S-glutathionylation impairs signal transducer and activator of transcription 3 activation and signaling.
S-glutathionylation is a physiological, reversible protein modification of cysteine residues with glutathione in response to mild oxidative stress. Because the key cell growth regulator signal transducer and activator of transcription (STAT) 3 is particularly susceptible to redox regulation, we hypothesized that oxidative modification of cysteine residues of STAT3 by S-glutathionylation may occur. Herein, we show that the cysteine residues of STAT3 are modified by a thiol-alkylating agent and are the targets of S-glutathionylation. STAT3 protein thiol reactivity was reversibly attenuated with concomitant increase in the S-glutathionylation of STAT3 upon treatment of human HepG2 hepatoma cells with pyrrolidine dithiocarbamate, glutathione disulfide, or diamide. Under these conditions there was a marked reduction in IL-6-dependent STAT3 signaling, including decreased STAT3 tyrosine phosphorylation, loss in nuclear accumulation of STAT3, and impaired expression of target genes, such as fibrinogen-gamma. In a cell-free system, diamide induced glutathionylation of STAT3, which was decreased upon addition of glutaredoxin (GRX)-1, a deglutathionylation enzyme, or the reducing agent, dithiothreitol. Glutathionylated STAT3 was a poor Janus protein tyrosine kinase 2 substrate in vitro, and it exhibited low DNA-binding activity. Cellular GRX-1 activity was inhibited by diamide and pyrrolidine dithiocarbamate treatment; however, ectopic expression of GRX-1 was accompanied by a modest increase in phosphorylation, nuclear translocation, and DNA-binding ability of STAT3 in response to IL-6. These results are the first to show S-glutathionylation of STAT3, a modification that may exert regulatory function in STAT3 signaling.
Xie
Yi
Y
Laboratories of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
Kole
Sutapa
S
Precht
Patricia
P
Pazin
Michael J
MJ
Bernier
Michel
M
D000975:Antioxidants / Q000494:pharmacology; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D003959:Diamines / Q000494:pharmacology; D054477:Glutaredoxins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D053614:Janus Kinase 2 / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D011759:Pyrrolidines / Q000494:pharmacology; D050796:STAT3 Transcription Factor / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D013859:Thiocarbamates / Q000494:pharmacology; D014162:Transfection
D000975:Antioxidants; D003959:Diamines; C516005:GLRX protein, human; D054477:Glutaredoxins; D015850:Interleukin-6; D016877:Oxidants; D011759:Pyrrolidines; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D013859:Thiocarbamates; C020972:pyrrolidine dithiocarbamic acid; D053614:Janus Kinase 2; D005978:Glutathione; D003545:Cysteine