14-3-3 Interacts directly with and negatively regulates pro-apoptotic Bax

Abstract

The Bcl-2 family of proteins comprises well characterized regulators of apoptosis, consisting of anti-apoptotic members and pro-apoptotic members. Pro-apoptotic members possessing BH1, BH2, and BH3 domains (such as Bax and Bak) act as a gateway for a variety of apoptotic signals. Bax is normally localized to the cytoplasm in an inactive form. In response to apoptotic stimuli, Bax translocates to the mitochondria and undergoes oligomerization to induce the release of apoptogenic factors such as cytochrome c, but it is still largely unknown how the mitochondrial translocation and proapoptotic activity of Bax is regulated. Here we report that cytoplasmic protein 14-3-3θ binds to Bax and, upon apoptotic stimulation, releases Bax by a caspase-independent mechanism, as well as through direct cleavage of 14-3-3θ by caspases. Unlike Bad, the interaction with 14-3-3θ is not dependent on the phosphorylation of Bax. In isolated mitochondria, we found that 14-3-3θ inhibited the integration of Bax and Bax-induced cytochrome c release. Bax-induced apoptosis was inhibited by over-expression of either 14-3-3θ or its mutant (which lacked the ability to bind to various phosphorylated targets but still bound to Bax), whereas overexpression of 14-3-3θ was unable to inhibit apoptosis induced by a Bax mutant that did not bind to 14-3-3θ. These findings indicate that 14-3-3θ plays a crucial role in negatively regulating the activity of Bax.