Su L K, Burrell M, Hill D E, Gyuris J, Brent R, Wiltshire R, Trent J, Vogelstein B, Kinzler K W
Johns Hopkins Oncology Center, Baltimore, Maryland 21231, USA.
Cancer Res. 1995 Jul 15;55(14):2972-7.
Mutations of the APC gene play a critical role in both sporadic and familial forms of colorectal cancer. The vast majority of these mutations result in the loss of the carboxyl terminus of the protein. To further elucidate the function of APC, we searched for cellular proteins that associate with its carboxyl terminus. One million human cDNA clones were screened with the use of the interaction trap two-hybrid system, and 67 clones were found to have a phenotype suggestive of an APC-interacting protein. Nucleotide sequence analysis revealed that 48 of these clones were derived from a single novel named EBI. The association of APC and EB1 proteins was confirmed with in vitro binding assays. mAbs against EB1 were then produced and used to demonstrate the association of APC and EB1 in vivo. The EB1 gene was predicted to encode a 268-amino acid protein without significant homology to proteins with known function. However, searches of nucleotide databases did identify evidence for at least two related human genes and a yeast homologue. This conservation suggests an essential function for EB1 that might provide clues to the mechanism through which APC suppresses colonic neoplasia.
APC基因的突变在散发性和家族性结直肠癌中均起着关键作用。这些突变绝大多数会导致该蛋白羧基末端的缺失。为了进一步阐明APC的功能，我们寻找了与其羧基末端相关的细胞蛋白。利用相互作用陷阱双杂交系统筛选了100万个人类cDNA克隆，发现67个克隆具有提示与APC相互作用蛋白的表型。核苷酸序列分析表明，这些克隆中有48个来自一个名为EBI的单一新基因。通过体外结合试验证实了APC与EB1蛋白的关联。然后制备了针对EB1的单克隆抗体，并用于证明APC与EB1在体内的关联。预计EB1基因编码一种268个氨基酸的蛋白，与已知功能的蛋白无明显同源性。然而，对核苷酸数据库的搜索确实发现了至少两个相关人类基因和一个酵母同源物的证据。这种保守性表明EB1具有重要功能，这可能为APC抑制结肠肿瘤形成的机制提供线索。
