Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

Simon Diering; Konstantina Stathopoulou; Mara Goetz; Laura Rathjens; Sönke Harder; Angelika Piasecki; Janice Raabe; Steven Schulz; Mona Brandt; Julia Pflaumenbaum; Ulrike Fuchs; Sonia Donzelli; Sakthivel Sadayappan; Viacheslav O. Nikolaev; Frederik Flenner; Elisabeth Ehler; Friederike Cuello

doi:10.1074/jbc.RA120.014467

Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

Simon Diering, Konstantina Stathopoulou, Mara Goetz, Laura Rathjens, Sönke Harder, Angelika Piasecki, Janice Raabe, Steven Schulz, Mona Brandt, Julia Pflaumenbaum, Ulrike Fuchs, Sonia Donzelli, Sakthivel Sadayappan, Viacheslav O. Nikolaev, Frederik Flenner, Elisabeth Ehler, Friederike Cuello

Research output: Contribution to journal › Article › peer-review

4 Citations (Scopus)

Abstract

The contraction and relaxation of the heart is controlled by stimulation of the b1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of b ₁-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

Original language	English
Pages (from-to)	15342-15365
Number of pages	24
Journal	The Journal of biological chemistry
Volume	295
Issue number	45
DOIs	https://doi.org/10.1074/jbc.RA120.014467
Publication status	Published - 6 Nov 2020

Keywords

cardiac myocyte
cardiomyocyte
cardiovascular disease
kinase
nitroxyl
oxidation
phosphatase
phosphorylation
protein kinase A (PKA)
redox regulation

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10.1074/jbc.RA120.014467

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Diering, S., Stathopoulou, K., Goetz, M., Rathjens, L., Harder, S., Piasecki, A., Raabe, J., Schulz, S., Brandt, M., Pflaumenbaum, J., Fuchs, U., Donzelli, S., Sadayappan, S., Nikolaev, V. O., Flenner, F., Ehler, E., & Cuello, F. (2020). Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents. The Journal of biological chemistry, 295(45), 15342-15365. https://doi.org/10.1074/jbc.RA120.014467

@article{8963e9e4b3734df19fbe48cde01d2a4d,

title = "Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents",

abstract = "The contraction and relaxation of the heart is controlled by stimulation of the b1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of b 1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance. ",

keywords = "cardiac myocyte, cardiomyocyte, cardiovascular disease, kinase, nitroxyl, oxidation, phosphatase, phosphorylation, protein kinase A (PKA), redox regulation",

author = "Simon Diering and Konstantina Stathopoulou and Mara Goetz and Laura Rathjens and S{\"o}nke Harder and Angelika Piasecki and Janice Raabe and Steven Schulz and Mona Brandt and Julia Pflaumenbaum and Ulrike Fuchs and Sonia Donzelli and Sakthivel Sadayappan and Nikolaev, {Viacheslav O.} and Frederik Flenner and Elisabeth Ehler and Friederike Cuello",

year = "2020",

month = nov,

day = "6",

doi = "10.1074/jbc.RA120.014467",

language = "English",

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Diering, S, Stathopoulou, K, Goetz, M, Rathjens, L, Harder, S, Piasecki, A, Raabe, J, Schulz, S, Brandt, M, Pflaumenbaum, J, Fuchs, U, Donzelli, S, Sadayappan, S, Nikolaev, VO, Flenner, F, Ehler, E & Cuello, F 2020, 'Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents', The Journal of biological chemistry, vol. 295, no. 45, pp. 15342-15365. https://doi.org/10.1074/jbc.RA120.014467

TY - JOUR

T1 - Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

AU - Diering, Simon

AU - Stathopoulou, Konstantina

AU - Goetz, Mara

AU - Rathjens, Laura

AU - Harder, Sönke

AU - Piasecki, Angelika

AU - Raabe, Janice

AU - Schulz, Steven

AU - Brandt, Mona

AU - Pflaumenbaum, Julia

AU - Fuchs, Ulrike

AU - Donzelli, Sonia

AU - Sadayappan, Sakthivel

AU - Nikolaev, Viacheslav O.

AU - Flenner, Frederik

AU - Ehler, Elisabeth

AU - Cuello, Friederike

PY - 2020/11/6

Y1 - 2020/11/6

N2 - The contraction and relaxation of the heart is controlled by stimulation of the b1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of b 1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

AB - The contraction and relaxation of the heart is controlled by stimulation of the b1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of b 1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

KW - cardiac myocyte

KW - cardiomyocyte

KW - cardiovascular disease

KW - kinase

KW - nitroxyl

KW - oxidation

KW - phosphatase

KW - phosphorylation

KW - protein kinase A (PKA)

KW - redox regulation

UR - http://www.scopus.com/inward/record.url?scp=85095861971&partnerID=8YFLogxK

U2 - 10.1074/jbc.RA120.014467

DO - 10.1074/jbc.RA120.014467

M3 - Article

C2 - 32868295

AN - SCOPUS:85095861971

SN - 0021-9258

VL - 295

SP - 15342

EP - 15365

JO - The Journal of biological chemistry

JF - The Journal of biological chemistry

IS - 45

ER -

Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

Abstract

Keywords

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