We continued to investigate the molecular mechanisms of PS-dependent Akt activation and its biological implication. Previously, We demonstrated that PS plays a crucial role in Akt translocation and subsequent conformational changes for phosphorylation and activation. We found that Akt interaction with R15 and K20 in the PH domain outside the PIP3 binding pocket is critical for Akt translocation and activation. PS also binds to the regulatory (RD) domain, augmenting Akt translocation, phosphorylation and activation triggered by PIP3 in living cells. We identified K419 and K420 in the RD as PS-interacting residues and found that Akt interaction with these residues is critical for Akt phosphorylation at S473 by mTORC2. It is well-known that S473 phosphorylation is an important modulator of Akt activation, and therefore can serve as a new target for drug development. During this review period we initiated efforts to identify inhibitors of Akt activation based on this new target that differs from the well-recognized PIP3- or ATP-binding sites. To this end, we have developed a cell-based assay targeting the S473 phosphorylation against the MLSCN (Molecular Libraries Screening Center Network) compound collection using homogeneous time-resolved fluorescence (HTRF) detection. The small molecules that inhibit specifically Akt S473 phosphorylation/mTORC2 activity thus identified will not only serve as valuable research tools but also may have significant therapeutic potential with fewer side effects, especially for conditions involving hyperactive Akt signaling such as cancer and Alzheimers disease. During this period, we also investigated the effects of membrane PS on the downstream events of Akt activation. We found that the membrane PS positively modulates the phosphorylation of Foxo1(T24), GSK-3(S9) and BAD(S136), consequently promoting cell survival. To identify interacting partners of Akt, we developed a novel approach by combining on-bead chemical cross-linking, co-immunoprecipitation (co-IP) and mass spectrometry. Akt antibody was immobilized on protein A/G beads by cross-linking with non-cleavable succinimidyl suberate (DSS). Subsequent treatment with dithiobissuccinimidylpropionate (DSP) after incubation of the immobilized Akt-antibody with cell lysates produced cleavable cross-linking between Akt and its binding partners. While the DSS cross-linking prevented the elution of antibody heavy and light chains, the DSP-cross-linking allowed Akt interacting proteins to remain on the beads during repeated wash steps. The co-IP products were eluted under a reducing condition that cleaves DSP cross-linking and subjected to SDS-PAGE/nanoLC-MS/MS analysis. This approach allowed us to identify minor Akt binding proteins including ERK, valosin-containing protein, alpha-actinin, 14-3-3, glycogen synthase kinase 3, cell cycling protein p38, and growth factor receptor-bound protein 10 from Neuro 2A cells using as little as 1.5 mg proteins. Several proteins were newly identified as potential Akt interacting proteins. This approach is being applied to investigate the effect of membrane modification by DHA or ethanol consumption on protein-protein interaction in Akt signaling cascades. As PS accumulation is an important aspect of DHA- or ethanol-mediated effects on Akt signaling, we continued to characterize the neuronal PS accumulation. We examined substrate preference and product inhibition in PS biosynthesis using microsomes and purified PSS2. To obtain sufficient amounts of PSS2 proteins for biochemical and structural characterization, we have successfully established a large-scale protein expression system using the mammalian HEK cell line. We have also established a reconstitution method to prepare functional PSS2-containing proteoliposomes by rapid dilution and ultrafiltration. We found that this integral protein uses DHA-containing PE as the best substrate for PS ssynthesis. We also found that microsomal PSS2 activity was inhibited by DHA-PS significantly less than oleic acid (OA)-containing PS (OA-PS), suggesting that the PS fatty acyl moiety affects the product inhibition. Reconstitution studies using functionally active purified PSS2 indicated that product inhibition in PS synthesis through the feedback mechanism depends on the PS molecular species and the composition of the membrane lipids with which PSS2 is reconstituted. To understand the underlying mechanism, mass spectrometric probing of the 3D-structure of PSS2 using PSS2-containing proteoliposomes is under progress. During this period, we have also developed a method to monitor PS synthesis and degradation simultaneously in living cells using stable isotope-labeled 13C3, 15N-serine. Newly synthesized PS and PE species were readily identified as they were detected at m/z values 4 and 3 daltons higher than their endogenous counterparts, respectively. Monitoring the time course of the production of labeled PS or PE in relation to endogenous species revealed the acyl chain preference of PS synthesis and degradation with a decreasing order of 22:6 n3 >18:1 n9 >20:4 n6-containining species in Neuro 2A cells. Furthermore, we found strong evidence that a significant portion of 18:0, 20:4- PE species, which is one of the major PE species present in Neuro 2A cells, was derived from deacylation / reacylation after the conversion of PS to PE by PS decarboxylation. Using this approach, we also found dose-dependent decreases in the rate of PS production from PE or PC as well as the overall PS level when Neuro 2A cells were treated chronically with ethanol for five weeks. These results were consistent with our previous findings of inhibited PS biosynthesis when tested in vitro using microsomes from ethanol-treated animals. To understand the role of DHA in neurodevelopment and function at the molecular level, we examined the protein composition of synaptic plasma membrane (SPM) in relation to the DHA status. The SPM proteins were obtained from DHA-adequate and deficient mouse brains by subcellular fractionation and analyzed by nanoLC- MS/MS after SDS PAGE and tryptic digestion. For quantification, the SPM proteins from DHA-adequate and deficient mice were differentially labeled with 16O/ 18O water, combined and analyzed by nanoLC-MS/MS. This strategy allowed us to detect more than 400 proteins from the SPM fraction including various receptors, kinases and transporter proteins. The analysis of the 18O/16O ratios revealed lower expression of several pre- and postsynaptic proteins involved in neurotransmission in the brains of DHA-deficient animals. Some of these include Synapsin 1, PSD-95, Syntaxin-1, Munc18-1, Glutamate receptors (GluR2, NR2), Dynamin-1 and Synaptic vesicle protein. The mass spectrometric results were validated by western blot analysis. Alteration of synaptic protein levels due to the DHA status suggests an important role of DHA in controlling synaptic protein expression and/or degradation. During this period, we have also extended our investigation of the role of DHA in neurodevelopment to the proliferation and differentiation of neural stem cells (NSCs) obtained from E14 rat embryos. When cultured with fibroblast growth factor (FGF)-containing medium, NSCs proliferated as neurospheres which were then dissociated and cultured repeatedly for several passages to enrich NSCs. Subsequently, NSCs were cultured in the medium without FGF to initiate differentiation, and supplemented with DHA to examine the effects of DHA on NSC differentiation. We found that DHA increases the percentage of Tuj1 (an immature neuronal marker)-positive cells, suggesting that DHA stimulates neuronal differentiation of NSCs. Cellular mechanisms for the observed effects of DHA on NSC differentiation are now under investigation.
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