MEMBRANE TARGETING OF FATTY ACYLATED PROTOONCOPROTEINS

这项研究的总体目标是了解蛋白质的作用 脂肪酰化在指导亚细胞定位和功能中的作用 正常和致癌信号蛋白。 Src家族成员 酪氨酸蛋白激酶将用作模型系统。 膜 这些蛋白质的附着对其功能至关重要， 负责膜靶向的机制涉及共价键， 用棕榈酸酯和/或肉豆蔻酸酯修饰。 提出的实验 在这个应用程序中，旨在解决3个关键问题：1）如何 脂肪酰化蛋白针对特定的膜亚结构域？ 的 双酰化Src家族激酶Fyn定位于非- 离子洗涤剂抗性膜域将在 体外 非酰化、肉豆蔻酰化和肉豆蔻酰化+的结合 棕榈酰化Fyn与具有特定磷脂组成的脂质体 将被定量;将小窝蛋白-1掺入到 将评估脂质体。 2)信号蛋白的功能可以 通过连接不同类型的膜靶向基序而改变？ 编码不同脂肪酰化/异戊二烯化信号的序列将 将所述构建体与c-Raf-1融合，并将所述构建体置于可诱导的 启动子 改性Raf启动下游的能力 将确定通过MAP激酶途径的信号传导。 3)什么 Fyn及其脂肪酰化在正常细胞功能中的作用？我们 最近确定少突胶质细胞，即产生髓鞘的细胞， 的CNS，依赖于Fyn的上调分化。 我们将测试wt，显性负性和脂肪酰化的能力 Fyn突变体促进少突胶质细胞分化过程。

The overall goal of this research is to understand the role of protein fatty acylation in directing subcellular localization and function of normal and oncogenic signaling proteins. Members of the Src family of tyrosine protein kinases will be used as model systems. Membrane attachment of these proteins is critical for their function, and the mechanism responsible for membrane targeting involves covalent modification with palmitate and/or myristate. The experiments proposed in this application are designed to address 3 key questions: 1) How are fatty acylated proteins targeted to specific membrane subdomains? The ability of the dually acylated Src family kinase Fyn to localize to non- ionic detergent-resistant membrane domains will be reconstituted in vitro. Binding of non-acylated, myristoylated, and myristoylated + palmitoylated Fyn to liposomes with specific phospholipid compositions will be quantitated; the effect of incorporating caveolin-1 into the liposomes will be assessed. 2) Can signaling protein function be altered by attachment of different types of membrane targeting motifs? Sequences encoding different fatty acylation/prenylation signals will be fused to c-Raf-1 and the constructs placed under an inducible promoter. The ability of the modified Raf to initiate downstream signaling through the MAP kinase pathway will be determined. 3) What is the role of Fyn and its fatty acylation in normal cell function? We recently determined that oligodendrocytes, the myelin producing cells of the CNS, are dependent on upregulation of Fyn for differentiation. We will test the abilities of wt, dominant negative and fatty acylation mutants of Fyn to promote the oligodendrocyte differentiation process.