REGULATION OF C-REL BY IKB-ALPH

大分子在细胞核和细胞质之间的转运是所有真核细胞的基本活动。 我们正在使用c-Rel转录因子及其抑制剂IkappaBalpha作为模型系统，以了解蛋白质在细胞核和细胞质之间的定向转运如何为基因表达的调控提供有效的机制。IkappaB α蛋白既可以抑制c-Rel的核输入，又可以指导c-Rel从核输出。 IkappaBalpha的这种核穿梭性质使得能够严格控制c-Rel的净核丰度。第一个假设是IkappaBalpha的核穿梭性质是核输入、核保留和核输出的净总和。 将使用体外和体内测定来确定负责这些功能中的每一个的IkappaB α中的氨基酸。将确定参与IkappaB α核输入的反式作用因子，并定义IkappaB α核输入途径的新机制特征。在这个提议中要检验的第二个假设是，IkappaBalpha通过抑制核输入和核输出来控制c-Rel的核丰度。 IkappaB α抑制c-Rel的核输入的能力将与IkappaB α不能抑制v-Rel癌蛋白的核输入形成对比。 IkappaB α作为衔接蛋白的功能，桥梁c-Rel与出口受体，Crm 1，将被检查。 将测定Crm 1区分游离IkappaB α和c-Rel相关IkappaB α的能力。 将开发一种创新的体外核输出测定法，并用于了解核输出如何在核孔复合物的细胞质面上终止。还将确定c-Rel：IkappaB α复合体在核出口终止后的命运。

The transport of macromolecules between the nucleus and the cytoplasm is an essential activity of all eukaryotic cells. We are using the c-Rel transcription factor and its inhibitor, IkappaBalpha, as a model system to understand how the directional transport of proteins between the nucleus and the cytoplasm can provide an effective mechanism for regulation of gene expression. The IkappaBalpha protein can both inhibit nuclear import of c-Rel and direct the export of c-Rel from the nucleus. This nuclear shuttling property of IkappaBalpha enables tight control over the net nuclear abundance of c-Rel. The first hypothesis to be addressed in this proposal is that the nuclear shuttling property of IkappaBalpha is the net sum of nuclear import, nuclear retention, and nuclear export. The amino acids in IkappaBalpha that are responsible for each of these functions will be defined using both in vitro and in vivo assays. Trans-acting factors that participate in nuclear import of IkappaBalpha will be identified and novel mechanistic features of IkappaBalpha nuclear import pathway will be defined. The second hypothesis to be tested in this proposal is that IkappaBalpha controls the nuclear abundance of c-Rel through both inhibition of nuclear import and through nuclear export. The ability of IkappaBalpha to inhibit nuclear import of c-Rel will be contrasted to the inability of IkappaBalpha to inhibit nuclear import of the v-Rel oncoprotein. The function of IkappaBalpha as an adaptor protein that bridges c-Rel with the export receptor, Crm1, will be examined. The ability of Crm1 to discriminate between free IkappaBalpha and c-Rel-associated IkappaBalpha will be determined. An innovative in vitro nuclear export assay will be developed and used to understand how nuclear export is terminated on the cytoplasmic face of the nuclear pore complex. The fate of the c-Rel: IkappaBalpha complex following termination of nuclear export will also be determined.