STRESS ACTIVATED PROTEIN KINASE PATHWAY

描述（改编自申请人摘要）：信号 介导对环境应激反应的转导机制 和炎症因子的认识还很有限。然而，这些刺激是 负责一些病理条件的临床 重要性包括急性期脓毒性休克、缺血性损伤（如 发生在心脏病发作和中风），发烧和辐射损伤。最近 申请人实验室的结果有助于划定 哺乳动物的信号通路， 环境应激、缺血性损伤与炎症因子 TNF-α和IL-1。 该信号通路使用三种顺序作用的蛋白激酶。 酶的这个模块类似于 哺乳动物促有丝分裂反应途径和几种酵母信号 途径。应激激活模块的三种酶是MEK 激酶1（MEKK 1）、SAPK/ERK激酶1（SEK 1）和应激活化蛋白 激酶（SAPK; ERK家族成员）。转录因子c-Jun是 SAPKs是这一通路的一个靶点，SAPKs是其主要介质。 磷酸化反应。 拟议研究的四个目标将剖析监管， SAPK通路的功能。目的1是鉴定 与MEKK 1 N-末端调节结构域相互作用，并表征 它们对SEK 1的MEKK 1活化的影响。这些MEKK 1结合物质 应用酵母双杂交和亲和层析技术进行鉴定 矩阵方法。 Aim 2采用酵母双杂交筛选， 生物化学测定以鉴定另外的SAPK底物。Aim 3采用 低严格性筛选方法和稳定和瞬时转染 来隔离MEKK家族的其他成员将进行各种分析， 以确定是否有任何新的MEKK成员激活SAPK 通路最后（目的4），哺乳动物Ste 20同源物的潜在作用 将测试SAPK激酶模块的激活。

DESCRIPTION (Adapted from the applicant's abstract): The signal transduction mechanisms mediating responses to environmental stresses and inflammatory agents are poorly understood. Yet, these stimuli are responsible for a number of pathological conditions of clinical importance including acute phase septic shock, ischemic injury (as occurs in heart attack and stroke), fever and radiation damage. Recent results from the applicant's laboratory contributed to the delineation of a mammalian signaling pathway that mediates responses to environmental stresses, ischemic injury and the inflammatory cytokines TNF- alpha and IL-1. This signaling pathway uses three sequentially acting protein kinases. This module of enzymes is analogous to the modules that are a part of the mammalian mitogenic response pathway and several yeast signal pathways. The three enzymes of the stress activated module are MEK kinase 1 (MEKK1), SAPK/ERK kinase 1 (SEK1) and stress activated protein kinase (SAPK; an ERK family member). The transcription factor, c-Jun is a target of this pathway and SAPKs are the primary mediators of its phosphorylation in response to stress. The four aims of the proposed research will dissect the regulation and function of the SAPK pathway. Aim 1 is to identify molecular species that interact with the MEKK1 N-terminal regulatory domain and characterize their effects on MEKK1 activation of SEK1. These MEKK1 binding species will be identified by application of yeast two hybrid and affinity matrix approaches. Aim 2 employs a yeast two hybrid screen and biochemical assays to identify additional SAPK substrates. Aim 3 employs low stringency screening methods and stable and transient transfection to isolate other members of the MEKK family. Various analyses will be applied to determine if any of the novel MEKK members activate the SAPK pathway. Finally (Aim 4), the potential role of mammalian Ste20 homologs in the activation of the SAPK kinase module will be tested.