|Budget Amount *help
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥2,600,000 (Direct Cost: ¥2,600,000)
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
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