Yamaji Satoshi, Suzuki Atsushi, Kanamori Heiwa, Mishima Wataru, Yoshimi Ryusuke, Takasaki Hirotaka, Takabayashi Maki, Fujimaki Katsumichi, Fujisawa Shin, Ohno Shigeo, Ishigatsubo Yoshiaki
The First Dept. of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan.
J Cell Biol. 2004 May 24;165(4):539-51. doi: 10.1083/jcb.200308141.
The linking of integrin to cytoskeleton is a critical event for an effective cell migration. Previously, we have reported that a novel integrin-linked kinase (ILK)-binding protein, affixin, is closely involved in the linkage between integrin and cytoskeleton in combination with ILK. In the present work, we demonstrated that the second calponin homology domain of affixin directly interacts with alpha-actinin in an ILK kinase activity-dependent manner, suggesting that integrin-ILK signaling evoked by substrate adhesion induces affixin-alpha-actinin interaction. The overexpression of a peptide corresponding to the alpha-actinin-binding site of affixin as well as the knockdown of endogenous affixin by small interference RNA resulted in the blockade of cell spreading. Time-lapse observation revealed that in both experiments cells were round with small peripheral blebs and failed to develop lamellipodia, suggesting that the ILK-affixin complex serves as an integrin-anchoring site for alpha-actinin and thereby mediates integrin signaling to alpha-actinin, which has been shown to play a critical role in actin polymerization at focal adhesions.
整合素与细胞骨架的连接是细胞有效迁移的关键事件。此前,我们报道了一种新型的整合素连接激酶(ILK)结合蛋白——亲和蛋白,它与ILK共同紧密参与整合素与细胞骨架之间的连接。在本研究中,我们证明亲和蛋白的第二个钙调蛋白同源结构域以依赖ILK激酶活性的方式直接与α-辅肌动蛋白相互作用,这表明由底物黏附引发的整合素-ILK信号传导诱导了亲和蛋白-α-辅肌动蛋白的相互作用。过表达对应于亲和蛋白α-辅肌动蛋白结合位点的肽段以及用小干扰RNA敲低内源性亲和蛋白均导致细胞铺展受阻。延时观察显示,在这两个实验中细胞均呈圆形,周边有小泡状突起,且无法形成片状伪足,这表明ILK-亲和蛋白复合物作为α-辅肌动蛋白的整合素锚定位点,从而介导整合素向α-辅肌动蛋白的信号传导,而α-辅肌动蛋白已被证明在粘着斑处的肌动蛋白聚合中起关键作用。