PKR AND A NOVEL IL-3 SIGNAL TRANSDUCTION PATHWAY

描述：（改编自研究者摘要）白细胞介素-3 IL-3是一种多能造血生长因子（HGF）， 介导靶细胞的生长和分化， 受体信号传导导致关键中间体的激活 蛋白激酶（图1）。 然而，生长因子的作用隐含着 合成结构和调节蛋白的需要 增长所必需的。因此，有效的增长来自于 协同刺激增殖和蛋白质合成， 抑制细胞凋亡。 关于IL-3是如何 结合并激活蛋白质合成。 初步数据表明 IL-3在一个新的信号通路中可逆地影响蛋白质合成 其特征在于快速调节双链RNA可激活的 蛋白激酶。 PKR是一种INF诱导基因， 在病毒诱导的蛋白质合成抑制中的作用， 宿主细胞抗病毒防御机制。 PKR广泛表达， 最近发现具有潜在的肿瘤抑制特性。 此外，激活的Ha-ras转化的3 T3细胞含有特异性的 PKR抑制剂。这些结果表明PKR在细胞凋亡中起着必要的作用。 增长 申请人发现IL-3的退出介导了 磷酸化和PKR的酶促活化， 其生理底物eIF 2a。 当被磷酸化时，eIF 2a已知 以抑制蛋白质合成的起始（图2）。 或者，IL-3 添加到因子剥夺细胞介导去磷酸化， PKR的失活与以下因素的快速相互作用密切相关： PKR和一个97 kDa的酪氨酸和丝氨酸含磷蛋白，pp 97。 IL-3除了在正常生长过程中调节PKR外， 在抗病毒宿主防御中的公认作用。 为了阐明本 这种新型IL-3通路的分子特征，研究人员将 重点关注IL-3如何可逆地调节PKR和蛋白质合成。 的 具体目标是确定：（1）IL-3的机制， 戒断激活PKR并抑制蛋白质合成;（2） IL-3加入剥夺细胞可抑制PKR 并抑制蛋白质合成;和（3）纯化，表征， 分子克隆和表达pp 97。 最先进的分子和 将采用生物化学方法，包括建筑和 催化失活PKR的强制表达，反义技术， 以及酵母双杂交系统用于相互作用克隆pp 97的用途。 预计PKR可能代表了一个独特的治疗靶点， 新的抗肿瘤策略。

DESCRIPTION: (Adapted from investigator's abstract) Interleukin-3 (IL-3) is a multipotential hematopoietic growth factor (HGF) that mediates growth and differentiation of target cells by triggering post- receptor signaling resulting in the activation of key intermediate protein kinases (Fig. 1). Implicit in growth factor action, however, is the requirement for the synthesis of structural and regulatory proteins necessary for growth. Thus, efficient growth results from the coordinated stimulation of proliferation and protein synthesis as well as the inhibition of apoptosis. Little is known about how IL-3 might couple to and activate protein synthesis. Preliminary data indicate that IL-3 reversibly affects protein synthesis in a novel signaling pathway featuring the rapid regulation of the double stranded RNA activatable protein kinase, PKR. PKR is an INF-inducible gene well known to play a role in viral-induced inhibition of protein synthesis as a part of the host-cell antiviral defense mechanism. PKR is ubiquitously expressed and has recently been found to have potential tumor-suppressor properties. Furthermore, activated Ha-ras transformed 3T3 cells contain a specific inhibitor of PKR. These findings suggest a necessary role for PKR in cell growth. The applicants find that IL-3 withdrawal mediates phosphorylation and enzymatic activation of PKR with phosphorylation of its physiologic substrate, eIF2a. When phosphorylated, eIF2a is known to inhibit initiation of protein synthesis (Fig 2). Alternatively, IL-3 addition to factor-deprived cells mediates dephosphorylation, and inactivation of PKR is closely associated with the rapid interaction of PKR and a 97 kDa tyrosine and serine-containing phosphoprotein, pp97. IL-3 can regulate PKR during normal growth in addition to its well recognized role in antiviral host defense. In order to elucidate the molecular features of this novel IL-3 pathway, the investigator will focus on how IL-3 reversibly regulates PKR and protein synthesis. The specific aims are to determine: (1) the mechanism by which IL-3 withdrawal activates PKR and inhibits protein synthesis; (2) the mechanism by which IL-3 addition to deprived cells can inactivate PKR and derepress protein synthesis; and (3) to purify, characterize, molecularly clone and express pp97. State of the art molecular and biochemical methodology will be employed, including construction and enforced expression of catalytically inactive PKR, antisense technology, and use of the yeast two-hybrid system for interactive cloning of pp97. It is expected that PKR may represent a unique therapeutic target for novel anti-neoplastic strategies.