Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27.

We have found that the small stress protein, hsp27, exists in extracts of U251 MG human glioma cells in two forms: a large or aggregated form (L-hsp27, 300-400 kDa) and a small or dissociated form (S-hsp27, < 70 kDa), as indicated by centrifugation on sucrose density gradients. Dissociation of L-hsp27 to S-hsp27 was enhanced by incubation of cells with phorbol 12-myristate-13 acetate, interleukin-1 alpha, tumor necrosis factor alpha, or okadaic acid, all of which are known to enhance or mimic the effects of phosphorylation of hsp27 without stimulation of its synthesis. Exposure of cells to chemical stressors, namely, NaAsO2 and CdCl2, also enhanced the dissociation of L-hsp27. hsp27 that had been labeled with [32P]H3PO4 in U251 MG cells was detected mostly in fractions that contained S-hsp27, and the incorporation of radioactivity to S-hsp27 was enhanced under conditions that stimulated the dissociation of L-hsp27. L-hsp27 present in the (NH4)2SO4 fraction (0-50% saturation) of cell extracts were dissociated to 32P-labeled hsp27 when incubated in the presence of [gamma-32P]ATP and Mg2+. These results indicate that the molecular configuration of hsp27 in cells is determined in part by phosphorylation and dephosphorylation of this protein by protein kinase(s) and phosphatase(s) and, moreover, that the rapid dissociation of the aggregated form of hsp27 by phosphorylation might be involved in a cellular defense mechanism for protection against stress.