Askham J M, Moncur P, Markham A F, Morrison E E
Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, UK.
Oncogene. 2000 Apr 6;19(15):1950-8. doi: 10.1038/sj.onc.1203498.
The interaction between the adenomatous polyposis coli (APC) tumour suppressor and the microtubule-associated protein EB1 was examined. Immunoprecipitation suggested that APC and EB1 were not associated in cultures of HCT116 cells arrested in mitosis. The C-terminal 170 amino acids of APC, purified as a bacterial fusion protein, precipitated EB1 from cell extracts, significantly refining the location of the EB1 interaction domain in APC. In vitro phosphorylation of this fusion protein by either protein kinase A or p34cdc2 reduced its ability to bind to EB1. Expression of GFP fusions to C-terminal APC sequences lacking or including the APC basic domain but encompassing the EB1 binding region in SW480 cells revealed a microtubule tip association which co-localized with that of EB1. Expression of the basic domain alone revealed a non-specific microtubule localization. In vitro interaction studies confirmed that the APC basic domain did not contribute to EB1 binding. These findings strongly suggest that the interaction between APC and EB1 targets APC to microtubule tips, and that the interaction between the two proteins is down-regulated during mitosis by the previously described mitotic phosphorylation of APC.
研究了腺瘤性息肉病大肠杆菌（APC）肿瘤抑制因子与微管相关蛋白EB1之间的相互作用。免疫沉淀表明，在有丝分裂停滞的HCT116细胞培养物中，APC和EB1不相关。作为细菌融合蛋白纯化的APC的C末端170个氨基酸从细胞提取物中沉淀出EB1，显著细化了EB1在APC中的相互作用结构域的位置。该融合蛋白被蛋白激酶A或p34cdc2体外磷酸化后，其与EB1结合的能力降低。在SW480细胞中，与缺乏或包含APC碱性结构域但包含EB1结合区域的C末端APC序列的GFP融合蛋白的表达显示出与EB1共定位的微管尖端关联。单独碱性结构域的表达显示出非特异性微管定位。体外相互作用研究证实，APC碱性结构域对EB1结合没有贡献。这些发现强烈表明，APC与EB1之间的相互作用将APC靶向微管尖端，并且这两种蛋白之间的相互作用在有丝分裂期间通过先前描述的APC的有丝分裂磷酸化而被下调。
