ERKL--NEGATIVE REGULATOR OF THE HEAT SHOCK RESPONSE

ERKL--热休克反应的负调节因子

• 批准号: 2733070
• 负责人: NAHID F MIVECHI
• 金额: $ 21.98万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2733070
• 关键词: 3T3 cells; DNA binding protein; T lymphocyte; aminophosphonate; chemical association; chemical kinetics; chimeric proteins; chloramphenicol acetyltransferase; enzyme activity; enzyme inhibitors; enzyme substrate; epitope mapping; gene expression; immune complex; immunoprecipitation; luciferin monooxygenase; mitogen activated protein kinase; molecular site; phosphoprotein phosphatase; phosphorylation; plasmids; protein purification; site directed mutagenesis; stress proteins; transcription factor

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.

描述：细胞对热、X射线照射等应激反应，通过 增强各种基因的转录以保护自己， 或修复因这种侮辱而造成的损害。 的转录因子 磷酸化是这种应激的主要反应者。 变化 转录因子的磷酸化受蛋白激酶调节 来传递来自环境的信号。 理解这些 这些变化应该会导致基因的表达被操纵， 断断续续。 因此，理解这一机制至关重要， 信号从膜传递到核。 研究最广泛的信号通路涉及生长因子 刺激和对各种应激的反应涉及MAP 激酶（ERK 1和2）。 MAP激酶已被证明激活转录 参与即刻早期基因表达的因素。 最近MAP激酶已被证明是由热休克激活。 的 MAP激酶可能参与HSF-1磷酸化 因此，这是允许的，并通过以下两者之间的密切联系得到加强： MAP激酶的活化和HSF-1的磷酸化。 使用两种基因 和药理学方法，我们表明，MAP激酶激活热， 休克不依赖于Ras和Raf-1激活。 我们还表明，RAS 通过ERK 1对热休克反应发挥负性调节作用。 这是因为ERK 1抑制突变体（ERK 1 KR）的过度表达， 使hsp 70-荧光素酶的表达增加30倍。 此外，委员会认为， 用钒酸钠预处理细胞，增加ERK 1激酶 活性，增加32 P掺入HSF-1。 这些数据表明 在生长因子刺激下被激活的MAp激酶 在非应激条件下，HSF-1可能磷酸化并抑制其活性 条件 MAP激酶通过热休克立即激活， 停用HSF-1 我们现在建议研究ERK 1在HSF-1磷酸化中的作用。 我们将用免疫复合物检测ERK 1是否磷酸化HSF-1 和凝胶内激酶测定。 为了进一步研究 在体内ERK 1对HSF-1负调控的意义，我们将 检查稳定过表达ERK 1的细胞是否具有减弱的热休克 通过分析HSF-1的各种特性来响应。 然后我们将绘制 通过胰蛋白酶作图和酶切位点分析确定HSF-1上ERK 1的磷酸化位点 定向诱变