Identification of a functionally critical protein kinase C phosphorylation residue of cardiac troponin T.
Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-alpha or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-alpha or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.
Sumandea
Marius P
MP
Department of Physiology and Biophysics, Program in Cardiovascular Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Pyle
W Glen
WG
Kobayashi
Tomoyoshi
T
de Tombe
Pieter P
PP
Solaro
R John
RJ
D000205:Actomyosin / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D018698:Glutamic Acid; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020107:Troponin T / Q000737:chemistry / Q000378:metabolism*; D016277:Ventricular Function, Left / Q000502:physiology*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D020107:Troponin T; D010769:Phosphothreonine; D010768:Phosphoserine; D018698:Glutamic Acid; D000205:Actomyosin; D011493:Protein Kinase C; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium