Figure 10. Schematic illustration showing that Akt can regulate caspase activation at a premitochondrial level (e.g., Kennedy et al., 1999), as well as at a postmitochondrial level downstream of cyto- chrome c release and before activation of caspase-9 (present study). solic cytochrome c to participate in other cellular process such as a-synuclein aggregation. Interestingly, expressing active Akt or treatment with zVAD-fmk inhibited chroma- tin condensation after cytochrome c injection, yet many rescued cells still shrank, suggesting that cytochrome c may cause cell shrinkage independent of caspase activation. It should be possible to examine additional roles of cyto- chrome c independent of its effects on apopsome complex formation in Apaf-1 or caspase-9–deficient neurons. A growing number of downstream targets of Akt have been identified, including glycogen synthase kinase-3, BAD, human caspase-9, and transcription factors such as CREB, Forkhead, and NFkB (Pap and Cooper, 1998; Khwaja, 1999). While each of these has been implicated as an important target for Akt to inhibit apoptosis, in the present case, caspase-9 appears to be the most likely can- didate since Akt phosphorylates human caspase-9 and in- hibits its activity (Cardone et al., 1998), which would pro- vide a potential mechanism for Akt to inhibit caspase activation at a postmitochondrial level. However, [32P]or- thophosphate labeling of cells expressing active Akt indi- cated that mouse caspase-9 is not phosphorylated by Akt (data not shown). This may be explained by recent reports that the consensus Akt phosphorylation sites on human caspase-9 are not conserved in caspase-9 from other spe- cies such as mouse caspase-9 in HMN1 cells (Fujita et al., 1999; Rodriguez et al., 2000). On the other hand, our data also suggest that there exists an additional, more general mechanism by which Akt can suppress activation of caspases by cytochrome c at a postmitochondrial stage. When added into vector-cell extracts, immunoisolated ac- tive Akt is sufficient to inhibit caspase activation induced by cytochrome c, suggesting that active Akt has a direct ef- fect on inhibiting cytochrome c–induced caspase activa- tion. One such direct target for Akt may be Apaf-1, which forms a holoenzyme with caspase-9 and regulates caspase-9 activity (Rodriguez and Lazebnik, 1999). Akt phosphory- lates Apaf-1 in vitro (our unpublished observations); however, further studies are needed to determine whether cellular Apaf-1 is a direct target of Akt, and how the phos- phorylation status of Apaf-1 may regulate caspase-9 acti- vation. In conclusion, our data based on subcellular fraction- ation, cell-free assays of apoptosis, and microinjection studies indicate that Akt inhibits apoptosis downstream of cytochrome c release and before activation of caspase-9 (Fig. 10). The ability of Akt to regulate apoptosis at a post- mitochondrial stage provides a potential molecular mech- anism allowing postmitotic cells like neurons to tolerate leakage of cytochrome c into the cytoplasm when growth factors are present. A key to understanding postmitochon- drial regulation of apoptosis by Akt will be future studies to identify the cellular target(s) of Akt. We thank Ms. Svetlana Savina for microinjection, Dr. Robert Siman for the caspase-3 antibody, and Dr. Morris Birnbaum (University of Pennsyl- vania) for the Akt constructs. We also thank members of the Pittman lab for support and helpful suggestions throughout the period of this work. This work was supported by NS32465 from the National Institutes of Health to R.N. Pittman. 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