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Cloning and characterization of a novel p21(Cip1/Waf1)-interacting zinc finger protein, ciz1
K Mitsui1, A Matsumoto, S Ohtsuka
1Institute of Life Science, Kurume University, Aikawa-machi 2432-3, Kurume, 839-0861, Japan.
Abstract:
p21(Cip1/Waf1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. Here, we report a novel p21(Cip1/Waf1)-interacting protein, Ciz1 (for Cip1 interacting zinc finger protein), which contains polyglutamine repeats and glutamine-rich region in the N-terminus as well as three zinc-finger motifs and one MH3 (matrin 3-homologous domain 3) in the C-terminal region. Ciz1 bound to the N-terminal, the CDK2-interacting part of p21(Cip1/Waf1), and the interaction was disrupted by the overexpression of CDK2. A region of about 150 amino acids containing the first zinc-finger motif in Ciz1 was the binding site for p21(Cip1/Waf1). When Ciz1 and p21(Cip1/Waf1) were individually overexpressed in U2-OS cells, they mostly localized in the nucleus. However, coexpression of Ciz1 induced cytoplasmic distribution of p21(Cip1/Waf1). These data indicate that Ciz1 is a unique nuclear protein that regulates the cellular localization of p21(Cip1/Waf1).
Insights
A newly discovered protein, Ciz1 (Cip1 interacting zinc finger protein), interacts with p21(Cip1/Waf1), a cell-cycle inhibitor. Ciz1 influences where p21(Cip1/Waf1) is located within the cell, impacting its function.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- p21(Cip1/Waf1) is a key regulator of the cell cycle, inhibiting progression by interacting with G1 cyclin/CDK complexes and PCNA.
- Understanding proteins that modulate p21(Cip1/Waf1) function is crucial for comprehending cell-cycle control.
Purpose of the Study:
- To identify and characterize novel proteins that interact with p21(Cip1/Waf1).
- To investigate the functional consequences of the interaction between p21(Cip1/Waf1) and its novel binding partner.
Main Methods:
- Co-immunoprecipitation assays to identify interacting proteins.
- Yeast two-hybrid screening to confirm interactions.
- Overexpression studies in U2-OS cells to analyze protein localization.
- Site-directed mutagenesis to map interaction domains.
Main Results:
- A novel p21(Cip1/Waf1)-interacting protein, Ciz1 (Cip1 interacting zinc finger protein), was identified.
- Ciz1 binds to the N-terminal CDK2-interacting domain of p21(Cip1/Waf1).
- Coexpression of Ciz1 alters the nuclear localization of p21(Cip1/Waf1) to the cytoplasm.
Conclusions:
- Ciz1 is a novel nuclear protein that interacts with p21(Cip1/Waf1).
- Ciz1 regulates the cellular localization of p21(Cip1/Waf1), suggesting a role in modulating cell-cycle progression.
- This interaction provides new insights into the regulatory mechanisms of cell-cycle control.