Importance of the ATP-binding domain and nucleolar localization domain of HSP72 in the protection of nuclear proteins against heat-induced aggregation.

Heat treatment of cells results in an increased protein content of nuclei when isolated after the heat treatment (intranuclear protein aggregation). In a previous study, the role of HSP72 was investigated using Rat-1 fibroblasts stably transfected with the human HSP72 gene. It was observed that the expression of human HSP72 in Rat-1 cells (HR cells) confers heat resistance. The initial heat-induced increase in the nuclear protein content was lower in HR cells as compared to the parent Rat-1 cells. In the present communication, the effects of overexpression of intact or mutant human HSP72 in Rat-1 cells on heat-induced increase in intranuclear protein aggregation and their relationship to cells' thermal sensitivity were examined. Four closely related cell lines were used for this study: Rat-1 cells which constitutively expressed the intact human HSP72, or mutant human HSP72 either missing its ATP-binding domain or nucleolar localization domain, and wild type Rat-1 cells. Our results show that expression of the intact human HSP72 or mutant human HSP72 missing its ATP-binding domain confers heat resistance and protects cells against heat-induced intranuclear protein aggregation. On the other hand, cells expressing mutant human HSP72 missing its nucleolar localization domain demonstrated heat shock responses similar to control Rat-1 cells.