Homma Miwako Kato, Li Dongxia, Krebs Edwin G, Yuasa Yasuhito, Homma Yoshimi
Department of Biomolecular Science, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan.
Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):5959-64. doi: 10.1073/pnas.092143199. Epub 2002 Apr 23.
Mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis coli and also sporadic colorectal cancer development. By using antibodies raised against the N-terminal region of APC protein, we have detected the variable masses of endogenous APC proteins in individual cell lines established from human colorectal carcinomas caused by nonsense mutations of the gene. Phosphorylation of immunoprecipitates of full-length and truncated APC were observed in in vitro kinase reaction, indicating association of APC with protein kinase activity. The kinase activity complexed with APC was sensitive to heparin and used GTP as phosphoryl donor, suggesting an involvement of casein kinase 2 (CK2). Both CK2alpha- and beta-subunits were found to associate with APC in immunoprecipitates as well as in pull-down assays, with preferential interaction of APC with tetrameric CK2 holoenzyme. In synchronized cell populations, the association of APC with CK2 was cell cycle dependent, with the highest association in G(2)/M. Unexpectedly, APC immunoprecipitates containing full-length APC protein inhibited CK2 in vitro, whereas immunoprecipitates of truncated APC had little effect. This was confirmed by using recombinant APC, and the inhibitory region was localized to the C terminus of APC between residues 2086 and 2394. Overexpression of this fragment in SW480 cells suppressed cell proliferation rates as well as tumorigenesis. These results demonstrate a previously uncharacterized functional interaction between the tumor suppressor protein APC and CK2 and suggest that growth-inhibitory effects of APC may be regulated by inhibition of CK2.
腺瘤性息肉病 coli（APC）基因的突变是家族性腺瘤性息肉病以及散发性结直肠癌发生的原因。通过使用针对 APC 蛋白 N 端区域产生的抗体，我们在由该基因无义突变导致的人结直肠癌建立的单个细胞系中检测到了内源性 APC 蛋白的可变质量。在体外激酶反应中观察到全长和截短的 APC 免疫沉淀物的磷酸化，表明 APC 与蛋白激酶活性相关。与 APC 复合的激酶活性对肝素敏感，并使用 GTP 作为磷酰供体，提示酪蛋白激酶 2（CK2）参与其中。在免疫沉淀物以及下拉试验中均发现 CK2α和β亚基与 APC 相关，APC 与四聚体 CK2 全酶优先相互作用。在同步化细胞群体中，APC 与 CK2 的关联是细胞周期依赖性的，在 G(2)/M 期关联最高。出乎意料的是，含有全长 APC 蛋白的 APC 免疫沉淀物在体外抑制 CK2，而截短的 APC 免疫沉淀物几乎没有作用。使用重组 APC 证实了这一点，并且抑制区域定位于 APC 的 C 末端，在残基 2086 和 2394 之间。该片段在 SW480 细胞中的过表达抑制了细胞增殖率以及肿瘤发生。这些结果证明了肿瘤抑制蛋白 APC 和 CK2 之间以前未被表征的功能相互作用，并表明 APC 的生长抑制作用可能通过抑制 CK2 来调节。
