title: Protein kinase D is a novel mediator of cardiac troponin I phosphorylation and regulates myofilament function.
abstract: Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown. Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins. In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin. Peptide mass fingerprint analysis of cTnI by liquid chromatography-coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets. Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T. PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD. Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (approximately 2 mol phosphate/mol cTnI) by both PKD and PKA. To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation. PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension-pCa relationship, indicating reduced myofilament Ca2+ sensitivity. At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics. Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.
authors.affiliation: Cardiovascular Division, King's College London, London, UK.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Haworth
authors.forename: Friederike
authors.initials: F
authors.lastname: Cuello
authors.forename: Todd J
authors.initials: TJ
authors.lastname: Herron
authors.forename: Gereon
authors.initials: G
authors.lastname: Franzen
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Kentish
authors.forename: Mathias
authors.initials: M
authors.lastname: Gautel
authors.forename: Metin
authors.initials: M
authors.lastname: Avkiran
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000328:Adult; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020013:Calcium Signaling; D002352:Carrier Proteins / Q000378:metabolism; D064211:Connectin; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007537:Isometric Contraction; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D000099163:Myosin Binding Protein C
chemical_list: D002352:Carrier Proteins; D064211:Connectin; D017868:Cyclic AMP-Dependent Protein Kinases; D018076:DNA, Complementary; D009124:Muscle Proteins; D010768:Phosphoserine; D011493:Protein Kinase C; D011993:Recombinant Fusion Proteins; D019210:Troponin I; D000099163:Myosin Binding Protein C; C577292:TCAP protein, human; C089212:protein kinase D