Translation of Ser16 and Thr17 phosphorylation of phospholamban into Ca2+-pump stimulation

Jackson, Wayne A.; Colyer, John

doi:10.1042/bj3160201

Cited by 47 publications

(53 citation statements)

References 39 publications

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“…Each phosphorylation is associated with stimulation of the apparent affinity of SERCA2 for Ca 2ϩ . In vivo studies have shown that only Ser 16 and Thr 17 are phosphorylated in cardiac myocytes or perfused hearts (11,12), whereas phosphorylation of PLB by protein kinase C has not been detected in vivo. Phosphorylation of PLB by PKA and CaMKII occurs during ␤-agonist exposure, although the relative contribution of each phosphorylation to the cardiac stimulatory effects is not presently clear.…”

Section: Phospholamban (Plb)mentioning

confidence: 99%

“…In vitro studies have shown that PLB can be phosphorylated on Ser 10 by protein kinase C, Ser 16 by cAMP-dependent protein kinase (PKA), and Thr 17 by Ca 2ϩ -calmodulin-dependent protein kinase (CaMKII) (1,9,10). Each phosphorylation is associated with stimulation of the apparent affinity of SERCA2 for Ca 2ϩ .…”

Section: Phospholamban (Plb)mentioning

confidence: 99%

“…However, those parameters were significantly attenuated in the S16A mutant myocytes. Thus, Ser 16 in PLB can be phosphorylated independently of Thr 17 in vivo, and phosphorylation of Ser 16 is sufficient for mediating the maximal cardiac responses to ␤-adrenergic stimulation.…”

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confidence: 99%

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A Single Site (Ser16) Phosphorylation in Phospholamban Is Sufficient in Mediating Its Maximal Cardiac Responses to β-Agonists

Chu

Lester

Young

et al. 2000

Journal of Biological Chemistry

146

117

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Phospholamban (PLB) can be phosphorylated at Ser 16by cyclic AMP-dependent protein kinase and at Thr 17 by Ca 2؉ -calmodulin-dependent protein kinase during ␤-agonist stimulation. A previous study indicated that mutation of S16A in PLB resulted in lack of Thr 17 phosphorylation and attenuation of the ␤-agonist stimulatory effects in perfused mouse hearts. To further delineate the functional interplay between dual-site PLB phosphorylation, we generated transgenic mice expressing the T17A mutant PLB in the cardiac compartment of the null background. Lines expressing similar levels of T17A mutant, S16A mutant, or wild-type PLB in the null background were characterized in parallel. Phospholamban (PLB)1 is a low molecular weight phosphoprotein in cardiac sarcoplasmic reticulum (SR). Dephosphorylated PLB is an inhibitor of the affinity of SERCA2 for Ca 2ϩ , and phosphorylation of PLB during ␤-adrenergic stimulation relieves its inhibitory effects on SERCA2 (1, 2). The physiological importance of PLB has been elucidated through the generation of genetically engineered mouse models with alterations in cardiac PLB expression levels (3, 4). Ablation of PLB was associated with significantly enhanced Ca 2ϩ affinity of SERCA2 and myocardial performance (3, 5, 6). The elevated basal contractile parameters could be minimally stimulated by ␤-agonists (3, 7), whereas there were no alterations in the ␤-receptor signaling pathway or the phosphorylation states of other major cardiac phosphoproteins (8). On the other hand, overexpression of PLB was associated with significant depression of contractile parameters, which could be reversed upon phosphorylation of PLB during ␤-agonist stimulation (4). These results indicate that PLB is a key regulator of cardiac function and a prominent mediator of the ␤-adrenergic effects in the myocardium.In vitro studies have shown that PLB can be phosphorylated on Ser 10 by protein kinase C, Ser 16 by cAMP-dependent protein kinase (PKA), and Thr 17 by Ca 2ϩ -calmodulin-dependent protein kinase (CaMKII) (1, 9, 10). Each phosphorylation is associated with stimulation of the apparent affinity of SERCA2 for Ca 2ϩ . In vivo studies have shown that only Ser 16 and Thr 17 are phosphorylated in cardiac myocytes or perfused hearts (11, 12), whereas phosphorylation of PLB by protein kinase C has not been detected in vivo. Phosphorylation of PLB by PKA and CaMKII occurs during ␤-agonist exposure, although the relative contribution of each phosphorylation to the cardiac stimulatory effects is not presently clear. Each phosphorylation appears to occur independently of the other (13-16). Some studies have reported additive effects of PKA and CaMKII phosphorylation of PLB on SR Ca 2ϩ transport (13,14,17,18), whereas others (16, 19) have proposed that maximal stimulation of the Ca 2ϩ pump occurs by phosphorylation at a single site, and additional phosphorylation of the other site does not further stimulate the pump activity.Several in vivo studies have shown that Ser 16 phosphorylation or dephosphorylation precede...

