TY - JOUR
T1 - Protein kinase D is a novel mediator of cardiac troponin I phosphorylation and regulates myofilament function
AU - Haworth, R S
AU - Cuello, F
AU - Herron, T J
AU - Franzen, G
AU - Kentish, J C
AU - Gautel, M
AU - Avkiran, M
PY - 2004/11/26
Y1 - 2004/11/26
N2 - 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 (approximate to 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.
AB - 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 (approximate to 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.
UR - http://www.scopus.com/inward/record.url?scp=9344230851&partnerID=8YFLogxK
U2 - 10.1161/01.RES.0000149299.34793.3c
DO - 10.1161/01.RES.0000149299.34793.3c
M3 - Article
VL - 95
SP - 1091
EP - 1099
JO - Circulation Research
JF - Circulation Research
IS - 11
ER -