Phosphorylation of cytochrome c threonine 28 regulates electron transport chain activity in kidney: Implications for amp kinase

Gargi Mahapatra, Ashwathy Varughese, Qinqin Ji, Icksoo Lee, Jenney Liu, Asmita Vaishnav, Christopher Sinkler, Alexandr A. Kapralov, Carlos T Moraes, Thomas H. Sanderson, Timothy L. Stemmler, Lawrence I. Grossman, Valerian E. Kagan, Joseph S. Brunzelle, Arthur R. Salomon, Brian F P Edwards, Maik Hüttemann

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (Δφm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissuespecific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing Δφm hyperpolarization, a known cause of ROS and trigger of apoptosis.

Original languageEnglish (US)
Pages (from-to)64-79
Number of pages16
JournalJournal of Biological Chemistry
Volume292
Issue number1
DOIs
StatePublished - Jan 6 2017

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Phosphorylation
Threonine
Electron Transport
Cytochromes c
Phosphotransferases
Kidney
Cytochrome c Group
Respiration
Apoptosis
Cell Respiration
Adenylate Kinase
Crystallography
Mitochondria
Protein Stability
Mitochondrial Membrane Potential
Electron Transport Complex IV
Molecular Dynamics Simulation
Heme
Computer Simulation
Molecular dynamics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Phosphorylation of cytochrome c threonine 28 regulates electron transport chain activity in kidney : Implications for amp kinase. / Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F P; Hüttemann, Maik.

In: Journal of Biological Chemistry, Vol. 292, No. 1, 06.01.2017, p. 64-79.

Research output: Contribution to journalArticle

Mahapatra, G, Varughese, A, Ji, Q, Lee, I, Liu, J, Vaishnav, A, Sinkler, C, Kapralov, AA, Moraes, CT, Sanderson, TH, Stemmler, TL, Grossman, LI, Kagan, VE, Brunzelle, JS, Salomon, AR, Edwards, BFP & Hüttemann, M 2017, 'Phosphorylation of cytochrome c threonine 28 regulates electron transport chain activity in kidney: Implications for amp kinase', Journal of Biological Chemistry, vol. 292, no. 1, pp. 64-79. https://doi.org/10.1074/jbc.M116.744664
Mahapatra, Gargi ; Varughese, Ashwathy ; Ji, Qinqin ; Lee, Icksoo ; Liu, Jenney ; Vaishnav, Asmita ; Sinkler, Christopher ; Kapralov, Alexandr A. ; Moraes, Carlos T ; Sanderson, Thomas H. ; Stemmler, Timothy L. ; Grossman, Lawrence I. ; Kagan, Valerian E. ; Brunzelle, Joseph S. ; Salomon, Arthur R. ; Edwards, Brian F P ; Hüttemann, Maik. / Phosphorylation of cytochrome c threonine 28 regulates electron transport chain activity in kidney : Implications for amp kinase. In: Journal of Biological Chemistry. 2017 ; Vol. 292, No. 1. pp. 64-79.
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abstract = "Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (Δφm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissuespecific manner and leads to regulation of electron transport chain flux via {"}controlled respiration,{"} preventing Δφm hyperpolarization, a known cause of ROS and trigger of apoptosis.",
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AU - Varughese, Ashwathy

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AU - Lee, Icksoo

AU - Liu, Jenney

AU - Vaishnav, Asmita

AU - Sinkler, Christopher

AU - Kapralov, Alexandr A.

AU - Moraes, Carlos T

AU - Sanderson, Thomas H.

AU - Stemmler, Timothy L.

AU - Grossman, Lawrence I.

AU - Kagan, Valerian E.

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AU - Salomon, Arthur R.

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