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1.  Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking, Co-Immunoprecipitation and Mass Spectrometry 
PLoS ONE  2013;8(4):e61430.
Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners.
doi:10.1371/journal.pone.0061430
PMCID: PMC3629208  PMID: 23613850
2.  Effects of Ethanol on Conformational Changes of Akt Studied by Chemical Cross-Linking, Mass Spectrometry, and 18O Labeling 
ACS chemical biology  2011;7(2):387-394.
Although PI3K/Akt signaling that regulates neuronal survival has been implicated in the deleterious effects of ethanol on the central nervous system, underlying molecular mechanisms have not been fully elucidated. Akt–membrane interaction is a prerequisite step for Akt activation since it induces interdomain conformational changes to an open conformer that allows Akt phosphorylation by upstream kinases. In this study, we investigated the effect of ethanol on Akt activation by quantitatively probing Akt conformation using chemical cross-linking, 18O labeling. and mass spectrometry. We found that ethanol at pharmacologically relevant concentrations (20 or 170 mM) directly interacts with Akt and alters the local pleckstrin homology domain configuration near the PIP3-binding site. We also found that ethanol significantly impairs subsequent membrane-induced interdomain conformational changes needed for Akt activation. The observed alteration of Akt conformation caused by ethanol during the activation sequence provides a new molecular basis for the effects of ethanol on Akt signaling. The in vitro conformation-based approach employed in this study should also be useful in probing the molecular mechanisms for the action of ethanol or drugs on other signaling proteins, particularly for those undergoing dramatic conformational change during activation processes such as members of AGC kinase super family.
doi:10.1021/cb2003237
PMCID: PMC3475205  PMID: 22129086
3.  Effects of docosahexaenoic acid on mouse brain synaptic plasma membrane proteome analyzed by mass spectrometry and 16O/18O labeling 
Journal of proteome research  2011;10(12):5472-5480.
Docosahexenoic acid (DHA, 22:6n-3) plays an important role in development of proper brain function in mammals. We have previously reported that DHA promotes synaptogenesis and synaptic function in hippocampal neurons while DHA-depletion in the brain due to n-3 fatty acid deficiency produces opposite effects. To gain insight into underlying molecular mechanisms, we investigated whether the brain DHA status affects the synaptic plasma membrane (SPM) proteome by using nanoLC/ESI-MS/MS and 16O/18O labeling. The DHA level in mouse brains was lowered by dietary depletion of n-3 fatty acids, and SPM was prepared by differential centrifugation followed by osmotic shock. SPM proteins from DHA-adequate and depleted brains were analyzed by nanoLC/ESI-MS/MS after SDS-PAGE, in-gel digestion and differential O18/O16 labeling. This strategy allowed comparative quantitation of more than 200 distinct membrane or membrane-associated proteins from DHA-adequate or depleted brains. We found that 18 pre- and postsynaptic proteins that are relevant to synaptic physiology were significantly down-regulated in DHA-depleted mouse brains. The protein network analysis suggests involvement of CREB and caspase-3 pathways in the DHA-dependent modulation of synaptic proteome. Reduction of specific synaptic proteins due to brain DHA-depletion may be an important mechanism for the suboptimal brain function associated with n-3 fatty acid deficiency.
doi:10.1021/pr2007285
PMCID: PMC3458425  PMID: 22003853
Synaptic plasma membrane (SPM); synaptic proteins; docosahexaenoic acid (DHA); 18O labeling; nano-LC/ESI-MS/MS; brain
4.  N-Docosahexaenoylethanolamide promotes development of hippocampal neurons 
The Biochemical journal  2011;435(2):327-336.
DHA (docosahexaenoic acid, C22:6,n−3) has been shown to promote neurite growth and synaptogenesis in embryonic hippocampal neurons, supporting the importance of DHA known for hippocampus-related learning and memory function. In the present study, we demonstrate that DHA metabolism to DEA (N-docosahexaenoylethanolamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. We found that a fatty acid amide hydrolase inhibitor URB597 potentiates DHA-induced neurite growth, synaptogenesis and synaptic protein expression. Active metabolism of DHA to DEA was observed in embryonic day 18 hippocampal neuronal cultures, which was increased further by URB597. Synthetic DEA promoted hippocampal neurite growth and synaptogenesis at substantially lower concentrations in comparison with DHA. DEA-treated neurons increased the expression of synapsins and glutamate receptor subunits and exhibited enhanced glutamatergic synaptic activity, as was the case for DHA. The DEA level in mouse fetal hippocampi was altered according to the maternal dietary supply of n−3 fatty acids, suggesting that DEA formation is a relevant in vivo process responding to the DHA status. In conclusion, DHA metabolism to DEA is a significant biochemical mechanism for neurite growth, synaptogenesis and synaptic protein expression, leading to enhanced glutamatergic synaptic function. The novel DEA-dependent mechanism offers a new molecular insight into hippocampal neurodevelopment and function.
doi:10.1042/BJ20102118
PMCID: PMC3169088  PMID: 21281269
docosahexaenoic acid (DHA); N-docosahexaenoylethanolamide (DEA); hippocampus; neurite growth; neuron; synaptogenesis
5.  Phosphatidylserine is a critical modulator for Akt activation 
The Journal of Cell Biology  2011;192(6):979-992.
