PHOSPHORYLATION AND SUPRAMOLECULAR ORGANIZATION OF MURINE SMALL HEAT-SHOCK PROTEIN HSP25 ABOLISH ITS ACTIN POLYMERIZATION-INHIBITING ACTIVITY

Characteristic features of mammalian small heat shock proteins are the ir rapid phosphorylation in response to stress and mitogenic signals a nd their ability to form multimeric particles of 200-700 kDa and large aggregates up to 5000 kDa. Recently, a chaperoning function and an ac tin polymerization-inhibiting activity were demonstrated for the recom binant murine and turkey small heat shock protein, respectively. In th is paper, we demonstrate that the actin polymerization-inhibiting acti vity of the murine small heat shock protein HSP25 is dependent on the degree of its phosphorylation and structural organization. Non-phospho rylated and phosphorylated HSP25 monomers, as well as non-phosphorylat ed multimeric HSP25 particles, were isolated from Ehrlich ascites tumo r cells by ammonium sulfate precipitation, column chromatography, and ultracentrifugation and tested for their actin polymerization-inhibiti ng activity. Fluorescence spectroscopy and electron microscopy were us ed to monitor actin polymerization. Non-phosphorylated HSP25 monomers were active in inhibiting actin polymerization with about 90% inhibiti on at a 1:1 ratio of actin to HSP25, while phosphorylated HSP25 monome rs and non-phosphorylated multimeric HSP25 particles were inactive. Fu rthermore, we present electron microscopic data on the structure of HS P25 particles.