From the Department of Pathology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and.
From the Department of Pathology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and; the Beijing Shijitan Hospital, Capital Medical University, Beijing 100069, China.
J Biol Chem. 2010 Feb 5;285(6):3554-3560. doi: 10.1074/jbc.M109.063354. Epub 2009 Dec 12.
IIp45 (aka MIIP) is a newly discovered gene whose protein product inhibits cell migration. HDAC6 is a class IIb deacetylase that specifically deacetylates alpha-tubulin, modulates microtubule dynamics, and promotes cell migration. A yeast two-hybrid assay using IIp45 as bait identified HDAC6 protein as a binding partner of IIp45. This physical interaction of the two functionally antagonistic proteins was confirmed by glutathione S-transferase pulldown assay and co-immunoprecipitation assay in human cells. Serial deletion constructs of HDAC6 were used to characterize the interaction of HDAC6 and IIp45, and this analysis found that the two catalytic domains of HDAC6 protein are required for IIp45 binding. We examined the protein expression patterns of IIp45 and HDAC6 in glioma tissues. Elevated protein levels of HDAC6 were found in high grade glioma samples, in contrast to the decreased protein expression of IIp45. The potential negative regulation of HDAC6 expression by IIp45 was confirmed in cell lines with altered IIp45 expression by constitutive overexpression or small interfering RNA knockdown. Protein turnover study revealed that overexpression of IIp45 significantly reduces the intracellular protein stability of endogenous HDAC6, indicating a possible mechanism for the negative regulation of HDAC6 by IIp45. Results from the HDAC activity assay demonstrated that overexpressed IIp45 effectively decreases HDAC6 activity, increases acetylated alpha-tubulin, and reduces cell migration. The increased cell migration resulting from siIIp45 knockdown was significantly reversed by co-transfection of siHDAC6. Thus, we report here for the first time a novel mechanism by which IIp45 inhibits cell motility through inhibition of HDAC6.
IIp45（又名 MIIP）是一种新发现的基因，其蛋白质产物抑制细胞迁移。HDAC6 是一种 IIb 类去乙酰化酶，特异性去乙酰化微管蛋白α，调节微管动力学，并促进细胞迁移。使用 IIp45 作为诱饵的酵母双杂交实验鉴定出 HDAC6 蛋白是 IIp45 的结合伴侣。这两种功能拮抗蛋白的物理相互作用通过谷胱甘肽 S-转移酶 pull-down assay 和人细胞中的共免疫沉淀assay 得到了证实。使用 HDAC6 的串联缺失构建体来表征 HDAC6 和 IIp45 的相互作用，该分析发现 HDAC6 蛋白的两个催化结构域是与 IIp45 结合所必需的。我们检查了胶质瘤组织中 IIp45 和 HDAC6 的蛋白表达模式。在高级别神经胶质瘤样本中发现 HDAC6 的蛋白水平升高，而 IIp45 的蛋白表达则降低。通过组成型过表达或小干扰 RNA 敲低改变 IIp45 表达的细胞系中，证实了 HDAC6 表达受 IIp45 负调控。蛋白周转研究表明，过表达 IIp45 显著降低内源性 HDAC6 的细胞内蛋白稳定性，表明 IIp45 对 HDAC6 负调控的可能机制。HDAC 活性测定结果表明，过表达的 IIp45 可有效降低 HDAC6 活性，增加乙酰化的微管蛋白α，并减少细胞迁移。siIIp45 敲低导致的细胞迁移增加，通过共转染 siHDAC6 可显著逆转。因此，我们首次报道了 IIp45 通过抑制 HDAC6 抑制细胞运动的新机制。
