with wild-type hearts. These attenuated responses were due to lack of phosphorylation of mutant phospholamban, assessed in 32 P labeling perfusion experiments. The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser 16 to Ala, since the mutated phospholamban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser 16 is a prerequisite for Thr 17 phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the ␤-agonist stimulatory responses in the mammalian heart. Phospholamban (PLB)1 is a regulator of the affinity of the cardiac sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase for Ca 2ϩ . Dephosphorylated PLB is an inhibitor, and phosphorylation of PLB removes its inhibitory effects on the SR Ca 2ϩ -ATPase. Recently, the critical role of PLB in the regulation of cardiac contractility has been defined through gene transfer (1) and knockout (2) technology in the mouse. Cardiac-specific overexpression of PLB was associated with decreases in the affinity of the SR Ca 2ϩ -ATPase for Ca 2ϩ and depressed cardiac function, whereas PLB deficiency resulted in increased Ca 2ϩ affinity of the Ca 2ϩ -ATPase and enhanced myocardial performance. Furthermore, the stimulatory effects to ␤-adrenergic agonists were more pronounced in the PLB-overexpressing hearts, whereas these effects were attenuated in the PLB-knockout hearts compared with wild types (1, 2). These studies suggested that PLB plays a prominent role in the heart's responses to ␤-agonists. However, PLB is phosphorylated on both Ser 16 and Thr 17 during isoproterenol stimulation (3) and the relative contribution of each site in the altered contractile responses of the heart is not presently well known. In vitro studies have shown that Ser 16 is phosphorylated by cAMP-dependent protein kinase, whereas Thr 17 is phosphorylated by Ca 2ϩ -calmodulin-dependent protein kinase (4). Phosphorylation of each site occurs in an independent manner, although it is not presently clear whether the stimulatory effects of the two phosphorylations on SR Ca...

Sites on the cytoplasmic region of phospholamban involved in interaction with the calcium‐activated ATPase of the sarcoplasmic reticulum

“…Although the detailed mechanism of this process is not understood, a current view is that in the unphosphorylated form PLB interacts with SERCA and inhibits pumping activity, possibly by reducing its affinity for calcium ions (compare [11] with [12,13]), and reducing the maximum turnover rate at saturating calcium ion concentration, which is reflected in the Ca/MgATPase activity rate (reviewed in [14,15]). Phosphorylation of Ser16 of PLB by cAMP or cGMP dependent protein kinases and/or phosphorylation of Thr17 by the multifunctional calmodulin dependent protein kinase [16,17] results in increased activity of the pump [18,19].The detailed mechanism by which PLB modulates the activity of the calcium pump is far from clear as the experimental findings of different investigators have not always been consistent (see [17,20] for discussion on this point). The fact that the sequence of PLB comprises two distinctive domains (an N-terminal hydrophilic region, residues 1±31, presumed to be cytoplasmic, and a C-terminal hydrophobic transmembrane region, residues 32±52), implies that the functional roles of these two domains in the interaction of PLB with the calcium pump are different.…”

“…The detailed mechanism by which PLB modulates the activity of the calcium pump is far from clear as the experimental findings of different investigators have not always been consistent (see [17,20] for discussion on this point). The fact that the sequence of PLB comprises two distinctive domains (an N-terminal hydrophilic region, residues 1±31, presumed to be cytoplasmic, and a C-terminal hydrophobic transmembrane region, residues 32±52), implies that the functional roles of these two domains in the interaction of PLB with the calcium pump are different.…”

“…Although the detailed mechanism of this process is not understood, a current view is that in the unphosphorylated form PLB interacts with SERCA and inhibits pumping activity, possibly by reducing its affinity for calcium ions (compare [11] with [12,13]), and reducing the maximum turnover rate at saturating calcium ion concentration, which is reflected in the Ca/MgATPase activity rate (reviewed in [14,15]). Phosphorylation of Ser16 of PLB by cAMP or cGMP dependent protein kinases and/or phosphorylation of Thr17 by the multifunctional calmodulin dependent protein kinase [16,17] results in increased activity of the pump [18,19].…”

Myotonic Dystrophy Protein Kinase Phosphorylates Phospholamban and Regulates Calcium Uptake in Cardiomyocyte Sarcoplasmic Reticulum

“…Under physiological conditions, PLN phosphorylation at Ser-16 by PKA is the predominant event so far described that leads to proportional increases in the rate of Ca 2ϩ uptake into SR and accelerates ventricular relaxation (40,41). DMPK phosphorylation of Ser-16-PLN emerges as a novel mechanism involved in the regulation of cardiac contractility that may be therapeutically relevant because of the preferential muscular expression pattern of DMPK.…”

The importance of the Thr17 residue of phospholamban as a phosphorylation site under physiological and pathological conditions

“…These findings suggested an interaction between the PKA and CaMKII pathways for PLN phosphorylation. The availability of transgenic models expressing wild-type PLN (PLN-WT), the Ser 16 →Ala mutant PLN (PLN-S16A) or the Thr 17 →Ala mutant PLN (PLN-T17A) in the cardiac compartment of PLN knock-out mice, and of phosphorylation sitespecific antibodies to PLN, which precisely discriminate between the Ser 16 and the Thr 17 phosphorylation sites, in combination with the quantification of 32 P incorporation into PLN, helped to clarify the relative role of Ser 16 and Thr 17 phosphorylation (9,13,14). Experiments in transgenic mice expressing either PLN-WT or the PLN-S16A, indicated that the phosphorylation of Ser 16 of PLN is a prerequisite for the phosphorylation of Thr 17 (13).…”