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Phosphorylation of troponin and the effects of interactions between the components of the complex.

ABSTRACT: 1. The troponin complex from skeletal muscle contains approximately 1 mol of phosphate/80000g of complex, covalently bound to the troponin T component. 2. On prolonged incubation of the troponin complex or troponin T with phosphorylase kinase the phosphate content of troponin T was increased to approx. 3mol/mol. 3. On prolonged incubation of troponin I with phosphorylase kinase up to 1.6mol of phosphate/mol were incorporated. 4. Phosphorylation of troponin I was greatly inhibited by troponin C owing to the strong interaction between these proteins. Thus in the troponin complex troponin T was the main substrate for phosphorylase kinase. The phosphorylation of isolated troponin T was also inhibited by troponin C. 5. Troponin I was phosphorylated when the troponin complex was incubated with a bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Troponin T either in its isolated form or in the troponin complex was not phosphorylated by bovine protein kinase to any significant extent under the conditions used. 6. If the troponin complex was dephosphorylated to 0.2mol/mol, or phosphorylated up to 2.5mol/mol there was no significant effect on the ability of normal concentrations to confer Ca(2+) sensitivity on the adenosine triphosphatase of densensitized actomyosin.

SUBMITTER: Perry SV

PROVIDER: S-EPMC1168180 | biostudies-other | 1974 Sep

REPOSITORIES: biostudies-other

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Project description:AimsProtein kinase C? (PKC?) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKC? is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKC? phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.Methods and resultsEndogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (?69%) with PKC?-treated recombinant human cTn (cTn (DD+PKC?)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKC?. Isometric force was measured at various [Ca(2+)] after exchange. The maximal force (Fmax) in the cTn (DD+PKC?) group (17.1±1.9 kN/m(2)) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m(2)). Exchange of endogenous cTn with cTn (DD+PKC?) increased Ca(2+)-sensitivity of force (pCa50?=?5.59±0.02) compared to cTn (DD) (pCa50?=?5.51±0.02). In contrast, subsequent PKC? treatment of the cells exchanged with cTn (DD+PKC?) reduced pCa50 to 5.45±0.02. Two PKC?-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKC? and showed the highest phosphorylation increase on Thr143.ConclusionPKC?-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca(2+)-sensitivity, while subsequent treatment with PKC? in situ decreased myofilament Ca(2+)-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKC? are phosphorylated in human cTn complex treated with PKC? with a high degree of specificity for Thr143.

Project description:BACKGROUND: The deleterious effect of a mutation can be reverted by a second-site interacting residue. This is an epistatic compensatory process explaining why mutations that are deleterious in some species are tolerated in phylogenetically related lineages, rendering evident that those mutations are, by all means, only deleterious in the species-specific context. Although an extensive and refined theoretical framework on compensatory evolution does exist, the supporting evidence remains limited, especially for protein models. In this current study, we focused on the molecular mechanism underlying the epistatic compensatory process in mammalian mitochondrial OXPHOS proteins using a combination of in-depth structural and sequence analyses. RESULTS: Modeled human structures were used in this study to predict the structural impairment and recovery of deleterious mutations alone and combined with an interacting compensatory partner, respectively. In two cases, COI and COIII, intramolecular interactions between spatially linked residues restore the folding pattern impaired by the deleterious mutation. In a third case, intermolecular contact between mitochondrial CYB and nuclear CYT1 encoded components of the cytochrome bc1 complex are likely to restore protein binding. Moreover, we observed different modes of compensatory evolution that have resulted in either a quasi-simultaneous occurrence of a mutation and corresponding compensatory partner, or in independent occurrences of mutations in distinct lineages that were always preceded by the compensatory site. CONCLUSION: Epistatic interactions between individual replacements involving deleterious mutations seems to follow a parsimonious model of evolution in which genomes hold pre-compensating states that subsequently tolerate deleterious mutations. This phenomenon is likely to have been constraining the variability at coevolving sites and shaping the interaction between the mitochondrial and the nuclear genome.

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Phosphorylation of troponin and the effects of interactions between the components of the complex.

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