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

从<Kip1>猪主动脉内皮细胞的cDNA文库中分离到一种新的细胞周期蛋白依赖性激酶抑制剂p27 α亚型。新亚型的N-末端162个氨基酸与p27 α的那些相同<Kip1>，而它含有独特的18个氨基酸的C-末端。发现新的同种型对蛋白酶介导的降解具有抗性，因此将其命名为p27 β<Kip1R>，p27 β的降解抗性同种型<Kip1>。153-168区域被确定为是其重要核定位所必需的。然而，该区域仅包含一个碱性氨基酸，并且发现脂肪族氨基酸在核定位信号中发挥功能性作用。也就是说，p27^<Kip1R>包含非典型的二分核定位信号。由于被脂肪族氨基酸的功能取代是不完全的，<Kip1R>除了强的核定位之外，p27 α还表现出在胞质溶胶中的弱但显著的定位。 ...更多信息 蛋白表达系统中，研究p27 ~+<Kip1R>对细胞生长的影响。发现其强烈抑制血管平滑肌细胞和HeLa细胞的细胞生长，如在p27的情况下<Kip1>。通过<Kip1R><Kip1>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，p27^和p27^的生长抑制作用被完全消除，而这些截短的突变体表现出显著的核定位。因此，p27 α抑制生长的机制<Kip1R>与p27 α相似<Kip1>。6周龄自发性高血压大鼠（SHR）主动脉主要表达p27 α<Kip1>，而正常大鼠（WKY）主动脉主要表达p27 α<Kip1R>。8周龄和13周龄SHR主动脉主要表达p27<Kip1R>。正常大鼠主动脉平滑肌细胞培养时主要表达p27<Kip1>。p27^的表达<Kip1R>似乎与平滑肌的增殖状态呈负相关。<Kip1>当细胞形成紧密的细胞-细胞接触时，发现p27 ^通过血管内皮细胞中的转录上调而上调。用克隆的Kip 1基因的启动子测定证明了在形成细胞接触后启动子活性的增加，从而表明启动子区域含有响应细胞接触的元件。少

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