Actin, a 42 kD ATP- or ADP-associated cytosolic protein, exists in monomeric (G) and polymeric (F) forms. Actin filaments in cells support cellular structures as well as play contractile roles to facilitate cell growth, division, and migration. Under certain stimuli, such as an increase in Ca(II)/Mg(II) concentration, G-actin polymerizes into F-actin to exert physiological functions. It was found by Western Blot assay that actin is moderately glutathionylated under normal cellular conditions. The level of glutathionylation was expected to be greatly elevated when cells (A431 and HeLa) were placed under oxidative stress, such as when treated with hydrogen peroxide because the higher oxidative potential caused by hydrogen peroxide led to an increased level of GSSG that oxidized actin by glutathionylation. In contrast to our common beliefs, however, when cells were treated with EGF, upon which the cellular oxidative potential increased, actin was deglutathionylated. We studied this phenomenon in some detail In collaboration with a mass spectroscopy (MS) laboratory here at NIH, we determined the glutathionylation site of actin (C286). This site lies within actin-actin interaction domain so its glutathionylation may disrupt actin polymerization in vivo. In an in vitro assay using light scattering, we found that the glutathionylation of actin did slow down its polymerization rate by several folds. We speculate that after treatment with EGF, the readiness of cell growth requires actin to be polymerized so as to expand the cell surface, etc. Thus, actin is deglutathionylated. Since this deglutathionylation is against redox potential change inside a cell, it should be an active process, such as the participation of a glutathione transferase. We considered thioltransferase (TTase) to be a primary candidate. We demonstrated by MS that the glutathione moiety on actin was transferred to TTase under physiological conditions. We also demonstrated that the TTase inhibitor, Cd(II), could inhibit deglutathionylation of actin upon EGF treatment. Focus has now been placed on whether post-translational modifications, such as phosphorylation, can affect the activity of TTase.
