Isoform-specific regulation of Akt by PDGF-induced reactive oxygen species

Abstract

Isoform-specific signaling of Akt, a major signaling hub and a prominent therapeutic target, remained poorly defined until recently. Subcellular distribution, tissue-specific expression, substrate specificity, and posttranslational modifications are believed to underlie isoform-specific signaling of Akt. The studies reported here show inhibition of Akt2 activity under physiologically relevant conditions of oxidation created by PDGF-induced reactive oxygen species. Combined MS and functional assays identified Cys124 located in the linker region between the N-terminal pleckstrin homology domain and the catalytic kinase domain as one of the unique regulatory redox sites in Akt2 with functional consequence on PDGF-stimulated glucose uptake. A model is proposed describing the consequence of increased endogenous oxidation induced by extracellular cues such as PDGF on Akt2 activity.disulfide | receptor tyrosine kinase | DCF | DCP-Bio1 P KB/Akt is a major signaling hub between cytokine, growth factor, and integrin signaling pathways of consequence to many biological processes. Energy storage, protein synthesis, cell survival and growth, cell cycle progression, and cell death are differentially regulated by the three known isoforms of Akt kinase: Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ (1). Although the molecular features underlying isoform-specific functional predominance are largely unknown, hypotheses to explain Akt isoform specificity include selective interactions with substrates and/ or binding partners, tissue specificity, subcellular location, and temporal changes in activation profiles of Akt isoforms (2). Posttranslational modifications may contribute to this isoform-specific signaling, but reports of modifications are lacking.Reactive oxygen species (ROS) are integral to cytokine and growth factor signaling; ROS generation in response to these extracellular cues is well documented (3). Earlier reports, in particular from Sundaresan et al. (4), showed ROS generation in response to PDGF stimulation of vascular smooth muscle cells and suggested that H 2 O 2 can relay redox signals to regulate physiological signaling in response to growth factors. More recently, it has been shown that PrxI phosphorylation at Y194 by Src family tyrosine kinases inhibited PrxI and resulted in H 2 O 2 accumulation at the cellular membrane where receptor tyrosine kinase activation occurs (5). Unanswered questions include which proteins are oxidized by receptor tyrosine kinase-induced ROS, which specific cysteine site(s) undergo oxidation, and the consequence of oxidation on activity of these proteins and propagation of receptor tyrosine kinase signaling. Recently, the synthesis of several dimedone-based chemoselective reagents capable of specific labeling of sulfenic acid oxidized proteins was reported; these reagents allow for specific enrichment and identification of oxidized proteins (6-10). In this study, we used a biotin-tagged 1,3-cyclohexadione derivative, DCP-Bio1 (Fig. 1A), to investigate isoform-specific effects of PDGF-induced ...

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

“…PDGF stimulation of fibroblasts induced H 2 O 2 production, which led to isoform-specific regulation of Akt2 (28). A cysteine (Cys-124) positioned in the linker region connecting the PH (pleckstrin homology) domain to the kinase domain was found to be susceptible to sulfenylation.…”

Iron overloaded polarizes macrophage to proinflammation phenotype through ROS/acetyl‐p53 pathway

“…Levels of ROS are mainly regulated by nonenzymatic antioxidants (eg, α‐tocopherol and glutathione) and antioxidant enzymes (eg, superoxide dismutase and catalase) 5. ROS physiologically regulates the synthesize of some kinds of proteins, such as phosphatase and tensin homologue, epidermal growth factor receptor, protein kinase B, and histone deacetylases,6, 7, 8, 9 and oxidizes DNA to promote transcription 4…”

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

“…Advances in the development of chemical probes have revolutionized the isolation and identification of other proteins that form a Cys sulfenic acid within cells exposed to a variety of stress conditions (6,(33)(34)(35). The reactivity of these probes is, however, not high enough to capture the transient Cys-S P OH intermediate during the Prx reaction cycle.…”