Evidence for protein phosphatase inhibitor‐1 playing an amplifier role in β‐adrenergic signaling in cardiac myocytes

El‐Armouche, Ali; Rau, Thomas; Zolk, Oliver; Ditz, Diana; Pamminger, Torsten; Zimmermann, WH; Jäckel, Elmar; Harding, Siân E.; Boknı́k, Peter; Neumann, Joachim; Eschenhagen, Thomas

doi:10.1096/fj.02-0057fje

Cited by 105 publications

(118 citation statements)

References 36 publications

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“…7 The recombinant I-1 protein was in vitro phosphorylated at Thr35 using the purified catalytic subunit of PKA (PKAc, specific activity ~10 U/µg protein; Sigma-Aldrich) to become activated. Phosphorylation/thiophosphorylation of I-1 protein was performed using a protocol described by Endo et al 17 The phosphorylation reaction was carried out in phosphatebuffered saline (PBS) at pH 7.4, containing 6 mM MgCl The reactions were terminated by incubating at 95 °C for 5 to 10 min and desalted in the enzyme dilution buffer using Amicon, 10K devices (Millipore, Billerica, MA) according to the manufacturer's guideline.…”

Section: Pka Phosphorylation/thiophosphorylation Of Recombinant I-1 Pmentioning

confidence: 99%

Development of a Colorimetric and a Fluorescence Phosphatase-Inhibitor Assay Suitable for Drug Discovery Approaches

Sotoud

Gribbon²,

Ellinger³

et al. 2013

SLAS Discovery

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Protein phosphatases (PP) are interesting drug targets. However, their ubiquitous presence and involvement in different, partially opposing signal pathways suggest that specificity may be achieved rather by targeting their interaction with subunits determining substrate specificity than the enzyme itself. An interesting subunit is phosphatase inhibitor-1 (I-1), which, in its protein kinase A–phosphorylated form (I-1P), inhibits the catalytic subunit of type 1 phosphatase (PP1c). In the current study, we established a colorimetric and a fluorescence-based assay system for the identification of compounds interfering with the inhibitory effect of I-1P on PP1c. The fluorescence assay exhibited 500-fold higher sensitivity toward PP1c. A nine-residue peptide containing the PP1c-binding motif (RVxF) of I-1 stimulated PP1c activity in the presence of I-1P (EC50 27 µM and 2.3 µM in the colorimetric and fluorescence assay, respectively). This suggests that the peptide interfered with the inhibitory effect of I-1P on PP1c and represents a proof-of-principle. The calculated Z′ factor for PP1c (0.84) and the PP1c–I-1P complex (0.73) confirmed the suitability of the fluorescence assay for high-throughput screenings (HTS). By testing several thousand small molecules, we suggest the advantages of kinetic measurements over single-point measurements using the fluorescence-based assay in an HTS format.

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Section: Pka Phosphorylation/thiophosphorylation Of Recombinant I-1 Pmentioning

confidence: 99%

Development of a Colorimetric and a Fluorescence Phosphatase-Inhibitor Assay Suitable for Drug Discovery Approaches

Sotoud

Gribbon²,

Ellinger³

et al. 2013

SLAS Discovery

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“…(761). In addition, this approach has been instrumental in identifying the relative contribution of individual targets to the inotropic and lusitropic effects of ␤-adrenergic signaling in cardiac myocytes (178,207,967). Most importantly, studies on the ␤-AR signaling pathway have provided insight into the paradoxical observation that ␤-AR agonists increase mortality by demonstrating that ␤-agonists activate a cascade of pathophysiological events in addition to the downstream increase in contractile function.…”

Section: ␤-Adrenergic Receptors and G Proteinsmentioning

confidence: 99%

Designing Heart Performance by Gene Transfer

Davis¹,

Westfall²,

Townsend³

et al. 2008

Physiological Reviews

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The birth of molecular cardiology can be traced to the development and implementation of high-fidelity genetic approaches for manipulating the heart. Recombinant viral vector-based technology offers a highly effective approach to genetically engineer cardiac muscle in vitro and in vivo. This review highlights discoveries made in cardiac muscle physiology through the use of targeted viral-mediated genetic modification. Here the history of cardiac gene transfer technology and the strengths and limitations of viral and nonviral vectors for gene delivery are reviewed. A comprehensive account is given of the application of gene transfer technology for studying key cardiac muscle targets including Ca2+handling, the sarcomere, the cytoskeleton, and signaling molecules and their posttranslational modifications. The primary objective of this review is to provide a thorough analysis of gene transfer studies for understanding cardiac physiology in health and disease. By comparing results obtained from gene transfer with those obtained from transgenesis and biophysical and biochemical methodologies, this review provides a global view of cardiac structure-function with an eye towards future areas of research. The data presented here serve as a basis for discovery of new therapeutic targets for remediation of acquired and inherited cardiac diseases.

