# Control of Cell Respiration and Apoptosis by Phosphorylation of Cytochrome c

> **NIH NIH R01** · WAYNE STATE UNIVERSITY · 2020 · $353,724

## Abstract

SUMMARY
 Cytochrome c (Cytc) plays a central role in mitochondrial respiration and type 2
apoptosis. We have developed new protocols to purify mitochondrial proteins while
maintaining their physiological regulatory properties and posttranslational modifications,
and we discovered that Cytc is phosphorylated on distinct tyrosine residues in heart and
liver tissue. Recently, we have mapped two more phosphorylation sites on Cytc purified
from cow and rat kidneys, Thr28 and Ser47, which are the primary focus of this grant
application. Supported by strong preliminary data it is our overall hypothesis that Cytc
phosphorylation regulates the two main functions of Cytc, mitochondrial respiration and
apoptosis. Our long term goal is to understand the regulation of Cytc by cell signaling
pathways under normal and pathological conditions. As a first step towards our goal we
will test four specific hypotheses: 1) to test the hypothesis that kidney Cytc can be
phosphorylated on Thr28 and Ser47, and that these modifications affect the basic
properties of the molecule; 2) to test the hypothesis that Cytc Thr28 and Ser47
phosphorylation affects mitochondrial respiration and apoptosis; 3) to identify kinases
and phosphatases that control Cytc phosphorylation; and 4) to demonstrate the
physiological effect in mice. Phosphorylated Cytc will be isolated from cow and rat
kidneys and phosphomimetic mutant Cytc will be overexpressed and purified from
bacteria, followed by structural characterization using mass spectrometry, spectroscopic
methods, and protein crystallography (Aim 1). Phosphorylated and phosphomimetic
mutant Cytc will be subjected to a comprehensive set of functional analyses including in
vitro respiration and apoptosis measurements, and by in vivo studies with
phosphomimetic Cytc stably expressed in Cytc knockout cells (Aim 2). We will next lay
the ground work to explore the signaling pathways and identify kinases and
phosphatases that act on Cytc using affinity purification/mass spectrometry techniques
(Aim 3). Finally, we will reintroduce phosphomimetic and non-phosphorylatable mutant
Cytc into Cytc knockout mice to study the effect of Cytc modification and regulation at
the animal level (Aim 4). We expect that this research will reveal that Cytc, a protein that
makes life and death decisions, is subject to regulation by cell signaling, opening new
opportunities for the understanding of mitochondrial respiration and apoptosis, and to
control it in pathological conditions in which respiration and apoptosis are dysregulated.

## Key facts

- **NIH application ID:** 9982332
- **Project number:** 5R01GM116807-04
- **Recipient organization:** WAYNE STATE UNIVERSITY
- **Principal Investigator:** MAIK HUETTEMANN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $353,724
- **Award type:** 5
- **Project period:** 2017-09-08 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9982332

## Citation

> US National Institutes of Health, RePORTER application 9982332, Control of Cell Respiration and Apoptosis by Phosphorylation of Cytochrome c (5R01GM116807-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9982332. Licensed CC0.

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