Differential contribution of troponin I phosphorylation sites to the endothelin-modulated contractile response.
Cardiac troponin I is a phosphorylation target for endothelin-activated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca2+ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca2+ transient could not explain the divergent relaxation response to endothelin in myocytes expressing cardiac versus slow skeletal troponin I nor could activation of Na+/H+ exchange. Three separate clusters within cardiac troponin I are phosphorylated by protein kinase C, and we set out to determine the contribution of the Thr144 and Ser23/Ser24 clusters to the endothelin-mediated contractile response. Myocyte replacement with a cardiac troponin I containing a Thr144 substituted with the Pro residue found in slow skeletal troponin I resulted in prolonged relaxation in response to acute endothelin compared with control myocytes. Ser23/Ser24 also is a target for protein kinase C phosphorylation of purified cardiac troponin I, and although this cluster was not acutely phosphorylated in intact myocytes, significant phosphorylation developed within 1 h after adding endothelin. Replacement of Ser23/Ser24 with Ala indicated that this cluster contributes significantly to relaxation during more prolonged endothelin stimulation. Overall, results with these mutants provide evidence that Thr144 plays an important role in the acute acceleration of relaxation, whereas Ser23/Ser24 contributes to relaxation during more prolonged activation of protein kinase C by endothelin.
Westfall
Margaret V
MV
Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109, USA. wfall@umich.edu
Lee
Adonia M
AM
Robinson
Dustin A
DA
D008841:Actin Cytoskeleton / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000409:Alanine / Q000737:chemistry; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D016000:Cluster Analysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D016232:Endothelins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015233:Models, Statistical; D032342:Muscle Cells / Q000166:cytology; D016296:Mutagenesis; D009200:Myocardial Contraction; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D012518:Sarcomeres / Q000187:drug effects / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D019210:Troponin I / Q000737:chemistry* / Q000378:metabolism
D018076:DNA, Complementary; D016232:Endothelins; D019210:Troponin I; D013912:Threonine; D012694:Serine; D000584:Amiloride; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000409:Alanine; D002118:Calcium; C039614:ethylisopropylamiloride