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“…6 When I-1's Thr35 residue is phosphorylated by protein kinase A (PKA), I-1 actively inhibits PP1 activity, potentiates PKA phosphorylation, and enhances cardiac contractility. On the other hand, when the Ser67 or Thr75 residues are phosphorylated by protein kinase C α (PKAα) 7,8 or the Thr35 residue is dephosphorylated by PP2B, 9 I-1 becomes inactive, resulting in an increase in PP1 activity and suppression of cardiac contractility.…”

Section: Article P 1133mentioning

confidence: 99%

Inhibitor-1 is Potential Target for Enhancing Sarcoplasmic Reticulum Ca2+ Loading in Failing Hearts

Ikeda

Yano

2009

Circ J

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ysregulated Ca 2+ homeostasis is a hallmark of heart failure that is directly linked to cardiac dysfunction and lethal arrhythmia. 1 It is associated with imbalance of kinases/phosphatase activities in the corresponding intracellular microdomains, leading to either hypophosphorylation of phospholamban (PLN), 2 hyperphosphorylation of ryanodine receptor (RyR) 3 or a combination of both in failing cardiomyocytes. It has been increasingly recognized that dysregulation of protein phosphatase 1 (PP1) signaling is associated with the imbalance of kinase/phosphatase in the failing heart. 3,4 Therefore, PP1 inhibition has been thought of as a potential molecular target for restoring impaired Ca 2+ cycling mediated by the sarcoplasmic reticulum (SR). 4 In this regard, the role of inhibitor-1 (I-1), an endogenous PP1 inhibitor, has been drawing attention, not only for understanding the mechanism of increased PP1 activity in failing hearts, but also for its application as a potential therapeutic target. 5 Article p 1133I-1 is a ubiquitously expressed acid-and heat-stable cytosolic protein that is primarily rich in skeletal and cardiac muscle. 6 When I-1's Thr35 residue is phosphorylated by protein kinase A (PKA), I-1 actively inhibits PP1 activity, potentiates PKA phosphorylation, and enhances cardiac contractility. On the other hand, when the Ser67 or Thr75 residues are phosphorylated by protein kinase C α (PKAα) 7,8 or the Thr35 residue is dephosphorylated by PP2B, 9 I-1 becomes inactive, resulting in an increase in PP1 activity and suppression of cardiac contractility. Therefore, I-1 is also recognized as an important element of β-adrenergic signaling, as well as Gq-receptor coupled signaling (angiotensin II, endothelin, and α-adrenergic signaling). 10 It has been reported that in heart failure, phosphorylation of I-1 at Thr35 is reduced compared with normal hearts, 9 and its protein expression level is significantly decreased, 11 thereby contributing to impaired β-adrenergic signaling and hence cardiac dysfunction in heart failure.In this issue, Kawashima et al 12 report a specific effect of I-1 on the SR Ca 2+ cycling in normal saponin-permeabilized rat cardiomyocytes. They found that administration of I-1 significantly potentiated PKA-mediated SR Ca 2+ load without affecting Ca 2+ release function, as assessed by caffeine-induced Ca 2+ transient and Ca 2+ spark frequency. The effects of I-1 on SR Ca 2+ load and Ca 2+ release function were also characterized as similar to nonspecific chemical inhibition of protein phosphatase by calyculin A. Indeed, there are at least 2 possible mechanisms by which Ca 2+ spark frequency increased in the permeabilized cardiomyocytes; namely, an increased gating property of the RyR, increased SR Ca 2+ loading (SR Ca 2+ content) or a combination of both. SR Ca 2+ loading seems to be a more important target for I-1 than Ca 2+ release, because I-1 had no appreciable effect on Ca 2+ spark frequency and caffeine-induced Ca 2+ transient after an abrupt inhibition of the SR Ca 2+ uptake by cyc...

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Evidence for protein phosphatase inhibitor‐1 playing an amplifier role in β‐adrenergic signaling in cardiac myocytes

Cited by 105 publications

References 36 publications

Development of a Colorimetric and a Fluorescence Phosphatase-Inhibitor Assay Suitable for Drug Discovery Approaches

Development of a Colorimetric and a Fluorescence Phosphatase-Inhibitor Assay Suitable for Drug Discovery Approaches

Designing Heart Performance by Gene Transfer

Inhibitor-1 is Potential Target for Enhancing Sarcoplasmic Reticulum Ca2+ Loading in Failing Hearts

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