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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Base Sequence
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Blotting, Northern
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CDC2-CDC28 Kinases*
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COS Cells
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Calcium-Binding Proteins*
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Carrier Proteins / chemistry
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Carrier Proteins / genetics*
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Carrier Proteins / metabolism
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Cell Cycle
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Cell Cycle Proteins*
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Cell Line
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Cell Nucleus / metabolism
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Cloning, Molecular
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Codon, Terminator
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclin-Dependent Kinases / metabolism*
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Cyclins / chemistry
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Cyclins / metabolism*
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DNA, Complementary / metabolism
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Fluorescent Antibody Technique, Indirect
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Glutathione Transferase / metabolism
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HeLa Cells
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Humans
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Models, Genetic
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Nuclear Proteins*
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Phosphotransferases / metabolism
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Plasmids / metabolism
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Proliferating Cell Nuclear Antigen / metabolism
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Protein Binding
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Protein Isoforms
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Protein Serine-Threonine Kinases / metabolism*
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Protein Structure, Tertiary
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Recombinant Proteins / metabolism
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Time Factors
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Tissue Distribution
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Two-Hybrid System Techniques
Substances
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BCCIP protein, human
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CDKN1A protein, human
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Calcium-Binding Proteins
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Carrier Proteins
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Cell Cycle Proteins
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Codon, Terminator
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins
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DNA, Complementary
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Nuclear Proteins
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Proliferating Cell Nuclear Antigen
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Protein Isoforms
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Recombinant Proteins
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Glutathione Transferase
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Phosphotransferases
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Protein Serine-Threonine Kinases
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CDC2-CDC28 Kinases
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CDK2 protein, human
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases
Associated data
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GENBANK/AB040450
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GENBANK/AB040451