title: Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.
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 (ΔΨm), 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, Thr28is 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 Thr28in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨmhyperpolarization, a known cause of ROS and trigger of apoptosis.
authors.affiliation: From the Center for Molecular Medicine and Genetics and.|the Departments of Biochemistry and Molecular Biology.
authors.forename: Gargi
authors.initials: G
authors.lastname: Mahapatra
authors.affiliation: From the Center for Molecular Medicine and Genetics and.|the Departments of Biochemistry and Molecular Biology.
authors.forename: Ashwathy
authors.initials: A
authors.lastname: Varughese
authors.affiliation: the Chemistry Department and.
authors.forename: Qinqin
authors.initials: Q
authors.lastname: Ji
authors.affiliation: From the Center for Molecular Medicine and Genetics and.|the College of Medicine, Dankook University, Cheonan-si, Chungcheongnam-do 31116, Republic of Korea.
authors.forename: Icksoo
authors.initials: I
authors.lastname: Lee
authors.affiliation: From the Center for Molecular Medicine and Genetics and.
authors.forename: Jenney
authors.initials: J
authors.lastname: Liu
authors.affiliation: the Departments of Biochemistry and Molecular Biology.
authors.forename: Asmita
authors.initials: A
authors.lastname: Vaishnav
authors.affiliation: From the Center for Molecular Medicine and Genetics and.
authors.forename: Christopher
authors.initials: C
authors.lastname: Sinkler
authors.affiliation: the Center for Free Radical and Antioxidant Health and the Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15219.
authors.forename: Alexandr A
authors.initials: AA
authors.lastname: Kapralov
authors.affiliation: the Department of Neurology, University of Miami School of Medicine, Miami, Florida 33136, and.
authors.forename: Carlos T
authors.initials: CT
authors.lastname: Moraes
authors.affiliation: Emergency Medicine, and.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Sanderson
authors.affiliation: Pharmaceutical Sciences, Wayne State University, Detroit, Michigan 48201.
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Stemmler
authors.affiliation: From the Center for Molecular Medicine and Genetics and.
authors.forename: Lawrence I
authors.initials: LI
authors.lastname: Grossman
authors.affiliation: the Center for Free Radical and Antioxidant Health and the Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15219.
authors.forename: Valerian E
authors.initials: VE
authors.lastname: Kagan
authors.affiliation: the Life Sciences Collaborative Access Team, Northwestern University, Center for Synchrotron Research, Argonne, Illinois 60439.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Brunzelle
authors.affiliation: the MCB Department, Brown University, Providence, Rhode Island 02912.
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Salomon
authors.affiliation: the Departments of Biochemistry and Molecular Biology.
authors.forename: Brian F P
authors.initials: BF
authors.lastname: Edwards
authors.affiliation: From the Center for Molecular Medicine and Genetics and mhuttema@med.wayne.edu.|the Departments of Biochemistry and Molecular Biology.
authors.forename: Maik
authors.initials: M
authors.lastname: Hüttemann
mesh_terms: D000263:Adenylate Kinase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D019069:Cell Respiration / Q000502:physiology*; D018360:Crystallography, X-Ray; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011487:Protein Conformation; D017382:Reactive Oxygen Species / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; D013912:Threonine; D045304:Cytochromes c; D003576:Electron Transport Complex IV; D000263:Adenylate Kinase
keywords: AMP-activated kinase (AMPK); apoptosis; cell signaling; cytochrome c; electron transport chain; electron transport system (ETS); kidney metabolism; mitochondria; oxidative phosphorylation; respiration