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

Abstract

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-1α and TOK-1β, 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-1α, but not TOK-1β, directly bound to the C-terminal proximal region of p21, and both were expressed at the G1/S boundary of the cell cycle. TOK-1α 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-1α enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1α is thus thought to be a new type of CDK2 modulator.