Dephosphorylation of the small heat shock protein Hsp27 in vivo by protein phosphatase 2A.

The phosphorylation of the Hsp27 complex is rapidly altered in MRC-5 cells when they are exposed to mitogens, cytokines, stress, or serine/threonine protein phosphatase inhibitors. Here we performed experiments to identify which cellular protein phosphatase (PP1, PP2A, or PP2B) is responsible for the in vivo phosphorylation/dephosphorylation of Hsp27. In their purified forms, PP2A dephosphorylates Hsp27 more effectively than PP2B, whereas PP1 is weakly active. Measurements of enzyme activity of lysates derived from inhibitor-treated cells indicated that Hsp27 phosphatase activity is equally sensitive to okadaic acid (PPI/PP2A inhibitor) and cyclosporin (PP2B inhibitor) and that both okadaic acid and cyclosporin treatment inhibited Hsp27 phosphatase activity additively. Together the in vitro data suggest that both PP2A and PP2B can dephosphorylate Hsp27. However, the phosphorylation of Hsp27 in vivo is only affected when cells are treated with PP1 and PP2A inhibitors (okadaic acid, calyculin A) or cantharidin (PP2A inhibitor), but not the PP2B inhibitor, cyclosporin A, suggesting PP2A to be the main enzyme dephosphorylating Hsp27 in the cells. Purification and immunoblotting of Hsp27 phosphatase from MRC-5 cells also suggest it to be PP2A and not PP1 or PP2B. The ability of PP2A to dephosphorylate Hsp27 is shown to be regulated by the phosphorylation state of PP2A itself.