ERKL--NEGATIVE REGULATOR OF THE HEAT SHOCK RESPONSE

描述：细胞对热、x射线等压力的反应，通过 增强各种基因的转录以保护自己， 或者弥补因这种侮辱而造成的损害。转录因子 变得磷酸化是对这种压力的主要反应。的变化 转录因子的磷酸化受蛋白激酶的调节 传输来自环境的信号。了解以下内容 改变应该导致对它们所转向的基因的表达的操纵 断断续续。因此，至关重要的是要了解 这些信号从细胞膜传递到细胞核。 生长因子参与的最广泛研究的信号通路 对各种压力的刺激和反应是涉及MAP的 激酶(ERK1和2)。已有研究表明，MAP激酶可以激活转录 参与即刻早期基因表达的因子。 最近，已有研究表明热休克可激活MAP激酶。这个 MAP激酶参与HSF-1磷酸化的可能性 因此，被允许和加强之间的密切关联 MAP激酶的激活和HSF-1的磷酸化。使用这两种基因 和药理学方法，我们证明了MAP激酶通过加热激活 休克不依赖于ras和RAF-1的激活。我们还展示了RAS 通过ERK1对热休克反应产生负性调节作用。 这是因为ERK1抑制突变体(ERK1KR)的过度表达。 使HSP70-荧光素酶的表达增加30倍。此外， 钒酸钠对细胞ERK1激酶活性的影响 活性，增加32P掺入HSF-1。这些数据表明 它们映射了在生长因子刺激下被激活的激活酶 可能在非应激状态下使HSF-1磷酸化并抑制其活性 条件。热休克立即激活MAP激酶有助于 停用HSF-1。 我们现在建议研究ERK1在HSF-1磷酸化中的作用。 我们将使用免疫复合体来确定HSF-1是否被ERK1磷酸化 和凝胶内激酶分析。为了进一步研究生物 ERK1在体内负调控HSF-1的意义，我们将 检查稳定过表达ERK1的细胞是否有减弱的热休克 通过分析HSF-1的各种特性做出回应。然后，我们将绘制 HSF-1上ERK1磷酸化位点(S)的胰酶定位研究 定向诱变。

DESCRIPTION: 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.