Coupling growth factors to the apoptotic machinery: Biochemical and genetic elucidation of the PI-3-kinase/Akt/BAD survival signaling cascade

Extracellular stimuli regulate a number of critical cellular phenotypes by activating signal transduction pathways that transmit information about the external environment to various effector machineries within the cell. One phenotype controlled by soluble trophic factors is cellular survival. However, the mechanisms by which extracellular survival signals promote cellular survival and block cell death are incompletely characterized.;The goals of this Thesis were threefold. First, to elucidate the signal transduction pathways that promote cellular survival. Second, to identify those targets within the cell-intrinsic death machinery whose activity is regulated by survival signals. Third, through reverse genetics, to establish the importance of the regulation of components of the cell-intrinsic death machinery by trophic factor-regulated survival kinases.;We demonstrate in cerebellar granule neurons that growth factors, acting through transmembrane receptor tyrosine kinases, promote cellular survival by activating the phosphoinositide-3-kinase (PI3K). The activity of the serine/threonine kinase Akt, a target of PI3K, was found to be both necessary and sufficient for growth factors to promote survival. These findings identify a critical signal transduction pathway whose activity mediates survival signals.;We further identify a component of the cell-intrinsic death machinery that is regulated by Akt-mediated phosphorylation and inactivation, the pro-apoptotic BCL-2 family member BAD. BAD promotes apoptosis by binding to and inactivating pro-survival BCL-2 family members such as BCL-XL. Akt phosphorylates BAD directly at Ser-136 both in vivo and in vitro, which promotes the interaction of BAD with 14-3-3 proteins and blocks the pro-apoptotic activity of BAD. We also demonstrate that Akt-mediated BAD Ser-136 phosphorylation inactivates BAD by a multi-step process that culminates in the phosphorylation of BAD at Ser-155, which prevents the interaction of BAD and pro-survival BCL-2 family members.;To assess the importance of the PI3K/Akt/BAD signaling cassette, we generate mice in which the BAD phosphoacceptor sites are mutated such that BAD can no longer be phosphorylated and inactivated. Analysis of these mice demonstrate that the PI3K/Akt pathway utilizes BAD phosphorylation to raise the threshold at which cells undergo cell death in response to an apoptotic stimulus, by increasing the resistance of mitochondria to death signals. Taken together, these results identify a survival signaling pathway whose activity is essential for the ability of soluble trophic factors to block apoptosis.