Characterization of 45‐kDa/54‐kDa HSP27 Kinase, a Stress‐Sensitive Kinase Which may Activate the Phosphorylation‐Dependent Protective Function of Mammalian 27‐kDa Heat‐shock Protein HSP27

Abstract

Heat‐shock protein 27 (HSP27) is a major target of phosphorylation upon cell stimulation with a variety of agents and has been suggested to have a phosphorylation‐regulated function at the level of actin filaments. Here we investigated comparatively the mechanisms of HSP27 phosphorylation by oxidative stresses, exposures to tumor necrosis factor (TNF), heat shock and growth factors. Extracts of Chinese hamster or human cells exposed to H2O2, xanthine/xanthine oxidase, menadione or TNF contained up to 15‐fold more HSP27 kinase activity than comparable extracts obtained from control cells. Induction of HSP27 kinase activity by TNF or H2O2 was completely inhibited by first treating the cells with the antioxidant N‐acetyl‐l‐cysteine, suggesting that generation of reactive oxygen metabolites was the key triggering element of this induction. In contrast, prior treatment with acetylcysteine had no or little effect on the induction by thrombin, serum and heat shock. The kinase activity in extracts of cells stimulated by healt shock, H2O2, sodium arsenite, TNF or growth factors was identified by in‐gel renaturation and purified ≈8000‐fold by sequential chromatography. In all cases, the induced kinase activity was entirely associated with two polypeptides of 45 kDa and 54 kDa, identified as mitogen‐activated‐protein kinase‐activated protein (MAPKAP) kinase‐2 based on its reactivation in vitro by 42/44‐kDa MAP kinases, its antigenic properties and its substrate specificity. The 45/54‐kDa HSP27 kinase may play an important role in the cell response to oxidative stress. Overexpression of the wild‐type HSP27 but not of a non‐phosphorylatable form of human HSP27 in Chinese hamster cells conferred resistance to actin fragmentation by oxidative stress generated by H2O2. It is concluded that activation of the 45/54‐kDa HSP27 kinase is a coimmon mechanism of HSP27 phosphorylation to which converge both oxyradical‐dependent and oxyradical‐independent pathways and which may participate in a homeostatic response to stress at the level of actin microfilament. Copyright © 1995, Wiley Blackwell. All rights reserved