title: Phosphorylation or glutamic acid substitution at protein kinase C sites on cardiac troponin I differentially depress myofilament tension and shortening velocity.
abstract: There is evidence that multi-site phosphorylation of cardiac troponin I (cTnI) by protein kinase C is important in both long- and short-term regulation of cardiac function. To determine the specific functional effects of these phosphorylation sites (Ser-43, Ser-45, and Thr-144), we measured tension and sliding speed of thin filaments in reconstituted preparations in which endogenous cTnI was replaced with cTnI phosphorylated by protein kinase C-epsilon or mutated to cTnI-S43E/S45E/T144E, cTnI-S43E/S45E, or cTnI-T144E. We used detergent-skinned mouse cardiac fiber bundles to measure changes in Ca(2+)-dependence of force. Compared with controls, fibers reconstituted with phosphorylated cTnI, cTnI-S43E/S45E/T144E, or cTnI-S43E/S45E were desensitized to Ca(2+), and maximum tension was as much as 27% lower, whereas fibers reconstituted with cTnI-T144E showed no change. In the in vitro motility assay actin filaments regulated by troponin complexes containing phosphorylated cTnI or cTnI-S43E/S45E/T144E showed both a decrease in Ca(2+) sensitivity and maximum sliding speed compared with controls, whereas filaments regulated by cTnI-S43E/S45E showed only decreased maximum sliding speed and filaments regulated by cTnI-T144E demonstrated only desensitization to Ca(2+). Our results demonstrate novel site specificity of effects of PKC phosphorylation on cTnI function and emphasize the complexity of modulation of the actin-myosin interaction by specific changes in the thin filament.
authors.affiliation: University of Illinois at Chicago, Department of Physiology and Biophysics, Program in Cardiovascular Sciences, College of Medicine, Chicago, Illinois 60612, USA.
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Burkart
authors.forename: Marius P
authors.initials: MP
authors.lastname: Sumandea
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Mahta
authors.initials: M
authors.lastname: Nili
authors.forename: Anne F
authors.initials: AF
authors.lastname: Martin
authors.forename: Earl
authors.initials: E
authors.lastname: Homsher
authors.forename: R John
authors.initials: RJ
authors.lastname: Solaro
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D019210:Troponin I; D018698:Glutamic Acid; D011493:Protein Kinase C