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...

HL-60 cells are an attractive model for studies of human myeloid cell differentiation. Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27. Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK). Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2 (MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation. As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation. We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology. These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation. However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.