Division of Cardiology, Department of Medicine, The Affiliated Hospital of Jiangsu University, Zhenjiang, People's Republic of China.
Mol Biol Rep. 2012 Jun;39(6):6521-7. doi: 10.1007/s11033-012-1480-8. Epub 2012 Feb 19.
Hck is the unique example among the Src PTKs to be expressed as two isoforms, which are generated by alternative translation. The two isoforms differs from each other by a 21 N-terminal amino acids sequence which supports myristoylation. Though it has been shown that these different acylation states govern the different subcellular localization of the isoforms and each Hck isoform could play a specific role, little study focus on the function of p56Hck. To investigated the role of p56Hck isoform in cell migration, GFP targeted p56Hck plasmid and its constitutively active form were constructed and transiently transfected into HeLa cells, F-actin staining and Indirect immunofluorescence for microtubules were then performed. Phagokinetic track motility assay and In vitro invasion assays were also investigated after transiently transfection respectively. In this study, we found ectopically expressing a constitutively active form of 56Hck will lead to membrane protrusion and F-actin reorganization in HeLa cells. Both 56Hck and its constitutive active form will lead to redistribution of microtubules and enhancement of cell motility and cell invasion. Hck inhibitor PP2 supplementation eliminated cell motility and cell invasion of p56Hck while PP3, a negative control of PP2 didn't eliminate cell motility and cell invasion of p56Hck. It is indicated that enhanced cell motility and cell invasion in p56Hck ectopically expressed HeLa cells are the results of reorganization of F-actin and microtubules.
Hck 是 Src PTKs 中唯一的一种能够通过选择性翻译产生两种异构体的蛋白。这两种异构体在 N 端 21 个氨基酸序列上存在差异，该序列支持豆蔻酰化。虽然已经表明这些不同的酰化状态决定了异构体的不同亚细胞定位，并且每种 Hck 异构体都可能发挥特定的作用，但很少有研究关注 p56Hck 的功能。为了研究 p56Hck 异构体在细胞迁移中的作用，构建了靶向 GFP 的 p56Hck 质粒及其组成型激活形式，并瞬时转染到 HeLa 细胞中，然后进行 F-肌动蛋白染色和微管间接免疫荧光。还分别进行了短暂转染后的趋化性轨迹运动分析和体外侵袭分析。在这项研究中，我们发现过表达组成型激活形式的 56Hck 会导致 HeLa 细胞的膜突起和 F-肌动蛋白重组。56Hck 和其组成型激活形式都会导致微管重排，并增强细胞迁移和细胞侵袭。Hck 抑制剂 PP2 的补充消除了 p56Hck 的细胞迁移和侵袭能力，而 PP3，作为 PP2 的阴性对照，并没有消除 p56Hck 的细胞迁移和侵袭能力。这表明，p56Hck 过表达 HeLa 细胞中增强的细胞迁移和侵袭是 F-肌动蛋白和微管重组的结果。
