Effects of phosphorylation and mutation R145G on human cardiac troponin I function.
We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin was reconstituted from recombinant human subunits including either wild-type or mutant cTnI and was used for reconstitution of thin filaments with skeletal muscle actin and tropomyosin. The Ca(2+)-dependent thin filament-activated myosin subfragment 1 ATPase (actoS1-ATPase) activity and the in vitro motility of these filaments driven by myosin were measured as a function of the cTnI phosphorylation state. Bisphosphorylation of wild-type cTnI decreases the Ca(2+) sensitivity of the actoS1-ATPase activity and the in vitro thin filament motility by about 0.15-0.21 pCa unit. The nonconservative replacement R145G in cTnI enhances the Ca(2+) sensitivity of the actoS1-ATPase activity by about 0.6 pCa unit independent of the phosphorylation state of cTnI. Furthermore, it mimics a strong suppressing effect on both the maximum actoS1-ATPase activity and the maximum in vitro filament sliding velocity which has been observed upon bisphosphorylation of wild-type cTnI. Bisphosphorylation of the mutant cTnI-R145G itself had no such suppressing effects anymore. Differential analysis of the effect of phosphorylation of each of the two serines, Ser23 in cTnI-S22A and Ser22 in cTnI-S23A, indicates that phosphorylation of Ser23 may already be sufficient for causing the reduction of maximum actoS1-ATPase activity and thin filament sliding velocity seen upon phosphorylation of both of these serines.
Deng
Y
Y
Institut für Physiologische Chemie, Abteilung für Biochemie Supramolekularer Systeme, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Schmidtmann
A
A
Redlich
A
A
Westerdorf
B
B
Jaquet
K
K
Thieleczek
R
R
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011817:Rabbits; D011994:Recombinant Proteins; D012694:Serine / Q000378:metabolism*; D019210:Troponin I / Q000502:physiology*
D000199:Actins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019210:Troponin I; C033025:actin subfragments; D012694:Serine; D001120:Arginine; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium