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 Ca(2+)/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 Ca(2+) 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 Ca(2+) transients.
authors.affiliation: From the Cardiovascular Division, King's College London British Heart Foundation Centre, London SE1 7EH, United Kingdom.
authors.forename: Alexandra J
authors.initials: AJ
authors.lastname: Candasamy
authors.forename: Robert S
authors.initials: RS
authors.lastname: Haworth
authors.forename: Friederike
authors.initials: F
authors.lastname: Cuello
authors.forename: Michael
authors.initials: M
authors.lastname: Ibrahim
authors.forename: Sriram
authors.initials: S
authors.lastname: Aravamudhan
authors.forename: Marcus
authors.initials: M
authors.lastname: Krüger
authors.forename: Mark R
authors.initials: MR
authors.lastname: Holt
authors.forename: Cesare M N
authors.initials: CM
authors.lastname: Terracciano
authors.forename: Manuel
authors.initials: M
authors.lastname: Mayr
authors.forename: Mathias
authors.initials: M
authors.lastname: Gautel
authors.forename: Metin
authors.initials: M
authors.lastname: Avkiran
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D064211:Connectin / Q000235:genetics / Q000378:metabolism*; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008870:Microtubules / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012518:Sarcomeres / Q000235:genetics / Q000378:metabolism
chemical_list: D064211:Connectin; D009124:Muscle Proteins; C577292:TCAP protein, human; C577294:Tcap protein, mouse; C507454:Tcap protein, rat; C089212:protein kinase D; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium
keywords: CaMKII; Cardiac Muscle; Cardiomyopathy; Excitation-Contraction Coupling; Protein Kinase D (PKD); Protein Phosphorylation