Phosphorylation of cardiac troponin I at protein kinase C site threonine 144 depresses cooperative activation of thin filaments.
There is evidence for PKC-dependent multisite phosphorylation of cardiac troponin I (cTnI) at Ser-23 and Ser-24 (also PKA sites) in the cardiac-specific N-terminal extension and at Thr-144, a unique residue in the inhibitory region. The functional effect of these phosphorylations in combination is of interest in view of data indicating intramolecular interaction between the N-terminal extension and the inhibitory region of cTnI. To determine the role of PKC-dependent phosphorylation of cTnI on sarcomeric function, we measured contractile regulation at multiple levels of complexity. Ca(2+) binding to thin filaments reconstituted with either cTnI(wild-type) or pseudo-phosphorylated cTnI(S23D/S24D), cTnI(T144E), and cTnI(S23D/S24D/T144E) was determined. Compared with controls regulated by cTnI(wild-type), thin filaments with cTnI(S23D/S24D) and cTnI(S23D/S24D/T144E) exhibited decreased Ca(2+) sensitivity. In contrast, there was no significant difference between Ca(2+) binding to thin filaments with cTnI(wild-type) and with cTnI(T144E). Studies of the pCa-force relations in skinned papillary fibers regulated by these forms of cTnI yielded similar results. However, in both the Ca(2+) binding measurements and the skinned fiber tension measurements, the presence of cTnI(S23D/S24D/T144E) induced a much lower Hill coefficient than either wild type, S23D/S24D, or T144E. These data highlight the importance of thin filament-based cooperative mechanisms in cardiac regulation, with implications for mechanisms of control of function in normal and pathological hearts.
Lu
Qun-Wei
QW
Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Hinken
Aaron C
AC
Patrick
Stacey E
SE
Solaro
R John
RJ
Kobayashi
Tomoyoshi
T
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009200:Myocardial Contraction; D009206:Myocardium / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000737:chemistry; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D019210:Troponin I; D013912:Threonine; D011493:Protein Kinase C; D002118:Calcium