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Section: Phospholamban (Plb)mentioning

confidence: 99%

Section: Phospholamban (Plb)mentioning

confidence: 99%

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A Single Site (Ser16) Phosphorylation in Phospholamban Is Sufficient in Mediating Its Maximal Cardiac Responses to β-Agonists

Chu

Lester

Young

et al. 2000

Journal of Biological Chemistry

146

117

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“…25,26 Phosphorylation site-specific PLB antibodies 27 were used in the present study to determine whether ␤-AR subtype stimulation differentially regulates PLB phosphorylation. In the absence of ␤-AR stimulation, only a minor PLB phosphorylation is detectable.…”

Section: Plb Phosphorylationmentioning

confidence: 99%

β ₂ -Adrenergic cAMP Signaling Is Uncoupled From Phosphorylation of Cytoplasmic Proteins in Canine Heart

et al. 1999

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Background —Recent studies of β-adrenergic receptor (β-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling is activated in the same manner in response to β 2 -AR versus β 1 -AR stimulation. Methods and Results —The present study compared, in the intact dog, the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects of cAMP accumulation, PKA activation, and PKA-dependent phosphorylation of key effector proteins in response to β-AR subtype stimulation. In addition, many of these parameters and L-type Ca 2+ current ( I Ca ) were also measured in single canine ventricular myocytes. The results indicate that although the cAMP/PKA-dependent phosphorylation cascade activated by β 1 -AR stimulation could explain the resultant modulation of cardiac function, substantial β 2 -AR–mediated chronotropic, inotropic, and lusitropic responses occurred in the absence of PKA activation and phosphorylation of nonsarcolemmal proteins, including phospholamban, troponin I, C protein, and glycogen phosphorylase kinase. However, in single canine myocytes, we found that β 2 -AR–stimulated increases in both I Ca and contraction were abolished by PKA inhibition. Thus, the β 2 -AR–directed cAMP/PKA signaling modulates sarcolemmal L-type Ca 2+ channels but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins. Conclusions —These results indicate that the dissociation of β 2 -AR signaling from cAMP regulatory systems is only apparent and that β 2 -AR–stimulated cAMP/PKA signaling is uncoupled from phosphorylation of nonsarcolemmal regulatory proteins involved in excitation-contraction coupling.

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“…Phosphorylation of phospholamban on one of a number of sites (Ser-16, cAMP-dependent protein kinase; Thr-17, CaMKII; Ref. 19) abrogates the inhibitory influence of phospholamban (20), to reveal enhanced Ca 2ϩ transport activity at all physiological Ca 2ϩ concentrations. The kinetic basis of inhibition and subsequent activation of Ca 2ϩ transport is complex; involving contributions from the acceleration of particular reaction steps in the catalytic cycle of SERCA (21) and an increase in the coupling efficiency between ATP hydrolysis and Ca 2ϩ movement (13).…”