Association of Akt with phosphatidylserine enhances binding to PIP3, inducing conformational changes in Akt that promote its phosphorylation-mediated activation.
Akt activation relies on the binding of Akt to phosphatidylinositol-3,4,5-trisphosphate (PIP3) in the membrane. Here, we demonstrate that Akt activation requires not only PIP3 but also membrane phosphatidylserine (PS). The extent of insulin-like growth factor–induced Akt activation and downstream signaling as well as cell survival under serum starvation conditions positively correlates with plasma membrane PS levels in living cells. PS promotes Akt-PIP3 binding, participates in PIP3-induced Akt interdomain conformational changes for T308 phosphorylation, and causes an open conformation that allows for S473 phosphorylation by mTORC2. PS interacts with specific residues in the pleckstrin homology (PH) and regulatory (RD) domains of Akt. Disruption of PS–Akt interaction by mutation impairs Akt signaling and increases susceptibility to cell death. These data identify a critical function of PS for Akt activation and cell survival, particularly in conditions with limited PIP3 availability. The novel molecular interaction mechanism for Akt activation suggests potential new targets for controlling Akt-dependent cell survival and proliferation.
doi:10.1083/jcb.201005100
PMCID: PMC3063130  PMID: 21402788
6.  Probing Akt-inhibitor interaction by chemical cross-linking and mass spectrometry 
The serine/threonine kinase Akt is a critical enzyme that regulates cell survival. As high Akt activity has been shown to contribute to the pathogenesis of various human malignancies, inhibition of Akt activation is a promising therapeutic strategy for cancers. We have previously demonstrated that changes in Akt interdomain arrangements from a closed to open conformation occur upon Akt-membrane interaction, which in turn allows Akt phosphorylation/activation. In the present study, we demonstrate a novel strategy to discern mechanisms for Akt inhibition based on Akt conformational changes using chemical cross-linking and 18O labeling mass spectrometry. By quantitative comparison of two interdomain cross-linked peptides which represent the proximity of the domains involved, we found that the binding of Akt to an inhibitor (PI analog) caused the open interdomain conformation where the PH and regulatory domains moved away from the kinase domain, even before interacting with membranes, subsequently preventing translocation of Akt to the plasma membrane. In contrast, the interdomain conformation remained unchanged after incubating with another type of inhibitor (peptide TLC1). Subsequent interaction with unilamellar vesicles suggested that TCL1 impaired particularly the opening of the PH domain for exposing T308 for phosphorylation at the plasma membrane. This novel approach based on the conformation-based molecular interaction mechanism should be potentially useful for drug discovery efforts for specific Akt inhibitors or anti-tumor agents.
doi:10.1016/j.jasms.2009.04.004
PMCID: PMC2750033  PMID: 19446470
Akt(PKB); conformation; mass spectrometry; inhibitor; chemical cross-linking
7.  Conformational changes in Akt1 activation probed by amide hydrogen/deuterium exchange and nano-electrospray ionization mass spectrometry† 
Amide hydrogen exchange coupled to nano-electrospray ionization mass spectrometry (nano-ESI-MS) has been used to identify and characterize localized conformational changes of Akt upon activation. Active or inactive Akt was incubated in D2O buffer, digested with pepsin, and analyzed by nano-ESI-MS to determine the deuterium incorporation. The hydrogen/deuterium (H/D) exchange profiles revealed that Akt undergoes considerable conformational changes in the core structures of all three individual domains after activation. In the PH domain, four β-strand (β1, β2 β5 and β6) regions containing membrane-binding residues displayed higher solvent accessibility in the inactive state, suggesting that the PH domain is readily available for the binding to the plasma membrane for activation. In contrast, these β-strands became less exposed or more folded in the active form, which is favored for the dissociation of Akt from the membrane. The beginning α-helix J region and the C-terminal locus (T450-470P) of the regulatory domain showed less folded structures that probably enable substrate entry. Our data also revealed detailed conformational changes of Akt in the kinase domain due to activation, some of which may be attributed to the interaction of the basic residues with phosphorylation sites. Our H/D exchange results indicating the conformational status of Akt at different activation states provided new insight for the regulation of this critical protein involved in cell survival.
doi:10.1002/rcm.4085
PMCID: PMC2752348  PMID: 19462409
8.  Multiple Pathways Involved in the Biosynthesis of Anandamide 
Neuropharmacology  2007;54(1):1-7.
Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by α,β-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5′ phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD−/− mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10 min) and the latter at longer incubations (60 min). In macrophages, the endotoxin-induced synthesis of anandamide proceeds uniquely through the phospholipase C/phosphatase pathway.
doi:10.1016/j.neuropharm.2007.05.020
PMCID: PMC2219543  PMID: 17631919

Results 1-8 (8)