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Cyclin-dependent kinases phosphorylate p73 at threonine 86 in a cell cycle-dependent manner and negatively regulate
Christian Gaiddon1, Maria Lokshin, Isabelle Gross
1Equipe d'Accecil Signalisations Moléculaires et Neurodégénéréscence, Université Louis Pasteur, Strasbourg 67000, France. gaiddon@neurochem.u-strasbg.fr
Abstract:
p73 transcription factors are members of the p53 family and participate in developmental processes and DNA damage response. p73 expression was shown to be regulated during the cell cycle, suggesting that p73 might play a role in cell growth and might be a target for cyclin-dependent kinases. Consistent with this hypothesis, we discovered that p73 interacts physically with various cyclins (A, B, D, and E). Furthermore, cyclin A/CDK1/2, cyclin B/CDK1/2, and cyclin E/CDK2 complexes can phosphorylate multiple p73 isoforms in vitro at threonine 86. A specific antibody directed against phosphorylated Thr86 showed that this site is phosphorylated in vivo and that such phosphorylation is regulated in a cell cycle-dependent manner. Thr86 phosphorylation is induced during S phase and is maximal in the G2/M phase. Accordingly inhibitors of cell growth, such as p16 and serum starvation, reduce Thr86 phosphorylation. Finally, we found that cyclin-dependent kinase (CDK)-dependent Thr86 phosphorylation represses the ability of p73 to induce endogenous p21 expression. Our results demonstrate that p73 proteins are targets of CDK complexes and that phosphorylation on Thr86 by CDKs regulates p73 functions.
Insights
p73 proteins are regulated by cyclin-dependent kinases (CDKs) through phosphorylation at Thr86. This phosphorylation impacts p73
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- p73 transcription factors are key players in the p53 family, involved in development and DNA damage response.
- p73 expression is cell cycle-regulated, hinting at a role in cell growth and potential regulation by cyclin-dependent kinases (CDKs).
Purpose of the Study:
- To investigate the interaction between p73 and cyclins/CDKs.
- To determine if CDKs phosphorylate p73 and the functional consequences of this modification.
Main Methods:
- Co-immunoprecipitation to assess physical interactions between p73 and cyclins.
- In vitro kinase assays using cyclin/CDK complexes and p73 isoforms.
- Western blotting with a phospho-specific antibody to detect Thr86 phosphorylation in vivo.
- Cell cycle analysis and treatment with growth inhibitors (p16, serum starvation).
Main Results:
- p73 physically interacts with cyclins A, B, D, and E.
- Cyclin/CDK complexes phosphorylate p73 at Threonine 86 (Thr86) in vitro.
- Thr86 phosphorylation occurs in vivo in a cell cycle-dependent manner, peaking in G2/M phase.
- Growth inhibitors reduce Thr86 phosphorylation.
- CDK-dependent Thr86 phosphorylation represses p73's ability to induce p21 expression.
Conclusions:
- p73 proteins are direct targets of CDK complexes.
- Phosphorylation of p73 at Thr86 by CDKs is a critical regulatory mechanism controlling p73 function, including its role in gene expression.