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Survival Signaling by C-RAF: Mitochondrial Reactive Oxygen Species and Ca²⁺ Are Critical Targets

Andrey V. Kuznetsov,^1, Julija Smigelskaite,^1, Christine Doblander,¹ Manickam Janakiraman,^1, Martin Hermann,² Martin Wurm,² Stefan F. Scheidl,¹ Robert Sucher,¹ Andrea Deutschmann,² and Jakob Troppmair^1*

Daniel Swarovski Research Laboratory,¹ KMT Laboratory, Department of General and Transplant Surgery, Innsbruck Medical University, 6020 Innsbruck, Austria²

Received 19 April 2007/ Returned for modification 23 July 2007/ Accepted 26 December 2007

Survival signaling by RAF occurs through largely unknown mechanisms. Here we provide evidence for the first time that RAF controls cell survival by maintaining permissive levels of mitochondrial reactive oxygen species (ROS) and Ca²⁺. Interleukin-3 (IL-3) withdrawal from 32D cells resulted in ROS production, which was suppressed by activated C-RAF. Oncogenic C-RAF decreased the percentage of apoptotic cells following treatment with staurosporine or the oxidative stress-inducing agent tert-butyl hydroperoxide. However, it was also the case that in parental 32D cells growing in the presence of IL-3, inhibition of RAF signaling resulted in elevated mitochondrial ROS and Ca²⁺ levels. Cell death is preceded by a ROS-dependent increase in mitochondrial Ca²⁺, which was absent from cells expressing transforming C-RAF. Prevention of mitochondrial Ca²⁺ overload after IL-3 deprivation increased cell viability. MEK was essential for the mitochondrial effects of RAF. In summary, our data show that survival control by C-RAF involves controlling ROS production, which otherwise perturbs mitochondrial Ca²⁺ homeostasis.

Published ahead of print on 22 January 2008.

These authors contributed equally to this work.

Present address: Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.