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TOK-1, a novel p21Cip1-binding protein that cooperatively enhances p21-dependent inhibitory activity toward CDK2
1Graduate School of Pharmaceutical Sciences, College of Medical Technology, Hokkaido University, Kita-ku, Sapporo 060, Japan.
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-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.
Insights
A novel protein, TOK-1alpha, binds to p21 and enhances its cell-cycle inhibitory activity. This discovery identifies TOK-1alpha as a potential modulator of cyclin-dependent kinase 2 (CDK2).
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- p21 (also known as Cip1/Waf1/Sdi1) is a key negative regulator of the cell cycle.
- p21 interacts with various proteins, including cyclin-dependent kinases (CDKs) and proliferating cell nuclear antigen (PCNA), through distinct regions.
- The C-terminal region of p21 binds to multiple proteins, suggesting diverse functional roles.
Purpose of the Study:
- To identify novel p21 C-terminal-binding proteins using a two-hybrid system.
- To characterize the interaction of the novel protein TOK-1 with p21 and its isoforms.
- To investigate the functional consequence of TOK-1 binding on p21's activity towards cyclin-dependent kinase 2 (CDK2).
Main Methods:
- Yeast two-hybrid screening to identify p21-interacting proteins.
- Cloning and characterization of TOK-1 splicing isoforms (TOK-1alpha and TOK-1beta).
- Co-localization studies in human cells.
- Analysis of protein-protein interactions using ternary complex formation.
- In vitro kinase assays to measure histone H1 kinase activity of CDK2.
Main Results:
- A novel p21 C-terminal-binding protein, TOK-1, was identified.
- Two splicing isoforms, TOK-1alpha and TOK-1beta, were characterized, both localizing to the nucleus and showing similar expression patterns to p21.
- TOK-1alpha, but not TOK-1beta, directly bound to the C-terminal region of p21.
- TOK-1alpha formed a ternary complex with p21 and an active form of CDK2.
- TOK-1alpha significantly enhanced the inhibitory effect of p21 on CDK2 histone H1 kinase activity.
Conclusions:
- TOK-1alpha is a novel binding partner of p21.
- TOK-1alpha modulates the interaction between p21 and CDK2.
- TOK-1alpha acts as a positive modulator of p21's cell-cycle inhibitory function, potentially serving as a new type of CDK2 modulator.