mentioning

confidence: 99%

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

Rodríguez¹,

Jackson²,

Colyer

2004

Journal of Biological Chemistry

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The phosphorylation of the cardiac muscle isoform of the sarcoplasmic reticulum (SR) Ca 2؉ -ATPase (SERCA2a) on serine 38 has been described as a regulatory event capable of very significant enhancement of enzyme activity (Hawkins, C., Xu, A., and Narayanan, N. (1994) J. Biol. Chem. 269, 31198 -31206). Independent confirmation of these observations has not been forthcoming. This study has utilized a polyclonal antibody specific for the phosphorylated serine 38 epitope on the Ca 2؉ -ATPase to evaluate the phosphorylation of SERCA2a in isolated sarcoplasmic reticulum vesicles and isolated rat ventricular myocytes. A quantitative Western blot approach failed to detect serine 38-phosphorylated Ca 2؉ -ATPase in either kinase-treated sarcoplasmic reticulum vesicles or suitably stimulated cardiac myocytes. Calibration standards confirmed that the detection sensitivity of assays was adequate to detect Ser-38 phosphorylation if it occurred on at least 1% of Ca 2؉ -ATPase molecules in SR vesicle experiments or on at least 0.1% of Ca 2؉ -ATPase molecules in cardiac myocytes. The failure to detect a phosphorylated form of the Ca 2؉ -ATPase in either preparation (isolated myocyte, purified sarcoplasmic reticulum vesicles) suggests that Ser-38 phosphorylation of the Ca 2؉ -ATPase is not a significant regulatory feature of cardiac Ca 2؉ homeostasis.Regulation of Ca 2ϩ sequestration by cardiac sarcoplasmic reticulum has been identified as a key control point in cardiac muscle contraction (1, 2). Stimulation of the rate of Ca 2ϩ uptake occurs during exercise, or following ␤-adrenergic stimulation (3) and is associated with an enhanced force of contraction and an increased rate of relaxation. This accounts for much of the positive inotropic and positive lusitropic effects of these interventions. In contrast, Ca 2ϩ sequestration by cardiac SR 1 is abnormally slow in the muscle of individuals with heart failure (4 -6). In animal studies, normalization of the rate of Ca 2ϩ sequestration has been shown to prevent progression of heart failure (7) and thus the molecular mechanism of control of Ca 2ϩ transport into the sarcoplasmic reticulum has become a focus for research into purposeful therapies to combat human heart failure (7,8).Ca 2ϩ transport into cardiac SR is an enzymatic process performed by the (Ca 2ϩ -Mg 2ϩ )-ATPase (9) (or SERCA2a, Ref. 10), a member of the P-type ATPase family (11). The transport process involves the movement of two Ca 2ϩ from the cytoplasm into the lumen of the SR following the hydrolysis of a single molecule of ATP (12), although significantly lower coupling efficiencies have been measured empirically (13). The reaction cycle is relatively slow and thus a large number of SERCA2 molecules are expressed in cardiac muscle (up to 45% of total SR protein content, 14) to achieve the rates of Ca 2ϩ sequestration required by the kinetics of contraction and relaxation.Regulation of Ca 2ϩ transport into the SR occurs on an acute time scale (seconds to minutes) through the transient modification of the proteins inv...

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Translation of Ser16 and Thr17 phosphorylation of phospholamban into Ca2+-pump stimulation

Cited by 47 publications

References 39 publications

A Single Site (Ser16) Phosphorylation in Phospholamban Is Sufficient in Mediating Its Maximal Cardiac Responses to β-Agonists

A Single Site (Ser16) Phosphorylation in Phospholamban Is Sufficient in Mediating Its Maximal Cardiac Responses to β-Agonists

β ₂ -Adrenergic cAMP Signaling Is Uncoupled From Phosphorylation of Cytoplasmic Proteins in Canine Heart

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

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Translation of Ser16 and Thr17 phosphorylation of phospholamban into Ca2+-pump stimulation

Cited by 47 publications

References 39 publications

A Single Site (Ser16) Phosphorylation in Phospholamban Is Sufficient in Mediating Its Maximal Cardiac Responses to β-Agonists

A Single Site (Ser16) Phosphorylation in Phospholamban Is Sufficient in Mediating Its Maximal Cardiac Responses to β-Agonists

β 2 -Adrenergic cAMP Signaling Is Uncoupled From Phosphorylation of Cytoplasmic Proteins in Canine Heart

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

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β ₂ -Adrenergic cAMP Signaling Is Uncoupled From Phosphorylation of Cytoplasmic Proteins in Canine Heart