caspase-11–, Bid-, or FADD-deficient mice do not show obvious defects in brain development (Los et al., 1999; Yin et al., 1999). These genetic studies confirm the critical roles for Apaf-1, caspase-9, and caspase-3 in regulating neuronal apoptosis. Neurons are dependent on neurotrophic factors for sur- vival, and removal of such factors results in apoptosis. Among the growth factor signaling molecules, phospho- inositide 3 (PI-3)1 kinase and mitogen-activated protein (MAP) kinase have been shown to be important for neu- ronal survival (Pettmann and Henderson, 1998). Recent studies indicate that the protective effects of PI 3-kinase are mediated primarily by one of its downstream targets— Akt (Franke et al., 1997). Upon activation by PI 3-kinase, Akt phosphorylates Bad at Ser136. This decreases the binding of Bad to Bcl-xL at the mitochondrial membrane and increases its binding to 14-3-3 in the cytosol (Zha et al., 1996; Datta et al., 1997). It has been speculated that Akt inhibits apoptosis by maintaining Bcl-x function and preventing cytochrome c release from mitochondria. How- ever, a direct effect of Akt in regulating cytochrome c translocation during apoptosis has not been shown. More- over, it remains to be explored whether Akt may also in- hibit apoptosis independent of cytochrome c release. In this report, we examine the cellular mechanism by which Akt inhibits apoptosis in hybrid motor neuron 1 (HMN1) cells, a neuronal cell line that requires PI 3-kinase but not MAP kinase for survival. By generating stable HMN1 lines overexpressing constitutively active Akt, sub- cellular fractionation, cell-free assays of apoptosis, and mi- croinjection, we investigated the effects of Akt on several critical apoptotic events, with particular focus on its effect on cytochrome c redistribution. Our data indicate that Akt inhibits apoptosis downstream of cytochrome c release based on the following observations: (a) Akt inhibits cell death but does not block release of cytochrome c, (b) Akt inhibits cytochrome c–induced caspase activation in a cell free assay, and (c) HMN1 cells expressing active Akt are resistant to apoptosis induced by microinjection of cyto- chrome c. In sum, our study indicates that Akt plays an im- portant role in suppressing neural apoptosis at a postmito- chondrial stage, downstream of cytochrome c release and before activating caspase-9. Materials and Methods Materials C2-ceramide, C2-dihydroceramide, PD 98059, and LY 294002 were ob- tained from Calbiochem. LipofectAMINE was obtained from Life Tech- nologies. Hygromycin B was from Boehringer. Fetal bovine serum was from HyClone. Polyvinylidene difluoride transfer membrane, ECL sub- strate, and [gamma-32P] ATP were from NEN Life Science Products. pCR3.1 vector was from Invitrogen. Caspase-9 colorimetric assay kit was from R&D Systems. Hoechst 33342 and antibody to cytochrome c oxidase (COX) subunit IV (clone 20E8-C12) were from Molecular Probes, Inc. Antibody to cytochrome c (clone 7H8.2C12), antibody to mouse BCL-2 (clone 3F11), and antibody to human BCL-2 (clone 6C8) were from PharMingen. Antibodies to caspase-9 were from Santa Cruz Biotechnol- ogy, Inc. and R&D Systems. Rabbit polyclonal antibody to Akt and phos- 1Abbreviations used in this paper: CEB, cell extract buffer; HA, hemagglu- tinin; HMN1, hybrid motor neuron 1; MAP, mitogen-activated protein; PARP, poly (ADP-ribose) polymerase; PI 3-kinase, phosphoinositide 3 kinase. phospecific antibody for Akt Serine-473 were from New England Biolabs, Inc., and a rabbit polyclonal phosphospecific antibody for MAP kinase threonine-202/tyrosine-204 was from Cell Signaling Technology. Fluores- cein isothiocyanate–conjugated secondary antibody was from Jackson Im- munoResearch Laboratories. Antibody to hemagglutinin (12CA5) was a generous gift from Dr. Jeffery Field (University