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Phosphorylation regulates the interaction and complex formation between wt p53 protein and PARP-1
Józefa Wesierska-Gadek1, Jacek Wojciechowski, Gerald Schmid
1Cell Cycle Regulation Unit, Institute of Cancer Research, University of Vienna, Vienna, Austria.
Journal of Cellular Biochemistry
|August 5, 2003
Summary
The interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the tumor suppressor p53 is regulated by p53 phosphorylation. PARP-1 presence controls basal p53 expression in unstressed cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The tumor suppressor p53 is a critical regulator of cellular responses to stress.
- Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair and other cellular processes.
- The interaction between p53 and PARP-1 is not fully understood, particularly the roles of specific protein domains and post-translational modifications.
Purpose of the Study:
- To identify the specific domains of PARP-1 and p53 involved in their protein-protein interaction.
- To investigate the role of p53 phosphorylation in regulating the binding between PARP-1 and p53.
- To elucidate the functional significance of the PARP-1 and p53 interaction on p53 expression.
Main Methods:
- Baculoviral expression systems were used to generate full-length and truncated versions of human PARP-1 and p53.
- Co-expression in insect cells followed by reciprocal immunoprecipitation to assess protein binding.
- Western blotting with phospho-specific antibodies and 2D gel electrophoresis to analyze p53 phosphorylation.
- Pull-down assays with dephosphorylated p53 to determine the necessity of phosphorylation for binding.
Main Results:
- The central and carboxy-terminal fragments of p53, but not the amino-terminal domain, were sufficient for PARP-1 binding.
- The amino-terminal and central fragments of PARP-1 were necessary for complex formation with p53.
- Phosphorylation of p53 at multiple sites, including Ser392, was essential for its binding to PARP-1.
- Dephosphorylation of p53 abolished its interaction with PARP-1.
- PARP-1 presence was shown to regulate the basal expression of wild-type p53 in unstressed cells.
Conclusions:
- The interaction between PARP-1 and p53 is mediated by specific domains of both proteins and is critically dependent on the phosphorylation status of p53.
- Phosphorylation of p53, particularly at Ser392, facilitates its interaction with PARP-1, potentially regulating p53 nuclear export.
- PARP-1 plays a regulatory role in controlling the basal expression levels of wild-type p53.