title: Nitric oxide activates intradomain disulfide bond formation in the kinase             loop of Akt1/PKBα after burn injury.
abstract: Severe burn injury is an acute inflammatory state with massive alterations             in gene expression and levels of growth factors, cytokines and free radicals.             During the catabolic processes, changes in insulin sensitivity and skeletal muscle             wasting (unintended loss of 5-15% of lean body mass) are observed clinically.             Here, we reveal a novel molecular mechanism of Akt1/protein kinase Bα (Akt1/PKBα)             regulated via cross-talking between dephosphorylation of Thr308 and S-nitrosylation             of Cys296 post severe burn injury, which were characterized using nano-LC interfaced             with tandem quadrupole time-of-fight mass spectrometry (Q-TOF)micro tandem mass             spectrometry in both in vitro and in vivo studies. For the in vitro studies, Akt1/PKBα             was S-nitrosylated with S-nitrosoglutathione and derivatized by three methods.             The derivatives were isolated by SDS-PAGE, trypsinized and analyzed by the tandem             MS. For the in vivo studies, Akt1/PKBα in muscle lysates from burned rats was             immunoprecipitated, derivatized with HPDP-Biotin and analyzed as above. The studies             demonstrated that the NO free radical reacts with the free thiol of Cys296 to             produce a Cys296-SNO intermediate which accelerates interaction with Cys310 to             form Cys296-Cys310 in the kinase loop. MS/MS sequence analysis indicated that             the dipeptide, linked via Cys296-Cys310, underwent dephosphorylation at Thr308.             These effects were not observed in lysates from sham animals. As a result of this             dual effect of burn injury, the loose conformation that is slightly stabilized             by the Lys297-Thr308 salt bridge may be replaced by a more rigid structure which             may block substrate access. Together with the findings of our previous report             concerning mild IRS-1 integrity changes post burn, it is reasonable to conclude             that the impaired Akt1/PKBα has a major impact on FOXO3 subcellular distribution             and activities.
authors: Surgical Service, Massachusetts General Hospital, Boston, MA,                     USA.
authors: X-M
authors: XM
authors: Lu
authors: R G
authors: RG
authors: Tompkins
authors: A J
authors: AJ
authors: Fischman
mesh_terms: D000818:Animals; D002056:Burns / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism; D015870:Gene Expression; D007249:Inflammation; D055504:Insulin Receptor Substrate Proteins / Q000378:metabolism; D007700:Kinetics; D018482:Muscle, Skeletal / Q000293:injuries / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D026422:S-Nitrosoglutathione / Q000737:chemistry / Q000494:pharmacology
chemical_list: D004220:Disulfides; C499515:FOXO3 protein, rat; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; C494920:Akt1 protein, rat; D051057:Proto-Oncogene Proteins c-akt; D003545:Cysteine