Abstract
Heat-shock stress causes inactivation and aggregation of various cellular proteins which become further insoluble. Previous studies have shown that the interferon-induced p68 kinase activity was greatly reduced in extracts of heat-shocked HeLa cells, and that the loss of activity was due to a decreased solubility of the enzyme. Here we show that the p68 kinase which is normally evenly distributed in the cytoplasm, aggregates as a thick ring around the nucleus in heat-shocked cells. The 70-kDa constitutive heat-shock proteins are major insolubilized proteins during stress and we find them to colocalize with the p68 kinase after stress. Treatments of cells with drugs which disrupt the cytoskeleton, such as colcemid and cytochalasin E, do not hinder the enzyme insolubilization during heat-shock. On the contrary, heat-protectors such as glycerol and deuterium oxide (D2O) keep the p68 kinase under a soluble and active form during heat-shock stress. Similarly, an attenuation of the insolubilization of this enzyme is observed in cells rendered thermo-tolerant by a previous heat-shock, suggesting that heat-shock proteins may also contribute to the protection. During the recovery period at normal temperature after heat-shock, resolubilization occurs and most of the enzyme is again recovered under an active soluble form.
Highlights
Heat-shock stress causes inactivation and aggrega- nucleoproteins has been described (14), but this might be a tion of various cellular proteins which become further consequence of the overall structural changes which lead to insoluble
Localization of thep68Kinase in Heat-Shocked HeLa CellsWe have previously shown that, during a 44 "C heat-shock, most of the interferon-induced p68 kinase present in HeLa cells becomes concomitantly insoluble in nonionic detergents and inactive
We show that the p68 kinase becomes resistant to nonionic detergenetxtraction during heat-shock while 2-5A synthetase remains mostly soluble
Summary
Reagents-[y-'"PIATP (>5000 Ci/mmola)nd [:"S]methionine lead totheinactivation of proteinfunction. Such a heat denaturation may be responsible for the loss of Cells-Human HeLa cells were grown at 37 "C in Dulbecco's modprotein solubility which has been shown to occur during heat ified Eagle's medium (GIBCO) containing 10% newborn calf serum. Indirect ImmunofluorescenceAnalysis-Cells were grown on tissue culture chamber/slides (Lab-TEK, Miles) and fixed with acetone/ methanol/formaldehyde (1919:2, v/v) for 5 min a t -20 "C. They were incubated with specific antibodies for 60 min with biotinylated anti-mouse Ig for 60 min and with fluorescein-labeled streptavidin for 15 min (Amersham).
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