Elucidation of the Mechanism for the cell cycle regulation by a novel isoform of p27^<Kip1> in the vascular cells

阐明血管细胞中新型 p27^<Kip1> 亚型调节细胞周期的机制

基本信息

批准号：
13670723
负责人：
HIRANO Katsuya
金额：
$ 2.37万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

A novel isoform of p27^<Kip1>, a cyclin-dependent kinase inhibitor, has been isolated from a cDNA library of porcine aortic endothelial cells. The N-terminal 162 amino acid of the new isoform was identical to those of p27^<Kip1>, while it contained a unique 18 amino acid C-terminus. The novel isoform was found to be resistant toward protease-mediated degradation, thus naming it p27^<Kip1R>, a degradation resistant sioform of p27^<Kip1>. The region 153-168 was determined to be necessary for its significant nuclear localization. However, this region contains only one basic amino acid, and an aliphatic amino acid was found to play a functional role in the nuclear localization signal. Namely, p27^<Kip1R> contains an atypical bipartite nuclear localization signal. Since functional substitution by an aliphatic amino acid was incomplete, p27^<Kip1R> also demonstrated a weak but significant localization in the cytosol, in addition to the strong nuclear localization.Using the green fluorescence … More protein expression system, the effect of p27^<Kip1R> on the cell growth was investigated. It was found to strongly inhibit the cell growth of vascular smooth muscle cells and HeLa cells, as in the case with p27^<Kip1>. The growth inhibitory effect of both p27^<Kip1R> and p27^<Kip1> was completely abolished by removing the N-terminal region that bind to cyclin and cyclin-dependent kinase, while these truncated mutants demonstrated a significant nuclear localization. As a result, the mechanism for growth inhibition by p27^<Kip1R> was similar to that of p27^<Kip1>. The structural difference between two isforms was thus not linked to the growth inhibition.The aorta of 6-week old spontaneously hypertensive rat (SHR) dominantly expressed p27^<Kip1>, while the aorta of normal rat (WKY) dominantly expressed p27^<Kip1R>. On the other hand, the aorta of 8 and 13-week old SHR dominantly expressed p27^<Kip1R>. When the aortic smooth muscle cells of normal rat were cultured, they dominanly expressed p27^<Kip1>. The expression of p27^<Kip1R> appeared to inversely correlate with proliferative state of the smooth muscle.p27^<Kip1> was found to be upregulated by transcriptional upregulation in the vascular endothelial cells, when the cells formed a tight cell-to-cell contact. The promoter assay with a cloned Kip1 gene demonstrated an increase in the promoter activity upon formation of cell contract, thus suggesting that the promoter region contains a element that respond to cell contact. Less

p27^<kip1>的一种新型亚型，一种依赖细胞周期蛋白的激酶抑制剂，已从猪主动脉内皮细胞的cDNA库中分离出来。新的同工型的N末端162氨基酸与p27^<kip1>相同，而它包含独特的18个氨基酸C-末端。发现新型的同工型对蛋白酶介导的降解具有抗性，因此将其命名为p27^<kip1r>，这是p27^<kip1>的抗降解剂。确定153-168区是其重大核定位所必需的。但是，该区域仅包含一个碱性氨基酸，发现脂肪族氨基酸在核定位信号中起功能。也就是说，p27^<kip1r>包含一个非典型的二分核定位信号。由于脂肪族氨基酸的功能取代是不完整的，因此除了强核定位外，P27^<kip1r>还显示出在细胞质中的弱但显着定位。使用绿色荧光……更多的蛋白质表达系统，P27^<kip1r>对细胞生长的影响。发现它强烈抑制血管平滑肌细胞和HeLa细胞的细胞生长，如p27^<kip1>。 p27^<kip1r>和p27^<kip1>的生长抑制作用通过去除与细胞周期蛋白和细胞周期蛋白依赖性激酶结合的N末端区域完全消除了，而这些截断的突变体则表现出显着的核定位。结果，p27^<kip1r>抑制生长的机制与p27^<kip1>相似。因此，两种ISFORM之间的结构差异与生长抑制无关。为期6周的自发性高血压大鼠（SHR）的主动脉主要表达了P27^<kip1>，而正常大鼠的主动脉（WKY）主动脉主动表达了P27^<kip1r>。另一方面，8周和13周的主动脉主要表达了p27^<kip1r>。当培养正常大鼠的主动脉平滑肌细胞时，它们会占主导地位，以p27^<kip1>。 p27^<kip1r>的表达似乎与平滑肌的增殖状态呈负相关。P27^<kip1>在血管内皮细胞中的转录上调（当细胞形成紧密的细胞对细胞对细胞对细胞接触）时，被发现通过转录上调进行更新。用克隆的KIP1基因的启动子测定法证明了形成细胞合同后启动子活性的增加，因此表明启动子区域包含对细胞接触反应的元素。较少的