HSP27 multimerization mediated by phosphorylation-sensitive intermolecularinteractions at the amino terminus

Distinct biochemical activities have been reported for small and large mole cular complexes of heat shock protein 27 (HSP27), respectively. Using glyce rol gradient ultracentrifugation and chemical cross-linking, we show here t hat Chinese hamster HSP27 is expressed in cells as homotypic multimers rang ing from dimers up to 700-kDa oligomers. Treatments with arsenite, which in duces phosphorylation on Ser(15) and Ser(90) provoked a major change in the size distribution of the complexes that shifted from oligomers to dimers, Ser(90) phosphorylation was sufficient and necessary for causing this chang e in structure. Dimer formation was severely inhibited by replacing Ser(90) with Ala(90) but not by replacing Ser(15) with Ala(15). Using the yeast tw o-hybrid system, two domains were identified that were responsible for HSP2 7 intermolecular interactions. One domain was insensitive to phosphorylatio n and corresponded to the C-terminal alpha-crystallin domain. The other dom ain was sensitive to serine 90 phosphorylation and was located in the N-ter minal region of the protein. Fusion of this N-terminal domain to firefly lu ciferase conferred luciferase with the capacity to form multimers that diss ociated into monomers upon phosphorylation. A deletion within this domain o f residues Arg(5)-Tyr(23), which contains a WDPF motif found in most protei ns of the small heat shock protein family, yielded a protein that forms onl y phosphorylation-insensitive dimers. We propose that HSP27 forms stable di mers through the alpha-crystallin domain. These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensiti ve N-terminal domain.