Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule
Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule
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DOI：
10.1038/sj.onc.1201580
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发表时间：
1998-02-05
期刊：
影响因子：
8
通讯作者：
Lane, DP
中科院分区：
文献类型：
--
作者：
Fåhraeus, R;Laín, S;Lane, DP
我们以前已经证明，来自p16(CDKN2/INK4a)(P16)肿瘤抑制蛋白(人p16蛋白的84-103个残基)第三个Ankyrin-like重复序列的20个氨基酸的多肽可以与CDK4和CDK6结合，在体外抑制CDK4-Cyclin D1的活性，并通过G1阻断细胞周期进程。与p16多肽的95和96位氨基酸(V95A和V96A)对应的两个Valine残基的替换减少了与CDK4和CDK6的结合，当连接到Antennapedia同源域载体序列时，其对激酶抑制的IC0.5增加了近三倍。同样的突变使p16蛋白中的IC0.5增加了大约五倍。天冬氨酸92被丙氨酸取代，反而增加了多肽与CDK4和CDK6的结合以及激酶抑制活性。在24 mM浓度下，p16多肽可阻断非同步化的人HaCaT细胞进入S相，阻断作用约为90%。V95A和V96A双取代使多肽的细胞周期抑制能力最小化，而D92A取代增加了其阻止细胞周期进展的能力。P16衍生多肽的缺失序列表明，10个残基的多肽仍然具有CDK4-细胞周期蛋白D1激酶和细胞周期抑制活性。与全长p16蛋白一样，p16多肽在体外不抑制细胞周期蛋白E依赖的细胞周期蛋白CDK2的活性。这些数据表明，来自INK4家族的一种多肽对CDK-Cyclin D活性的急性抑制以一种pRb依赖的方式阻止细胞在G1晚期。
We have previously shown that a 20 amino acid peptide derived from the third ankyrin-like repeat of the p16(CDKN2/INK4a) (p16) tumour suppressor protein (residues 84-103 of the human p16 protein) can bind to cdk4 and cdk6 and inhibit cdk4-cyclin D1 kinase activity in vitro as well as block cell cycle progression through G1. Substitution of two valine residues corresponding to amino acids 95 and 96 (V95A and V96A) of the p16 peptide reduces the binding to cdk4 and cdk6 and increases its IC0.5 for kinase inhibition approximately threefold when linked to the Antennapedia homeodomain carrier sequence. The same mutations increase the IC0.5 approximately fivefold in the p16 protein. Substitution of aspartic acid 92 by alanine instead increases the binding of the peptide to cdk4 and cdk6 and the kinase inhibitory activity. The p16 peptide blocks S-phase entry in nonsynchronized human HaCaT cells by approximately 90% at a 24 mu M concentration. The V95A and V96A double substitution minimizes the cell cycle inhibitory capacity of the peptide whereas the D92A substitution increases its capacity to block cell cycle progression. A deletion series of the p16 derived peptide shows that a 10 residue peptide still retains cdk4-cyclin D1 kinase and cell cycle inhibitory activity. The p16 peptide inhibited S-phase entry in five cell lines tested, varying between 47-75%, but had only a limited (11%) inhibitory effect in the pRb negative Saos-2 cells at a concentration of 24 mu M. Like the full length p16 protein, the p16 peptide does not inhibit cyclin E dependent cdk2 kinase activity in vitro. These data suggest that acute inhibition of CDK-cyclin D activity by a peptide derived from the INK4 family mill stop cells in late G1 in a pRb dependent fashion.