Phosphomimetic Substitution of Cytochrome c Tyrosine 48 Decreases Respiration and Binding to Cardiolipin and Abolishes Ability to Trigger Downstream Caspase Activation

Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Biochemistry (Impact Factor: 3.02). 08/2010; 49(31):6705-14. DOI: 10.1021/bi100486s
Source: PubMed


Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.

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    • "). A recent discovery of tyrosine phosphorylation sites in cyt c implies that its peroxidase function may be also regulated through this type of signaling (Pecina et al., 2010). Indeed, it has been established that Tyr 48 phosphorylation might serve as an anti-apoptotic switch, possibly via its effects on CL peroxidation during apoptosis. "
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    ABSTRACT: Cardiolipins (CLs) are ancient and unusual dimeric phospholipids localized in the plasma membrane of bacteria and in the inner mitochondrial membrane of eukaryotes. In mitochondria, two types of asymmetries-trans-membrane and molecular - are essential determinants of CL functions. In this review, we describe CL-based signaling mitochondrial pathways realized via modulation of trans-membrane asymmetry and leading to externalization and peroxidation of CLs in mitophagy and apoptosis, respectively. We discuss possible mechanisms of CL translocations from the inner leaflet of the inner to the outer leaflet of the outer mitochondrial membranes. We present redox reaction mechanisms of cytochrome c-catalyzed CL peroxidation as a required stage in the execution of apoptosis as well as a possible source of lipid mediators. We also emphasize the significance of CL-related metabolic pathways as new targets for drug discovery. Finally, a remarkable diversity of polyunsaturated CL species and their oxidation products have evolved in eukaryotes vs. prokaryotes. This diversity - associated with CL molecular asymmetry - is presented as the basis for mitochondrial communications language.
    Chemistry and Physics of Lipids 11/2013; 179. DOI:10.1016/j.chemphyslip.2013.11.010 · 2.42 Impact Factor
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    • "Traditional studies of isolated Cytc did not preserve the in vivo phosphorylation state during protein purification. Recent studies by our group have demonstrated that mammalian Cytc can be post-translationally modified by tyrosine phosphorylation at two distinct residues in a tissue-specific manner [13]–[15]. These results introduced a novel concept, that Cytc is targeted by cell signaling by yet unidentified mitochondrial tyrosine kinases. "
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    ABSTRACT: Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.
    PLoS ONE 11/2013; 8(11):e78627. DOI:10.1371/journal.pone.0078627 · 3.23 Impact Factor
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    • "Speci fi cally, Cytc might be targeted for phosphorylation for two reasons: since the functionally studied Cytc phosphorylations on tyrosines 48 and 97 both lead to an inhibition of respiration as discussed above, increased phosphorylation would contribute to the Warburg effect. In addition, Cytc phosphorylation may interfere with apoptosis as suggested by studies with phosphomimetic Cytc, which was not able to trigger any measureable caspase activation (Pecina et al. 2010 ) . Since cancers manage to evade apoptosis, increased phosphorylation of Cytc via cancer signaling might provide a mechanism for the suppression of apoptosis. "
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    ABSTRACT: The mitochondrial oxidative phosphorylation (OxPhos) system not only generates the vast majority of cellular energy, but is also involved in the generation of reactive oxygen species (ROS), and apoptosis. Cytochrome c (Cytc) and cytochrome c oxidase (COX) represent the terminal step of the electron transport chain (ETC), the proposed rate-limiting reaction in mammals. Cytc and COX show unique regulatory features including allosteric regulation, isoform expression, and regulation through cell signaling pathways. This chapter focuses on the latter and discusses all mapped phosphorylation sites based on the crystal structures of COX and Cytc. Several signaling pathways have been identified that target COX including protein kinase A and C, receptor tyrosine kinase, and inflammatory signaling. In addition, four phosphorylation sites have been mapped on Cytc with potentially large implications due to its multiple functions including apoptosis, a pathway that is overactive in stressed cells but inactive in cancer. The role of COX and Cytc phosphorylation is reviewed in a human disease context, including cancer, inflammation, sepsis, asthma, and ischemia/reperfusion injury as seen in myocardial infarction and ischemic stroke.
    Advances in Experimental Medicine and Biology 06/2012; 748:237-64. DOI:10.1007/978-1-4614-3573-0_10 · 1.96 Impact Factor
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