PKR AND A NOVEL IL-3 SIGNAL TRANSDUCTION PATHWAY

描述：(改编自调查人员摘要)白介素3 白介素3(IL-3)是一种多潜能的造血生长因子(HGF) 通过触发后转录因子介导靶细胞的生长和分化 导致关键中间体激活的受体信号转导 蛋白激酶(图1)。然而，隐含在生长因子作用中的是 结构蛋白和调节蛋白合成的要求 对于成长来说是必要的。因此，高效的增长来自于 对增殖和蛋白质合成的协同刺激 作为对细胞凋亡的抑制。关于IL-3如何 偶联并激活蛋白质合成。初步数据显示， IL-3通过一条新的信号通路可逆地影响蛋白质合成 具有快速调节可激活的双链RNA的特点 蛋白激酶，PKR。PKR是一种已知发挥作用的干扰素诱导基因 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普遍表达，并且 最近被发现具有潜在的肿瘤抑制特性。 此外，活化的Ha-ras转化的3T3细胞含有特异性的 蛋白酪氨酸激酶受体抑制剂。这些发现提示了PKR在细胞中的必要作用。 成长。申请者发现IL-3的停用在 蛋白酪氨酸激酶受体的磷酸化和酶激活 其生理底物为eIF2a。当磷酸化时，eIF2a是已知的 以抑制蛋白质合成的启动(图2)。或者，IL-3 添加到缺乏因子的细胞中介导去磷酸化，并且 蛋白激酶受体的失活与蛋白的快速相互作用密切相关 PKR和一个含有97 kDa酪氨酸和丝氨酸的磷酸蛋白pp97。 IL-3在PKR的正常生长过程中起着重要的调节作用。 在抗病毒宿主防御中的公认作用。为了澄清这个问题 这一新的IL-3途径的分子特征，研究人员将 重点研究IL-3如何可逆地调节PKR和蛋白质合成。这个 具体目的是确定：(1)IL-3的作用机制 戒断激活PKR并抑制蛋白质合成；(2) IL-3加入剥夺细胞失活PKR的机制 抑制蛋白质合成；(3)纯化、鉴定、 分子克隆和表达pp97。最先进的分子和 将采用生化方法，包括施工和 强制表达催化失活的PKR，反义技术， 并利用酵母双杂交系统进行pp97的交互克隆。 预计PKR可能代表着一种独特的治疗靶点。 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.