Phosphorylation at tyrosine-524 influences nuclear accumulation of HSP72 with heat stress

Nuclear accumulation of heat shock protein (HSP) 72 occurs after cardiac is chemia. This nuclear accumulation of HSP72 with stress occurs in other tiss ues and species. We postulated that nuclear accumulation of HSP72 was impor tant for the protective effect of HSP72 and that phosphorylation of a singl e tyrosine (Y-524) regulated nuclear accumulation of HSP72. Western blots o f immunoprecipitated HSP72 from Cos-1 cells demonstrated that tyrosine beco mes phosphorylated after heat shock. Treatment with the tyrosine kinase inh ibitor geldanamycin blocked nuclear accumulation of HSP72 with heat shock. Two epitope-tagged constructs were made: M17 converting Y-524 to aspartic a cid (pseudophosphorylation) and M18 converting Y-524 to phenylalanine. When transfected into Cos-1 cells, M17 accumulates more rapidly and M18 less ra pidly than wild-type (WT) HSP72 in the nucleus following heat shock. Cells expressing M18 had less viability after heat shock at 43.5 degrees C than o ther constructs. After heat shock at 45 degrees C, cells expressing M17 had superior survival compared with WT and M18. These data suggest that phosph orylation at Y-524 facilitates nuclear accumulation of HSP72 following heat stress, and substitution of aspartic acid at Y-524 enhances resistance to heat-shock injury.