Abstract
Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.
Highlights
The role of the individual phosphorylation sites was evaluated by determining the protective activity of Hsp27 double mutants having one phosphorylation changed to Ala and the other to Glu (Hsp27-AE and Hsp27-EA)
Hsp27 is expressed in cells as homopolymers of sizes ranging from 2- to 24-mers depending on the phosphorylation state of the protein
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
Summary
Overexpression of wild type or pseudo-phosphorylated Hsp but not a non-phosphorylatable mutant induced an enhanced accumulation of stress fibers in response to growth factors or mild cytotoxic stress (6, 26 –29), an effect that in the case of oxidative stress or cisplatin treatment can lead to actin filament-dependent cell blebbing [30] In other contexts, this activity results into a phosphorylation-dependent protection of the actin filaments against severe stress such as cell treatment with the actin polymerization inhibitor cytochalasin-D. Hsp with Ser replaced by Glu to mimic phosphorylation or Hsp in which Ser is replaced by Ala so that only Ser is phosphorylatable, are expressed in cells as high molecular oligomers only slightly smaller that the wild type protein in the presence or absence of stress, suggesting that the major role of this phosphorylation site is not to regulate oligomerization [17].
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