Phosphoregulation of the Titin-cap Protein Telethonin in Cardiac Myocytes

Alexandra J. Candasamy; Robert S. Haworth; Friederike Cuello; Michael Ibrahim; Sriram Aravamudhan; Marcus Krueger; Mark R. Holt; Cesare M. N. Terracciano; Manuel Mayr; Mathias Gautel; Metin Avkiran

doi:10.1074/jbc.M113.479030

Phosphoregulation of the Titin-cap Protein Telethonin in Cardiac Myocytes

Alexandra J. Candasamy, Robert S. Haworth, Friederike Cuello, Michael Ibrahim, Sriram Aravamudhan, Marcus Krueger, Mark R. Holt, Cesare M. N. Terracciano, Manuel Mayr, Mathias Gautel, Metin Avkiran^*

^*Corresponding author for this work

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

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Abstract

Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca2+/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca2+ transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca2+ transients.

Original language	English
Pages (from-to)	1282-1293
Number of pages	12
Journal	Journal of Biological Chemistry
Volume	289
Issue number	3
Early online date	1 Jan 2014
DOIs	https://doi.org/10.1074/jbc.M113.479030
Publication status	Published - 17 Jan 2014

Keywords

CaMKII
Cardiac Muscle
Cardiomyopathy
Excitation-Contraction Coupling
Protein Kinase D (PKD)
Protein Phosphorylation
SARCOMERIC PROTEIN
KINASE-D
Z-DISC
DILATED CARDIOMYOPATHY
MUSCULAR-DYSTROPHY
TUBULE STRUCTURE
MAMMALIAN HEART
TROPONIN-I
T-CAP
PHOSPHORYLATION

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@article{0c98f70be1ed406f81dd231fcab7e483,

title = "Phosphoregulation of the Titin-cap Protein Telethonin in Cardiac Myocytes",

abstract = "Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca2+/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca2+ transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca2+ transients.",

keywords = "CaMKII, Cardiac Muscle, Cardiomyopathy, Excitation-Contraction Coupling, Protein Kinase D (PKD), Protein Phosphorylation, SARCOMERIC PROTEIN, KINASE-D, Z-DISC, DILATED CARDIOMYOPATHY, MUSCULAR-DYSTROPHY, TUBULE STRUCTURE, MAMMALIAN HEART, TROPONIN-I, T-CAP, PHOSPHORYLATION",

author = "Candasamy, {Alexandra J.} and Haworth, {Robert S.} and Friederike Cuello and Michael Ibrahim and Sriram Aravamudhan and Marcus Krueger and Holt, {Mark R.} and Terracciano, {Cesare M. N.} and Manuel Mayr and Mathias Gautel and Metin Avkiran",

year = "2014",

month = jan,

day = "17",

doi = "10.1074/jbc.M113.479030",

language = "English",

volume = "289",

pages = "1282--1293",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology Inc.",

number = "3",

}

TY - JOUR

T1 - Phosphoregulation of the Titin-cap Protein Telethonin in Cardiac Myocytes

AU - Candasamy, Alexandra J.

AU - Haworth, Robert S.

AU - Cuello, Friederike

AU - Ibrahim, Michael

AU - Aravamudhan, Sriram

AU - Krueger, Marcus

AU - Holt, Mark R.

AU - Terracciano, Cesare M. N.

AU - Mayr, Manuel

AU - Gautel, Mathias

AU - Avkiran, Metin

PY - 2014/1/17

Y1 - 2014/1/17

N2 - Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca2+/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca2+ transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca2+ transients.

AB - Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca2+/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca2+ transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca2+ transients.

KW - CaMKII

KW - Cardiac Muscle

KW - Cardiomyopathy

KW - Excitation-Contraction Coupling

KW - Protein Kinase D (PKD)

KW - Protein Phosphorylation

KW - SARCOMERIC PROTEIN

KW - KINASE-D

KW - Z-DISC

KW - DILATED CARDIOMYOPATHY

KW - MUSCULAR-DYSTROPHY

KW - TUBULE STRUCTURE

KW - MAMMALIAN HEART

KW - TROPONIN-I

KW - T-CAP

KW - PHOSPHORYLATION

U2 - 10.1074/jbc.M113.479030

DO - 10.1074/jbc.M113.479030

M3 - Article

SN - 0021-9258

VL - 289

SP - 1282

EP - 1293

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 3

ER -

Phosphoregulation of the Titin-cap Protein Telethonin in Cardiac Myocytes

Abstract

Keywords

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