Phosphorylation is not essential for protection of L929 cells by Hsp25 against H2O2-mediated disruption actin cytoskeleton, a protection which appears related to the redox change mediated by Hsp25.

Small stress proteins protect against the cytotoxicity mediated by oxidative stress. The relationship between Hsp25 expression and the integrity of the actin network was studied in H2O2-treated murine L929 fibrosarcoma cells overexpressing endogenous wild-type (wt-) or non-phosphorylatable mutant (mt-) Hsp25. We show here that both proteins prevented actin network disruption induced by a 1 h treatment with 400 microM H2O2. In contrast, SB203580, a p38 MAPkinase inhibitor which suppresses Hsp25 phosphorylation, abolished the protective activity conferred by both wt- and mt-Hsp25. Hence, phosphorylation does not appear essential for Hsp25 protective activity against H2O2-induced actin disruption, and SB203580-sensitive events other than Hsp25 phosphorylation may be important for actin network regulation. Since, in L929 cells, wt- or mt-Hsp25 expression modulates intracellular glutathione levels, analyses were performed which revealed a direct correlation between glutathione and the integrity of the actin network. Moreover, laser scanning confocal immunofluorescences revealed that only a small fraction of wt- or mt-Hsp25 colocalized with actin microfilaments. Taken together, our results suggest that, in L929 cells, the protection against actin network disruption is probably a consequence of the redox change mediated by Hsp25 rather than a direct effect of this stress protein towards actin.