We recently discovered that cytochrome c (Cytc) is directly regulated by cell signaling via tyrosine phosphorylation on two distinct sites in mammalian liver and heart, a finding made possible by isolation techniques developed by us that preserve the physiological phosphorylation status of the protein. It is our overall hypothesis that Cytc phosphorylation regulates the two main functions of Cytc, mitochondrial respiration and Cytc release during apoptosis. Our long term goal is to understand the effect of Cytc phosphorylation on respiration and apoptosis and to identify the cell signaling pathways directed to Cytc. Supported by preliminary data, three specific aims will be investigated: 1) to test the hypotheses that Cytc is phosphorylated in vivo in a distinct tissue-specific manner and that this phosphorylation results in structural changes of Cytc;2) to test the hypotheses that phosphorylation of Cytc leads to controlled respiration and prevents its participation in apoptosis;and 3) to systematically identify mitochondrial tyrosine kinases that phosphorylate Cytc. Phosphorylated Cytc will be isolated from cow liver and heart tissue and structurally characterized using mass spectrometry and protein crystallography (Aim 1). Phosphorylated Cytc will be subjected to functional assays including in vitro respiration and apoptosis measurements, accompanied by mutagenesis studies in vivo with Cytc constructs in a Cytc knockout cell line (Aim 2). Experiments to identify kinases that act on Cytc will include a proteomic approach, kinase localization, and yeast two-hybrid studies (Aim 3). This research is expected to reveal that Cytc, long recognized as a central molecule in respiration and apoptosis, is subject to regulation by cell signaling, opening new opportunities for the understanding and control of those two key biological processes.

Public Health Relevance

Our laboratory discovered that Cytochrome c (Cytc), which plays a key role in programmed cell death (apoptosis) and cellular respiration, is regulated by cell signaling networks that reversibly phosphorylate Cytc. In this study, we will analyze the structural changes brought about by phosphorylation, determine whether and to what extent phosphorylation affects the functions of Cytc in apoptosis and respiration, and identify the kinases that phosphorylate Cytc. Since apoptosis and cellular respiration are involved in many diseases such as cancer and neurodegeneration, this study is expected to lead to better understanding and control of such diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Maas, Stefan
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Wayne State University
Schools of Medicine
United States
Zip Code
Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin et al. (2017) Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE. J Biol Chem 292:64-79
Shay, Joseph; Elbaz, Hosam A; Lee, Icksoo et al. (2015) Molecular Mechanisms and Therapeutic Effects of (-)-Epicatechin and Other Polyphenols in Cancer, Inflammation, Diabetes, and Neurodegeneration. Oxid Med Cell Longev 2015:181260
Elbaz, Hosam A; Lee, Icksoo; Antwih, Deborah A et al. (2014) Epicatechin stimulates mitochondrial activity and selectively sensitizes cancer cells to radiation. PLoS One 9:e88322
Lee, Icksoo; Hüttemann, Maik (2014) Energy crisis: the role of oxidative phosphorylation in acute inflammation and sepsis. Biochim Biophys Acta 1842:1579-86
Sanderson, Thomas H; Reynolds, Christian A; Kumar, Rita et al. (2013) Molecular mechanisms of ischemia-reperfusion injury in brain: pivotal role of the mitochondrial membrane potential in reactive oxygen species generation. Mol Neurobiol 47:9-23
Sanderson, Thomas H; Mahapatra, Gargi; Pecina, Petr et al. (2013) Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment. PLoS One 8:e78627
Hüttemann, Maik; Lee, Icksoo; Perkins, Guy A et al. (2013) (-)-Epicatechin is associated with increased angiogenic and mitochondrial signalling in the hindlimb of rats selectively bred for innate low running capacity. Clin Sci (Lond) 124:663-74
Hüttemann, Maik; Helling, Stefan; Sanderson, Thomas H et al. (2012) Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation. Biochim Biophys Acta 1817:598-609
Hüttemann, Maik; Lee, Icksoo; Grossman, Lawrence I et al. (2012) Phosphorylation of mammalian cytochrome c and cytochrome c oxidase in the regulation of cell destiny: respiration, apoptosis, and human disease. Adv Exp Med Biol 748:237-64
Helling, Stefan; Huttemann, Maik; Ramzan, Rabia et al. (2012) Multiple phosphorylations of cytochrome c oxidase and their functions. Proteomics 12:950-9

Showing the most recent 10 out of 16 publications