During this period we continued to investigate the biological implication of membrane phosphotidylserine (PS) on Akt signaling that regulates cell survival, using various cellualr and in vitro models where PS levels were altered. Mass spectrometric analysis of conformational changes coupled with biomolecular interaction analysis based on surface plasmon resonance revelaed that PS promotes Akt-membrane binding and conformational changes for activation through electrostatic interaction. We found that both PH and regulatory domains of Akt interact with PS. When Neuro 2A cells were expressed with Akt mutated at the potential PS-binding residues (R15A or K20A) in the PH domain of Akt, in vitro Akt-PS binding as well as in vivo membrane translocation triggered by IGF was greatly diminished. Furthermore, IGF-induced phosphorylation of both T308 and S473 was abolished in these mutants, strongly indicating that PS is necessary for Akt activation in vivo. When basic residues in the regulatory domain were mutated with alanine, T308 phosphorylation significantly decreased and S-473 phosphorylation was abolished, indicating that PS-regulatory interaction is also important for phosphorylation of Akt, especially for S473 for optimal activation. We have also established during this period a novel strategy to study Akt-inhibitor interaction by probing Akt conformation altered by inhibitors. We have previously identified two inter-domain cross-linked lysine pairs, K30-K389 (PH-kinase) and K426-K284 (regulatory-kinase), in inactive Akt molecules, indicating a folded structure with the PH and regulatory domains covering the kinase domain. These cross-linked pairs were not observed within 24 angstrom spatial distance constraint upon interaction with membranes containing PIP3 and PS, suggesting that changes to an open conformation occurred after the membrane interaction. Using this strategy, we demonstrated that when Akt interacted with a PI analog, an open inter-domain conformation necessary for Akt activation occurred, even before Akt interacted with membranes. The Akt-PI analog interaction presumably prevented the Akt-PIP3 binding and hence blocked Akt membrane translocation and activation. In contrast, the folded inter-domain conformation was unchanged by interacting with Inhibitor VI, a peptide supposedly binding to the PH domain. Upon subsequent interaction with the membrane, the extent of PH-kinase cross-linking in comparison to the case without the inhibitor VI increased, indicating that the inhibitor impaired the opening of the PH domain for exposing T308 for phosphorylation at the plasma membrane. Our results demonstrated that molecular mechanisms for Akt inhibition can be deduced by monitoring the Akt conformational changes probed by mass spectrometry. During this report period we have also established quantitative mass spectrometric analysis of phosphoproteins. Proteins were first labeled with O-18 by tryptic digestion in either regular or O-18 labeled water, differentially labeled peptides were mixed together and phosphopeptides were enriched using titanium oxide prior to mass spectrometric analysis. This approach enabled us to unveil minor phosphopeptides generated during stimulation of the cells. This approach is now being applied to investigate the Akt interacting phosphoproteins during stimulation. Our previous findings suggested that DHA-induced neurite outgrowth may not be mediated by direct activation of RXR, suggesting other mediators are involved. During this period, we have also established an HPLC/ESI-MS/MS method for quantitative profiling of lipid mediators derived from DHA. Using this method, we found that DHA metabolizes to N-docosahexaenoylethanolamide (DEA) in developing hippocampi. We subsequently demonstrated that DEA is a novel and effective ligand to RXR and promotes neurite development, synaptogenesis and synaptic protein expression through activating RXR. We found in mouse embryonic hippocampal neuronal cultures that DHA at low micromolar concentrations uniquely promotes not only neurite development but also the expression of synaptic proteins such as synapsins and glutamate receptors and improves excitatory synaptic activity. DHA supplementation of E-18 hippocampal neuronal culture increased DEA content. Indeed, DEA at submicromolar concentrations activated RXR and promoted neurite growth and synaptic protein expression in an RXR-dependent manner. Conversely, depleting DHA by feeding mice with n-3 fatty acid deficient diet during pregnancy resulted in a marked reduction of DEA in E-18 fetal hippocampi as well as significant inhibition of neurite development and synaptogenesis in E-18 hippocampal cultures. Furthermore, hippocampal slices obtained from the deficient animals at a later stage of development exhibited retarded long-term potentiation (LTP) while the expression of hippocampal synapsins and glutamate receptors was significantly decreased, supporting a significant role of DHA in promoting synapse development and learning and memory. As the DEA production is directly linked to the DHA level, DEA- and RXR-dependent hippocampal neurite growth and synaptic protein expression provide a novel mechanism for the role of DHA in hippocampal development and hippocampus-related cognitive function. Based on these data, we suggest that DHA metabolism to DEA and RXR activation emerge as potential new targets for regulating physiological and pathophysiological processes of neurodevelopment and function.