FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation.
FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca(2+) and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca(2+) and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca(2+) sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca(2+)-sensitivity. The rate of Ca(2+)-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca(2+)-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca(2+)-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca(2+)-sensitivity and likely play an important role in cardiac regulation.
The Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA.
William
W
Schlecht
The Department of Integrated Neuroscience and Physiology, Washington State University, Pullman, WA 99164, USA.
Zhiqun
Z
Zhou
The Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA.
King-Lun
KL
Li
The Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA.
Daniel
D
Rieck
The Department of Integrated Neuroscience and Physiology, Washington State University, Pullman, WA 99164, USA.
Yexin
Y
Ouyang
The Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA; The Department of Integrated Neuroscience and Physiology, Washington State University, Pullman, WA 99164, USA. Electronic address: wdong@vetmed.wsu.edu.
Wen-Ji
WJ
Dong
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D009210:Myofibrils / Q000235:genetics / Q000378:metabolism*; D015879:Myosin Subfragments / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D020107:Troponin T / Q000235:genetics / Q000378:metabolism*
D015879:Myosin Subfragments; D011994:Recombinant Proteins; D020107:Troponin T; D018698:Glutamic Acid; D011493:Protein Kinase C; D002118:Calcium
Cardiac troponin T; FRET; PKC phosphorylation; Thin filament regulation