Akt Regulates Cell Survival and Apoptosis at a Postmitochondrial Level Honglin Zhou, Xin-Ming Li, Judy Meinkoth, and Randall N. Pittman Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084 Abstract. Phosphoinositide 3 kinase/Akt pathway plays an essential role in neuronal survival. However, the cellular mechanisms by which Akt suppresses cell death and protects neurons from apoptosis remain un- clear. We previously showed that transient expression of constitutively active Akt inhibits ceramide-induced death of hybrid motor neuron 1 cells. Here we show that stable expression of either constitutively active Akt or Bcl-2 inhibits apoptosis, but only Bcl-2 prevents the release of cytochrome c from mitochondria, suggesting that Akt regulates apoptosis at a postmitochondrial level. Consistent with this, overexpressing active Akt rescues cells from apoptosis without altering expression levels of endogenous Bcl-2, Bcl-x, or Bax. Akt inhibits apoptosis induced by microinjection of cytochrome c Introduction Apoptosis plays an important role in the development and homeostasis of multicellular organisms (Vaux and Kors- meyer, 1999). In the nervous system, apoptosis is required for normal development (Oppenheim, 1991; Jacobson et al., 1997) and has been implicated in the pathogenesis of various neurodegenerative conditions, including acute in- sults to the central nervous system such as head trauma and spinal cord injury, and chronic degeneration such as Alzhei- mer’s disease and Huntington’s disease (Thompson, 1995). Genetic studies in the nematode Caenorhabditis elegans have provided an understanding of apoptosis at the molec- ular level that show that three genes, ced-3, ced-4, and ced-9, play critical roles in regulating the cell death pro- gram. ced-3 and ced-4 were identified as proapoptotic genes, whereas ced-9 was identified as an antiapoptotic gene (Hengartner and Horvitz, 1994). Mammalian homologues of CED-3 have been identified as the caspase family. Caspases are activated in response to apoptotic stimuli and subsequently cleave cellular proteins to cause cell death (Salvesen and Dixit, 1997; Thornberry and Lazebnik, 1998). The mammalian homologue of CED-4 is Apaf-1 Address correspondence to Randall N. Pittman, Ph.D., Dept. Pharmacol- ogy, Room 154 John Morgan, Univ. of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084. Tel.: (215) 898-9736. Fax: (215) 573-2236. E-mail: pittman@pharm.med.upenn.edu and lysates from cells expressing active Akt inhibit cy- tochrome c induced caspase activation in a cell-free as- say while lysates from Bcl-2–expressing cells have no effect. Addition of cytochrome c and dATP to ly- sates from cells expressing active Akt do not activate caspase-9 or -3 and immunoprecipitated Akt added to control lysates blocks cytochrome c–induced activation of the caspase cascade. Taken together, these data sug- gest that Akt inhibits activation of caspase-9 and -3 by posttranslational modiﬁcation of a cytosolic factor downstream of cytochrome c and before activation of caspase-9. Key words: protein serine-threonine kinase • cyto- chrome c • apoptosis • neuron • mitochondria (apoptotic protease activating factor 1), which is important for initiating a cytochrome c–dependent caspase activation cascade (Zou et al., 1997; Slee et al., 1999). Mammalian ho- mologues of CED-9 are members of the Bcl-2 family, which includes both positive and negative regulators of cell survival (Adams and Cory, 1998; Gross et al., 1999). Recent biochemical studies have revealed that caspase activation during apoptosis is a tightly regulated process (Salvesen and Dixit, 1997; Thornberry and Lazebnik, 1998; Budihardjo et al., 1999). Apoptotic stimuli such as activation of cell surface receptors or environmental stress can induce cytochrome c release from mitochondria (Green and Reed, 1998). Once in the cytosol, cytochrome c binds to Apaf-1 and induces its oligomerization. Oligo- merization of Apaf-1 recruits procaspase-9 and results in subsequent caspase-9 activation (Srinivasula et al., 1998; Zou et al., 1999). Active caspase-9 cleaves procaspase-3 and generates active caspase-3. Active caspase-3 cleaves a number of important cellular proteins to execute cell death and activate additional downstream caspases (Slee et al., 1999). Phenotypes of caspase-3–, caspase-9–, or Apaf-1–deficient mice are very similar to each other in that all these mice manifest brain overgrowth due to re- duced apoptosis during brain development (Cecconi et al., 1998; Hakem et al., 1998; Kuida et al., 1998; Yoshida et al., 1998). On the other hand, caspase-1–, caspase-2–, caspase-8–, ª The Rockefeller University Press, 0021-9525/2000/10/483/12 $5.00 The Journal of Cell Biology, Volume 151, Number 3, October 30, 2000 483–494 http://www.jcb.org/cgi/content/full/151/3/483 483