Reversible glutathionylation regulates actin polymerization in A431 cells.

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Growth factor stimulation induces reactive oxygen species (ROS), leading to protein tyrosine phosphorylation and glutathionylation in mammalian cells.
• Actin, a key cytoskeletal protein, undergoes post-translational modifications influencing its polymerization and cellular functions.

Purpose of the Study:

• To investigate the role of epidermal growth factor (EGF)-induced protein glutathionylation in A431 cells.
• To elucidate the mechanism regulating actin polymerization via reversible glutathionylation.

Main Methods:

• Mass spectrometry to identify glutathionylation sites on actin.
• In vivo and in vitro assays to measure actin polymerization rates and glutaredoxin activity.
• Inhibition studies using cadmium (Cd(II)) to assess glutaredoxin's role in deglutathionylation.

Main Results:

• Epidermal growth factor (EGF) stimulation paradoxically led to deglutathionylation of actin at Cysteine-374.
• Actin deglutathionylation increased G-actin polymerization rate by approximately sixfold.
• In vivo studies showed increased F-actin content and peripheral localization after EGF treatment, suggesting growth factor-mediated regulation.
• Glutaredoxin (thioltransferase) was identified as the likely catalyst for actin deglutathionylation, supported by inhibition studies and direct GSH transfer assays.

Conclusions:

• Actin polymerization is dynamically regulated by reversible glutathionylation of its penultimate cysteine residue.
• This novel glutathionylation mechanism is mediated by growth factor stimulation and likely catalyzed by glutaredoxin.
• The findings reveal a new layer of physiological control over the actin cytoskeleton.