Ono T, Kitaura H, Ugai H, Murata T, Yokoyama K K, Iguchi-Ariga S M, Ariga H
Graduate School of Pharmaceutical Sciences, College of Medical Technology, Hokkaido University, Kita-ku, Sapporo 060, Japan.
J Biol Chem. 2000 Oct 6;275(40):31145-54. doi: 10.1074/jbc.M003031200.
A p21(Cip1/Waf1/Sdi1) is known to act as a negative cell-cycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a two-hybrid system. Two splicing isoforms of TOK-1, TOK-1alpha and TOK-1beta, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1alpha, but not TOK-1beta, directly bound to the C-terminal proximal region of p21, and both were expressed at the G(1)/S boundary of the cell cycle. TOK-1alpha also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1alpha enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1alpha is thus thought to be a new type of CDK2 modulator.
已知p21（Cip1/Waf1/Sdi1）通过抑制多种细胞周期蛋白依赖性激酶的激酶活性来充当负性细胞周期调节因子。除了细胞周期蛋白依赖性激酶与p21的N端区域结合外，p21在其C端区域还与增殖细胞核抗原（PCNA）、SET/TAF1和钙调蛋白结合，这表明p21具有多种功能。在本研究中，我们通过双杂交系统克隆了编码一种名为TOK-1的新型蛋白的cDNA，该蛋白作为p21的C端结合蛋白。TOK-1的两种剪接异构体TOK-1α和TOK-1β分别由322和314个氨基酸组成，它们与p21在细胞核中共定位，并且在人体组织中的表达谱与p21相似。TOK-1α而非TOK-1β直接与p21的C端近端区域结合，并且两者均在细胞周期的G(1)/S边界表达。TOK-1α还通过p21优先与活性形式的细胞周期蛋白依赖性激酶2（CDK2）结合，并且它们在人体细胞中形成三元复合物。此外，三种不同类型实验的结果表明，TOK-1α增强了p21对CDK2组蛋白H1激酶活性的抑制活性。因此，TOK-1α被认为是一种新型的CDK2调节剂。
