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 H202, 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 H,O, 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, Hz02, 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 kinaseactivated 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 nonphosphorylatable form of human HSP27 in Chinese hamster cells conferred resistance to actin fragmentation by oxidative stress generated by H,O,. 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.

Natural annual cycle of heat shock protein expression in land snails: desertversusMediterranean species ofSphincterochila

“…In the surf clam Donax variabilis, hyperoxia induced the expression of Hsp70, whereas sulfide-induced oxidative damage induced the expression of sHSPs (Joyner-Matos et al, 2006). Hsp70 and Hsp27 have potential roles against cellular damage induced by oxidative stress Giffard et al, 2004;Huot et al, 1995;Mehlen et al, 1995), Hsp70 probably by acting as a molecular chaperone, whereas the protective effects of sHSPs were related to Z. Arad and others Hsp level (% change from aestivation) Fig.4. The change in small HSP levels after arousal in the foot, kidney and hepatopancreas tissues of S. zonata and S. cariosa.…”

Essential Role of the NH2-terminal WD/EPF Motif in the Phosphorylation-activated Protective Function of Mammalian Hsp27

“…In unstressed cells, Hsp27 is mostly found unphosphorylated and assembled in 24-mers (17). Upon stress that activates the p38 kinase pathway and leads to Hsp27 phosphorylation, Hsp27 dissociates into dimers (9,12,17). Our results show that the pseudo-phosphorylated Hsp27EE mutant, an almost exclusive dimeric molecule, provides full thermoprotection, whereas the non-phosphorylatable Hsp27AA, found mostly as large 24-mers, provides very little protection, suggesting that phosphorylation-induced dissociation is an important step in the activation of the thermoprotective activity of Hsp27.…”

Protein phosphatase inhibitors and heat preconditioning prevent Hsp27 dephosphorylation, F‐actin disruption and deterioration of morphology in ATP‐depleted endothelial cells

“…On the other hand, the artificial maintenance of the cytosolic (soluble) pool of phosphorylated Hsp27 within ATP-depleted EC may by itself contribute to the microfilament stability. In support of the latter, phosphorylated Hsp27 only was shown to be involved in protection of F-actin under heat shock [14,15], oxidative stress [16,17], and treatment with cytochalasin D [14,18], whereas non-phosphorylated Hsp27 promotes the reduction of F-actin in cells [12,13]. The precise mechanism of the protective action of sodium orthovanadate on ATP-depleted EC is unclear.…”

“…The main in vivo functions of this protein are actin reorganization in response to cell-activating stimuli [12,13] and protection of F-actin under stresses [14][15][16][17][18]. In cells overexpressing Hsp27, the actin networks were more resistant to heating [14,15], oxidants [16,17] and cytochalasin D [14,18] that was associated with improved survival of the treated cells. The protective potential of human Hsp27 toward microfilaments is up-regulated by phosphorylation of its serine residues Ser 15, Ser 7s and Ser ~2 in a stressand mitogen-sensitive signaling pathway [15 18].…”