TOK-1, a novel p21(Cip1)-binding protein that cooperatively enhances p21-dependent inhibitory activity toward CDK2 kinase

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 ad dition to binding of the cyclin-dependent kinase to the N-terminal region o f p21, p21 is also bound at its C-terminal region by proliferating cell nuc lear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile fun ction 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 splic ing isoforms of TOK-1, TOK-1 alpha and TOK-1 beta, comprising 322 and 314 a mino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1 alpha, b ut not TOK-1 beta, 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-1 al pha also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furtherm ore, the results of three different types of experiments showed that TOK-1 alpha enhanced the inhibitory activity of pal toward histone Ill kinase act ivity of CDK2. TOK-1 alpha is thus thought to be a new type of CDK2 modulat or.