Phosphoregulation of the Titin-cap Protein Telethonin in Cardiac MyocytesThis work was supported by British Heart Foundation (BHF) 4-year Ph.D. Program FS/06/079, Centre of Research Excellence Award RE/08/003, and Medical Research Council Grant G0800206.▪▪This article contains supplemental Figs. S1–S5, Experimental Procedures, and references

• Published in: The Journal of biological chemistry Vol. 289; no. 3; pp. 1282 - 1293
• Main Authors: Candasamy, Alexandra J., Haworth, Robert S., Cuello, Friederike, Ibrahim, Michael, Aravamudhan, Sriram, Krüger, Marcus, Holt, Mark R., Terracciano, Cesare M.N., Mayr, Manuel, Gautel, Mathias, Avkiran, Metin
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.01.2014
• Subjects: CaMKII; Cardiac Muscle; Cardiomyopathy; Excitation-Contraction Coupling; Protein Kinase D (PKD); Protein Phosphorylation; Cardiac Muscle; CaMKII; Cardiomyopathy; Excitation-Contraction Coupling; Protein Phosphorylation; Protein Kinase D (PKD)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.479030

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. Background: Telethonin mutations are associated with cardiomyopathy through unknown mechanisms. Results: Telethonin is a substrate for CaMK family kinases and exists in a bis-phosphorylated state in cardiomyocytes, in which non-phosphorylated telethonin disrupts transverse tubule organization and intracellular calcium transients. Conclusion: Telethonin phosphorylation is critical for the maintenance of normal cardiomyocyte morphology and calcium handling. Significance: Disruption of phospho-telethonin functions may contribute to pathogenesis in cardiomyopathy.