of Pennsylvania, Philadel- phia, PA). Antibody to activated caspase-3 was a generous gift from Dr. Robert Siman (University of Pennsylvania, Philadelphia, PA). All other reagents were obtained from Sigma-Aldrich. Cell Viability, Internucleosomal DNA Fragmentation, and Nuclear Condensation HMN1 cells were washed with serum-free DMEM. LY 294002, PD 98059, or vehicle was diluted into serum-free DMEM at the indicated concentra- tions. Cell viability and DNA fragmentation were analyzed as described previously (Zhou et al., 1998). For C2-ceramide–induced apoptosis, HMN1 cells were maintained in serum-free DMEM for 24 h before exper- iments. Staining nuclei with Hoechst 33342 was performed as described previously (Zhou et al., 1998). Cell Transfection and Generation of Stable Cell Lines HMN1 cells were plated in six-well plates and cotransfected with 0.5 mg of pREP-4 encoding a hygromycin resistance gene and 4.5 mg of pCMV, pCMVhBcl-2, pCR3.1, or pCR3.1myr-AktD4-129 (a constitutively active Akt containing a consensus myristoylation site and lacking the PH do- main; Kohn et al., 1996; Zhou et al., 1998). 36 h after transfection, cells were replated into 10-cm plates and selected in medium containing 350 mg/ml hygromycin for 4 wk to select stable clones. Clones expressing hu- man Bcl-2 were screened with monoclonal antibody, 6C8, for human Bcl-2 (PharMingen). Clones expressing constitutively active Akt were screened with anti-hemagglutinin (anti–HA) monoclonal antibody (12CA5) and verified by anti–Akt antibody as well as anti–Akt (Ser473) phosphospe- cific antibody (New England Biolabs, Inc.). SDS-PAGE and Western Blot Analysis Proteins were separated by SDS-PAGE and transferred onto PVDF membranes, followed by probing with various antibodies. Bound antibod- ies were detected using appropriate peroxidase-coupled secondary anti- bodies, followed by detection using the Enhanced Chemiluminescence system (NEN Life Science Products). Subcellular Fractionation The cytosolic fraction for detecting cytochrome c was prepared as de- scribed by Erhardt et al., (1999). In brief, HMN1 cells were harvested by centrifugation at 600 g for 5 min at 48C. The cell pellet was washed once with ice-cold phosphate-buffered saline and resuspended in 5 vol of buffer (20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 10 mg/ml leupeptin, 2 mg/ml aprotinin, and 0.1 mM PMSF) containing 250 mM sucrose. Cells were ho- mogenized with 20 strokes of a Dounce homogenizer, and the homoge- nates were centrifuged at 900 g for 5 min at 48C to remove nuclei. Super- natants were centrifuged at 10,000 g for 15 min at 48C. The resulting supernatants were used as the cytosolic fraction for detecting cytochrome c and the resulting pellets were used as a positive control for mitochon- drial protein cytochrome c oxidase (COX) subunit IV. Preparation of Cell-free Extracts and Cell-free Reactions Cell-free extracts were generated from HMN1 cells as described by Slee et al. (1999) with some modifications. Cells were pelleted and washed twice with PBS, pH 7.2, followed by washing once with 5 ml of ice-cold cell ex- tract buffer (CEB; 20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM okadaic acid, 1 mM dithiothreitol, 100 mM PMSF, 10 mg/ml leupeptin, 2 mg/ml aprotinin). The cell pellet was resuspended in 2 vol of ice-cold CEB and incubated on ice for 15 min. Cells in CEB were then transferred to a 2-ml Dounce ho- mogenizer and disrupted with 20 strokes of a B-type pestle. Lysates were transferred to Eppendorf tubes and centrifuged at 15,000 g for 15 min at 48C. The resulting supernatants were obtained as postnuclear extracts and frozen in aliquots at 2808C until required. Cell-free reactions were set up in a 120-ml reaction volume. Cell extracts were brought to a final volume The Journal of Cell Biology, Volume 151, 2000 484