Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells

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Abstract
The small molecular weight heat shock protein HSP27 was recently shown to confer a stable thermoresistant phenotype when expressed constitutively in mammalian cells after structural gene transfection. These results suggested that HSP27 may also play an important role in the development of thermotolerance, the transient ability to survive otherwise lethal heat exposure after a mild heat shock. In Chinese hamster O23 cells increased thermoresistance is first detected at 2 h after a triggering treatment of 20 min at 44°C, attains a maximum at 5 hours, and decays thereafter with a half‐life of 10 h. We found that the development and decay of transient thermotolerance cannot be solely explained on the basis of changes in the cellular concentration of HSP27. The cellular HSP27 concentration is not increased appreciably at 2 h after heat shock and attains a maximum at 14 h. Similar results were obtained in the case of another heat shock protein, HSP70. HSP70 follows slightly faster kinetics of accumulation (peaks at 10 h) and decays much more rapidly (t1/2 = 4h) than HSP27 (t1/2 = 13h). HSP27 has 3 isoelectric variants A, B, and C of which B and C are phosphorylated. In cells maintained at normal temperature, HSP27A represents more than 90% of all HSP27. Shifting the cell culture temperature from 37 to 44°C induces the incorporation of 32P into the more acidic B and C forms, a process that occurs very rapidly since the reduction in the concentration of the A form and a corresponding increase in the level of B and C is detectable by immunoblot analysis within 2.5 min at 44°C. Analyses performed at various times during development and decay of transient thermo‐tolerance revealed a close relationship between the effect of heat shock on HSP27 phosphorylation and cell ability to survive. For example, fully thermotolerant cells (5 h post‐induction) are refractory to induction of HSP27 phosphorylation by a 20‐min heat shock. The induction of HSP27 phosphorylation was also studied in a family of clonal cell lines of O23 cells that are thermoresistant as a result of the constitutive expression of a transfected human HSP27 gene. In these thermore‐sistant cells, phosphorylation of the endogenous hamster HSP27 is induced to a level comparable to that found in the thermosensitive parental cells. However, phosphorylation of the exogencus human protein, which represents more than 80% of total HSP27 in these cells, was much less induced. Although different mechanisms are involved in the maintenance of a high level of unphosphorylated HSP27 in induced‐thermotolerant and thermoresistant transfectant O23 cells, our results suggest that the degree of HSP27 phosphorylation could be a determinant of the ability of cells to survive hyperthermia.