Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress tumor necrosis factor alpha by phosphorylation

The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and pro tect cells from heat shock and oxidative stress when overexpressed. In addi tion, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at tw o or three serine residues in response to various extracellular stresses. H ere we analyze the effect of sHsp phosphorylation on its quaternary structu re, chaperone function, and protection against oxidative stress. We show th at in vitro phosphorylation of recombinant sHsp as well as molecular mimicr y 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 molec ular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were anal yzed for their ability to confer resistance against oxidative stress when o verexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers res istance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are n ecessary for chaperone action and resistance against oxidative stress where as phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.