Nitric oxide activates intradomain disulfide bond formation in the kinase loop of Akt1/PKBα after burn injury

Lu,X.-M.; Tompkins,R.  G.; Fischman,A. J.

doi:10.3892/ijmm.2013.1241

Nitric oxide activates intradomain disulfide bond formation in the kinase loop of Akt1/PKBα after burn injury

Authors:
- X.-M. Lu
- R. G. Tompkins
- A. J. Fischman
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Affiliations: Surgical Service, Massachusetts General Hospital, Boston, MA, USA, Shriners Hospital for Children, Boston, MA 02114, USA
Published online on: January 11, 2013 https://doi.org/10.3892/ijmm.2013.1241
Pages: 740-750
Copyright: © Lu et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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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.

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