Molecular and Functional Regulation of Akt2 by Oxidation

Phosphorylation is well-known to regulate intracellular signaling pathways; however, the functional contribution of oxidation in signaling regulation under physiologically-relevant conditions of growth factor/cytokine stimulation has not been fully demonstrated, despite the emerging knowledge of redox-sensitive protein targets. In the following study, chemoselective reagents for -SOH oxidation were applied in conjunction with mass spectrometry to identify the linker region cysteine, Cys124, in the Akt2 kinase as a target of PDGF-stimulated oxidation. In vitro studies shown here demonstrate selective inactivation of the Akt2 isoform following direct oxidation with H₂O₂ which is shown to occur through a reversible intramolecular disulfide between the linker region Cys124 and either of the activation site cysteines, Cys297 or Cys311. Endogenous accumulation of PDGF-derived ROS was shown to inactivate a fraction of Akt2 kinase selectively and thus suggested to allow for distinct regulation of Akt1 and Akt2 signaling in the NIH 3T3 cells. The role of NOX-derived and mitochondrial-based ROS in regulating Akt2 activity following PDGF stimulation is also elucidated. Finally, functional consequence of Cys124 oxidation on modulating Akt-regulated biological processes of glucose uptake, cell migration and cell cycle distribution were studied and shown to be regulated by the oxidation state of Akt2 kinase. Specifically, PDGF-induced oxidation inactivated Akt2 and led to reduced glucose uptake, increased migration and G₁-S transition in the Akt2 KO MEFs that were transduced with the WT Akt2 relative to the Cys124Ser Akt2 mutant. In summary, the mechanistic and functional studies of Akt2 oxidation presented here underscore the role of posttranslational oxidation in regulating molecular and functional outcomes in an isoform-selective manner under physiological settings.

Grantor dc:publisher

Wake Forest University

Year dc:date.issued

2011

Author dc:creator

• Wani, Revati

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/10339/36431

OAI identifier oai:identifier

oai:wakespace.lib.wfu.edu:10339/36431