S-Glutathionylation at Cys328 and Cys542 impairs STAT3 phosphorylation

ACS Chem Biol. 2014 Aug 15;9(8):1885-93. doi: 10.1021/cb500407d. Epub 2014 Jun 27.


STAT3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in cancers. Although many reports provide evidence that STAT3 is a direct target of oxidative stress, its redox regulation is poorly understood. Under oxidative conditions STAT3 activity can be modulated by S-glutathionylation, a reversible redox modification of cysteine residues. This suggests the possible cross-talk between phosphorylation and glutathionylation and points out that STAT3 is susceptible to redox regulation. Recently, we reported that decreasing the GSH content in different cell lines induces inhibition of STAT3 activity through the reversible oxidation of thiol groups. In the present work, we demonstrate that GSH/diamide treatment induces S-glutathionylation of STAT3 in the recombinant purified form. This effect was completely reversed by treatment with the reducing agent dithiothreitol, indicating that S-glutathionylation of STAT3 was related to formation of protein-mixed disulfides. Moreover, addition of the bulky negatively charged GSH moiety impairs JAK2-mediated STAT3 phosphorylation, very likely interfering with tyrosine accessibility and thus affecting protein structure and function. Mass mapping analysis identifies two glutathionylated cysteine residues, Cys328 and Cys542, within the DNA-binding domain and the linker domain, respectively. Site direct mutagenesis and in vitro kinase assay confirm the importance of both cysteine residues in the complex redox regulatory mechanism of STAT3. Cells expressing mutant were resistant in this regard. The data presented herein confirmed the occurrence of a redox-dependent regulation of STAT3, identified the more redox-sensitive cysteines within STAT3 structure, and may have important implications for development of new drugs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cysteine / metabolism*
  • Glutathione / metabolism*
  • HeLa Cells
  • Humans
  • Oxidation-Reduction
  • Phosphorylation
  • STAT3 Transcription Factor / chemistry
  • STAT3 Transcription Factor / metabolism*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • STAT3 Transcription Factor
  • STAT3 protein, human
  • Glutathione
  • Cysteine