Isshiki Kinuka, Hirase Taishi, Matsuda Shinya, Miyamoto Kenji, Tsuji Akihiko, Yuasa Keizo
Department of Biological Science and Technology, Tokushima University Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Department of Biological Science and Technology, Tokushima University Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Biochem Biophys Res Commun. 2015;468(1-2):113-8. doi: 10.1016/j.bbrc.2015.10.151. Epub 2015 Oct 31.
Death-associated protein kinase 2 (DAPK2) is a positive regulator of apoptosis. Although we recently reported that 14-3-3 proteins inhibit DAPK2 activity and its subsequent apoptotic effects via binding to DAPK2, the molecular mechanisms underlying the DAPK2-mediated apoptotic pathway remain unclear. Therefore, we attempted to further identify DAPK2-interacting proteins using pull-down assays and mass spectrometry. The microtubule β-tubulin was identified as a novel DAPK2-binding protein in HeLa cells. Pull-down assays revealed that DAPK2 interacted with the α/β-tubulin heterodimer, and that the C-terminal region of DAPK2, which differs from that of other DAPK family members, was sufficient for the association with β-tubulin. Although the microtubule-depolymerizing agent nocodazole induced apoptosis in HeLa cells, the level of apoptosis was significantly decreased in the DAPK2 knockdown cells. Furthermore, we found that treatment with nocodazole resulted in an increased binding of DAPK2 to β-tubulin. These findings indicate that DAPK2 mediates nocodazole-induced apoptosis via binding to tubulin.
死亡相关蛋白激酶2（DAPK2）是细胞凋亡的正向调节因子。尽管我们最近报道14-3-3蛋白通过与DAPK2结合来抑制其活性及其随后的凋亡效应，但DAPK2介导的凋亡途径的分子机制仍不清楚。因此，我们试图通过下拉实验和质谱法进一步鉴定与DAPK2相互作用的蛋白。微管β-微管蛋白被鉴定为HeLa细胞中一种新的DAPK2结合蛋白。下拉实验显示DAPK2与α/β-微管蛋白异二聚体相互作用，并且DAPK2不同于其他DAPK家族成员的C末端区域足以与β-微管蛋白结合。尽管微管解聚剂诺考达唑可诱导HeLa细胞凋亡，但在DAPK2敲低的细胞中凋亡水平显著降低。此外，我们发现用诺考达唑处理会导致DAPK2与β-微管蛋白的结合增加。这些发现表明DAPK2通过与微管蛋白结合介导诺考达唑诱导的细胞凋亡。
