PKC-betaII sensitizes cardiac myofilaments to Ca2+ by phosphorylating troponin I on threonine-144.
Ventricular myocytes express Galphaq-coupled receptors that can mediate enhanced contractility by increasing the sensitivity of the contractile apparatus to Ca(2+). The precise mechanisms underlying this change have been difficult to define, in part because myofilament regulatory proteins contain multiple phosphorylation sites for protein kinase C (PKC), protein kinase A (PKA) and myosin light chain kinase (MLCK), with potentially opposing effects. MLCK increases whereas PKC and PKA have a strong tendency to decrease myofilament Ca(2+) sensitivity in myocardium. Here we show in mouse cardiac myocytes that PKC-betaII can increase Ca(2+) sensitivity of tension by a similar magnitude to MLCK but via a distinct mechanism. For PKC-betaII (32)P-incorporation occurred primarily into cardiac troponin I (cTnI) and functional effects were highly dependent upon mutations in phosphorylation sites of cTnI. Replacement of serines-23/24 (PKA sites) with alanine prevented cross-phosphorylation of these sites, reduced (32)P-incorporation into cTnI by half and resulted in myofilament Ca(2+) sensitization rather than desensitization in response to PKC-betaII. Replacement of three additional sites on cTnI, serines-43/45 and threonine-144, eliminated PKC-betaII-mediated Ca(2+) sensitization and the remaining (32)P-incorporation into cTnI. A preference for PKC-betaII phosphorylation of threonine-144 in the intact filament lattice was revealed by differential stable isotope labeling and supported by an analysis of peptide phosphorylation. The results suggest that threonine-144 within the critical inhibitory domain of cTnI represents a novel site of regulation of myofilament Ca(2+) sensitivity by PKC-betaII, with possible implications for chronically stressed or diseased hearts.
Wang
Hao
H
Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA.
Grant
Jennifer E
JE
Doede
Christopher M
CM
Sadayappan
Sakthivel
S
Robbins
Jeffrey
J
Walker
Jeffery W
JW
D008841:Actin Cytoskeleton / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003285:Contractile Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000187:drug effects*; D009210:Myofibrils / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism* / Q000502:physiology; D064546:Protein Kinase C beta; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry; D019210:Troponin I / Q000235:genetics / Q000378:metabolism*
D003285:Contractile Proteins; D018994:Myosin Light Chains; D010455:Peptides; D019210:Troponin I; D013912:Threonine; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D002118:Calcium