The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers. Small heat shock proteins (sHsps)1 are constitutively expressed in virtually all organisms and exhibit a monomeric molecular mass of 15-42 kDa (for a recent review see Ref. 1). Within the cell they can form oligomeric complexes of up to 1 MDa (2). Overexpression of different mammalian sHsps increases cellular thermoresistance to a significant degree (3, 4). sHsps can, furthermore, function in different, seemingly unrelated processes like RNA stabilization (5), interaction with the cytoskeleton (6, 7), or apoptosis (8, 9). In vitro sHsps act as molecular chaperones preventing unfolded proteins from irreversible aggregation (10 -12) and, in cooperation with other factors, e.g. Hsp70 and ATP, facilitating productive refolding of unfolded proteins (13,14).In mammalian cells certain sHsps, e.g. mouse Hsp25 or human Hsp27, form a converging element of the cellular stress response since they show both a stress-induced increase in expression and phosphorylation. Under heat shock conditions increased phosphorylation can be detected after several minutes while changes in expression are detected after several hours (15). The rapid stress-induced phosphorylation is the result of stimulation of the p38 MAP kinase cascade and subsequent activation of MAPKAP kinases 2 and 3 which directly phosphorylate mammalian sHsps (16, 17) at several distinct sites (18,19). Since sHsp phosphorylation and stress-induced expression show different kinetics, it is assumed that phosphorylation of the pre-existing constitutively expressed sHsps is a first phase of the stress response while the elevated expression at a time w...

Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.

The small stress protein Hsp27 is expressed during mammalian neural development. We have analyzed the role of this protein in immortalized rat olfactory neuroblasts. In the presence of dopamine a fraction of these cells differentiate into neurons while the remaining cells undergo apoptosis. We report here that the dopamine induced differentiation and apoptosis are associated with a transient and specific accumulation of Hsp27. Moreover, transfection experiments have shown that Hsp27 overexpression drastically decreases the fraction of cells undergoing apoptosis. In contrast, reduction of the endogenous level of Hsp27 led to abortion of differentiation and, therefore, drastically increased the number of apoptotic cells. Furthermore, in the normal cell population we show that Hsp27 accumulation takes place only in differentiating cells that were not undergoing apoptosis. We therefore conclude that Hsp27 may represent a key protein that controls the decision of olfactory precursor cells to undergo either differentiation or cell death.