Transient activation of a distinct serine protein kinase is responsible for 27-kDa heat shock protein phosphorylation in mitogen-stimulated and heat-shocked cells.

We have investigated the phosphorylation of HSP27, a 27-kDa heat shock protein which is involved in cellular thermoresistance and is also an early target of phosphorylation during heat shock and cell stimulation by a variety of growth and differentiation factors. HSP27 is transiently phosphorylated after shifting Chinese hamster cells from their normal temperature of 37 to 44 degrees C. The phosphorylation correlated in time with the transient activation of specific HSP27 protein kinase activities. HSP27 kinase was also induced to maximal levels within 5-15 min following stimulation of quiescent cells with heat shock, serum, thrombin, or basic fibroblast growth factor. Extracts from quiescent cells stimulated by heat shock or serum were analyzed after sequential chromatography on cation exchange and hydroxylapatite columns. In both cases, a single and identical peak of HSP27 kinase activity was obtained, suggesting that the same protein kinase was induced. The HSP27 kinase efficiently phosphorylated recombinant Chinese hamster HSP27 or the synthetic peptide RALNRQLSSGV containing the major in vivo phosphorylation site of rodent HSP27. The kinase was inactive toward the ribosomal S6 protein, the peptide RALSSLRA from S6 protein, or the mutant HSP27 proteins with in vivo phosphorylation sites altered to glycine. The partially purified HSP27 kinase had no kinase C, kinase A, or S6 kinase activities; conversely, HSP27 was not a good substrate for these kinases. HSP27 kinase was rapidly inactivated in the presence of acid phosphatase, suggesting that its activity was regulated by phosphorylation. It is suggested that this heat shock- and serum-induced HSP27 kinase is a novel serine kinase which is linked to a major signal transduction cascade.