Osmotic stress inhibits p70/85 s6 kinase through activation of a protein phosphatase

While studying the stress regulation of p70/85 S6 kinase (S6K), we observed that anisomycin and UV light stimulated S6K activity, but that sorbitol in activated S6K, Pretreatment with hyperosmotic stress also prevented the act ivation of S6K by both 12-O-tetradecanoylphorbol-13-acetate and anisomycin, Comparison of sorbitol and rapamycin revealed that both agents inactivated S6K and caused dephosphorylation of Ser/Thr-Pro sites in the COOH terminus of S6K, including Thr(412) a residue essential to S6K regulation, as deter mined by phospho-specific antibodies. Rapamycin-resistant S6K truncation mu tants were similarly resistant to deactivation by sorbitol, Additionally, t he PHAS-1 mobility shift, which is sensitive to rapamycin, was also found t o be sensitive to osmotic stress. Experiments using the p38 inhibitor SB203 580 and dominant negative mutants involving both stress-activated protein k inase/c-Jun NH2-terminal kinase and p38 stress pathways indicated that thes e pathways are probably not involved in osmotic stress inhibition of S6K. E xamining the potential involvement of a phosphatase, we found that sodium p yrophosphate, sodium vanadate, cyclosporin A, tautomycin, and okadaic acid had no effect on osmotic stress inhibition of S6K, However, calyculin A pre vented both rapamycin- and sorbitol-mediated deactivation of S6K, Our resul ts suggest that osmotic stress and rapamycin act through a calyculin A-sens itive phosphatase to cause dephosphorylation and deactivation of S6K.