@article {Lemieux151480, author = {H{\'e}l{\`e}ne Lemieux and Patrick Subarsky and Christine Doblander and Martin Wurm and Jakob Troppmair and Erich Gnaiger}, title = {Impairment of mitochondrial respiratory function as an early biomarker of apoptosis induced by growth factor removal}, elocation-id = {151480}, year = {2017}, doi = {10.1101/151480}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Intracellular signaling pathways not only control cell proliferation and survival, but also regulate the provision of cellular energy and building blocks through mitochondrial and non-mitochondrial metabolism. Wild-type and oncogenic RAF kinases have been shown to prevent apoptosis following the removal of interleukin 3 (IL-3) from mouse pro-myeloid 32D cells by reducing mitochondrial reactive oxygen species production. To study primary effects of RAF on mitochondrial energy metabolism, we applied high-resolution respirometry after short-term IL-3 deprivation (8 h), before 32D cells show detectable signs of cell death. Respiration in intact 32D cells was suppressed as an early event following removal of IL-3, but remained more stable in 32D cells expressing the v-RAF oncogene. In permeabilized 32D cells deprived of IL-3, respiratory capacities of the NADH-pathway, the convergent NADH\&succinate-pathway, and Complex IV activity were decreased compared to cells grown in the presence of IL-3, whereas succinate-supported respiration remained unchanged, consistent with control by Complex IV. The apparent Complex IV excess capacity was zero above NADH\&succinate-pathway capacity reconstituting tricarboxylic acid cycle function. In comparison, electron flow reached only 60\% when supported by succinate alone through Complexes II, III and IV, and was therefore relatively insensitive to Complex IV injuries up to a threshold of 40\% inhibition. A slight increase in respiration following addition of cytochrome c, a marker of mitochondrial outer membrane leakage, was present after IL-3 depletion, indicating mitochondrial fragility. Our results highlight a novel link between the key mitogenic and survival kinase CRAF and mitochondrial energy homeostasis.}, URL = {https://www.biorxiv.org/content/early/2017/06/18/151480}, eprint = {https://www.biorxiv.org/content/early/2017/06/18/151480.full.pdf}, journal = {bioRxiv} }