REGULATION OF C-REL BY IKB-ALPH

大分子在细胞核和细胞质之间的运输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作为模型系统来了解蛋白质在细胞核和细胞质之间的定向运输如何为基因表达的调控提供有效的机制。IkappaBalpha蛋白既能抑制c-Rel的细胞核输入，又能指导c-Rel从细胞核输出。IkappaBalpha的这种核穿梭特性可以严格控制c-Rel的净核丰度。本提案要解决的第一个假设是，IkappaBalpha的核穿梭特性是核进口、核保留和核出口的净总和。IkappaBalpha中负责这些功能的氨基酸将通过体外和体内测定来确定。将确定参与IkappaBalpha核进口的反式作用因子，并定义IkappaBalpha核进口途径的新机制特征。本提案要检验的第二个假设是，IkappaBalpha通过抑制核进口和核出口来控制c-Rel的核丰度。IkappaBalpha抑制c-Rel核输入的能力将与IkappaBalpha无法抑制v-Rel癌蛋白核输入的能力进行对比。IkappaBalpha作为连接c-Rel和输出受体Crm1的衔接蛋白的功能将被研究。将确定Crm1区分游离IkappaBalpha和c- rel相关IkappaBalpha的能力。将开发一种创新的体外核输出测定方法，并用于了解核输出如何在核孔复合物的细胞质表面终止。核出口终止后，c-Rel: IkappaBalpha复合体的命运也将决定。

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.