We investigated the regulation of Hsp27 phosphorylation by protein kinase C delta (PKC delta) during etoposide-induced apoptosis. The phosphorylation of Hsp27 at Ser78 was temporally correlated with the proteolytic activation of PKC delta during apoptosis. Hsp27 phosphorylation was dependent on the activity of PKC delta since treatment with rottlerin, a chemical inhibitor of PKC delta, or overexpression of a PKC delta dominant negative mutant abolished the phosphorylation. In addition, recombinant PKC delta phosphorylated Hsp27 at Ser78 in vitro. Moreover, caspase-3 was specifically activated following Hsp27 phosphorylation at Ser78. Pull-down assays using a phosphomimetic Hsp27 mutant revealed that binding between Hsp27 and cytochrome c was abolished by the phosphorylation. These results suggest that Hsp27 dissociates from cytochrome c following PKC delta-mediated phosphorylation at Ser78, which allows formation of the apoptosome and stimulates apoptotic progression. (C) 2012 Elsevier Inc. All rights reserved.