Cells respond to stresses such as heat, x-irradiation etc., by enhancing the transcription of various genes in order to protect themselves, or repair the damage ensuing from such insults. Transcription factors which become phosphorylated are major responders to such stresses. Changes in the phosphorylation of transcription factors are regulated by protein kinases that transmit the signals coming from the environment. Understanding such changes should lead to the manipulation of expression of the genes they turn on and off. It is, therefore, crucial to understand the mechanism by which the signals are transmitted from membrane to the nucleus. The most widely studied signaling pathway involved in growth factor stimulation and response to various stresses is that involving the MAP kinases (ERK1 and 2). MAP Kinases have been shown to activate transcription factors that are involved in the expression of the immediate early genes. Recently MAP kinases have been shown to be activated by heat shock. The possibility that MAP kinases are involved in HSF-1 phosphorylation therefore, is permissible and strengthened by a close correlation between activation of MAP kinases and phosphorylation of HSF-1. Using both genetic and pharmacological approaches, we show that MAP kinase activation by heat shock is independent of ras and raf-1 activation. We also show that ras exert a negative regulatory effect on the heat shock response through ERK1. This is because the overexpression of the inhibitory mutant of ERK1 (ERK1KR) increases the expression of hsp70-luciferase by 30 fold. Furthermore, pretreatment of cells with sodium vanadate, which increased ERK1 kinase activity, increases the incorporation of 32P into HSF-1. These data suggest that MAp kinases that are activated in response to growth factor stimulation may phosphorylate and repress the activity of HSF-1 under unstressed conditions. MAP kinases activation immediately by heat shock serves to deactivate HSF-1. We now propose to study the role of ERK1 in the phosphorylation of HSF-1. We will determine if HSF-1 is phosphorylated by ERK1 by using immune complex and in-gel kinase assays. To further investigate the biological significance of the negative regulation of HSF-1 by ERK1 in vivo, we will examine if cells stably overexpressing ERK1 have an attenuated heat shock response by analyzing various properties of HSF-1. We will then map the phosphorylation site(s) of ERK1 on HSF-1 by tryptic mapping and site directed mutagenesis.
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