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Expression and cell transformation activity of dynactin-associated protein isoforms
FEBS Open Bio ( IF 2.8 ) Pub Date : 2021-05-27 , DOI: 10.1002/2211-5463.13202
Xiaobo Yin 1 , Shota Yamada 1 , Hiroaki Kobayashi 1 , Ryota Tanaka 1 , Yuki Togo 1 , Miho Hosoi 1, 2 , Mie Tsuchida 1, 2 , Tatsuki Kunoh 1 , Shuichi Wada 1 , Toshinobu Nakamura 1 , Ryuzo Sasaki 1, 2 , Tamio Mizukami 1, 2 , Makoto Hasegawa 1
Affiliation  

Overexpression of human dynactin-associated protein isoform a (dynAPa) transforms NIH3T3 cells. DynAPa is a single-pass transmembrane protein with a carboxy-terminal region exposed to the outside of cells. According to the NCBI RefSeq database, there may be two other splicing variants of the encoding gene (dynAPb and c). DynAPa and c differ in some amino-terminal residues (NH2-MVA in dynAPa and NH2-MEYQLL in dynAPc). DynAPb has the same amino-terminal residues as dynAPc, but lacks 55 residues in the intracellular region. All three isoforms have the same carboxy-terminal region, including the transmembrane domain. Expression of mRNAs of three splicing variants was found in human cancer cell lines ACHN and Caki-1. The subcellular localization and in vitro cell transformation ability of the three isoforms were examined using NIH3T3 cells overexpressing each respective isoform. All isoforms were found to be localized to the Golgi apparatus and plasma membrane, where the carboxy-terminal region was exposed to the outside of cells. Cell transformation was tested using focus formation due to loss of contact inhibition of cell proliferation, and colony formation was examined on soft agar and spheroid formation in ultralow U-bottomed wells. DynAPa robustly formed foci and colonies on soft agar and spheroid, whereas these abilities were considerably decreased for dynAPb and completely lost in dynAPc. These findings warrant dissection studies to identify the dynAP domain that is required for cell transformation.

中文翻译:

dynactin 相关蛋白亚型的表达和细胞转化活性

人动力蛋白相关蛋白亚型 a (dynAPa) 的过度表达可转化 NIH3T3 细胞。DynAPa 是一种单次跨膜蛋白,其羧基末端区域暴露于细胞外部。根据NCBI RefSeq数据库,编码基因可能还存在另外两种剪接变体(dynAPb和c)。DynAPa和c在一些氨基末端残基上不同(dynAPa中的NH 2 -MVA和dynAPc中的NH 2 -MEYQLL)。DynAPb 具有与 dynAPc 相同的氨基末端残基,但胞内区域缺少 55 个残基。所有三种亚型都具有相同的羧基末端区域,包括跨膜结构域。在人类癌细胞系 ACHN 和 Caki-1 中发现了三种剪接变体 mRNA 的表达。亚细胞定位和体外使用过度表达各自同种型的 NIH3T3 细胞检查了三种同种型的细胞转化能力。发现所有亚型都位于高尔基体和质膜,其中羧基末端区域暴露于细胞外部。由于失去了细胞增殖的接触抑制,使用焦点形成来测试细胞转化,并在超低 U 形底孔中检查软琼脂上的集落形成和球体形成。DynAPa 在软琼脂和球体上牢固地形成病灶和集落,而 dynAPb 的这些能力显着降低,而 dynAPc 则完全丧失。这些发现需要解剖研究来确定细胞转化所需的 dynAP 结构域。
更新日期:2021-08-03
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