Birchenall-Roberts Maria C, Fu Tao, Kim Soo-Gyung, Huang Ying K, Dambach Michael, Resau James H, Ruscetti Francis W
Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 2170, USA.
Biochem Biophys Res Commun. 2006 Sep 22;348(2):540-9. doi: 10.1016/j.bbrc.2006.07.094. Epub 2006 Jul 28.
Kirsten Ras4B (K-Ras4B) is a potent onco-protein that is expressed in the majority of human cell types and is frequently mutated in carcinomas. K-Ras4B, like other members of the Ras family of proteins, is considered to be a cytoplasmic protein that must be localized to the plasma membrane for activation. Here, using confocal microscopy and biochemical analysis, we show that K-Ras4B, but not H-Ras or the closely related K-Ras4A, is also present in the nucleoli of normal and transformed cells. Subcellular fractionation and immunostaining show that K-Ras4B is located not only in the cytoplasm, but also in the nucleolar compartment. Modification of a C-terminal hexa-lysine motif unique to K-Ras4B results in exclusively cytoplasmic forms of the protein. Nucleolin, a pleiotropic regulator of cellular processes, including transcriptional regulation, is also characterized by a nucleolar-like nuclear appearance. We show that K-Ras4B and nucleolin co-localize within the nucleus and that nucleolin physically associates with K-Ras4B. Inhibition of K-Ras4B/nucleolin association blocked nucleolar localization of K-Ras4B. Using siRNA to knockdown the expression of nucleolin eliminated the nucleolar localization of K-Ras4B and significantly repressed the activation of the well-characterized K-Ras4B transcriptional target Ap-1, but stimulated Elk1. These data provide evidence of a nucleolar localization of K-Ras4B and describe a functional association between K-Ras4B and nucleolin.
Kirsten Ras4B（K-Ras4B）是一种强效癌蛋白，在大多数人类细胞类型中表达，且在癌症中经常发生突变。与Ras蛋白家族的其他成员一样，K-Ras4B被认为是一种细胞质蛋白，必须定位于质膜才能被激活。在此，我们利用共聚焦显微镜和生化分析表明，K-Ras4B而非H-Ras或密切相关的K-Ras4A，也存在于正常细胞和转化细胞的核仁中。亚细胞分级分离和免疫染色显示，K-Ras4B不仅位于细胞质中，也存在于核仁区室。对K-Ras4B特有的C端六赖氨酸基序进行修饰会导致该蛋白仅以细胞质形式存在。核仁素是细胞过程（包括转录调控）的多效性调节因子，其特征也是具有类似核仁的核外观。我们表明，K-Ras4B和核仁素在细胞核内共定位，且核仁素与K-Ras4B存在物理关联。抑制K-Ras4B/核仁素的关联会阻止K-Ras4B的核仁定位。使用小干扰RNA（siRNA）敲低核仁素的表达消除了K-Ras4B的核仁定位，并显著抑制了特征明确的K-Ras4B转录靶点Ap-1的激活，但刺激了Elk1。这些数据提供了K-Ras4B核仁定位的证据，并描述了K-Ras4B与核仁素之间的功能关联。
