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Published on: June 27, 2017
Two distinct signaling pathways activate the latent DNA binding function of p53 in a casein kinase II-independent
Abstract:
Post-translational modification of a carboxyl-terminal negative regulatory domain in vitro by either casein kinase II or protein kinase C allosterically activates the latent sequence-specific DNA binding function of p53. Reported here is a biochemical approach to determine the types of signaling pathways and enzymes that are involved in p53 activation in cells. Using a novel chromatographic method, we have been able to separate three distinct biochemical forms of p53 that have been synthesized in vivo; two are in an activated state, and one is in a latent state for sequence-specific DNA binding. The two activated forms of p53 appear to be controlled individually by either a constitutive or a UV-inducible signaling pathway. p53 lacking the COOH-terminal casein kinase II site (p53 delta 4) was characterized biochemically and used to determine the affects of deletion of the casein kinase II motif on the production of the two activated forms of p53 in vivo. As observed with full-length p53, the production of two distinct chromatographic forms of activated p53 delta 4 occurs in vivo, indicating that p53 activation can occur through a casein kinase II-independent pathway and suggesting that two other factors are involved in activation of p53 in vivo.
Insights
p53 protein activation involves post-translational modifications. This study identifies distinct biochemical forms of p53 and reveals casein kinase II-independent pathways contributing to its activation in cells.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Signaling
Background:
- The tumor suppressor protein p53 plays a critical role in cellular responses to stress.
- p53's sequence-specific DNA binding activity is regulated by post-translational modifications, particularly phosphorylation.
- Casein kinase II (CK2) and protein kinase C (PKC) have been implicated in the in vitro activation of p53.
Purpose of the Study:
- To biochemically investigate the signaling pathways and enzymes responsible for p53 activation in vivo.
- To characterize the different biochemical forms of p53 synthesized within cells.
- To determine the role of the carboxyl-terminal CK2 phosphorylation site in p53 activation.
Main Methods:
- Development of a novel chromatographic method for separating p53 protein forms.
- Biochemical characterization of wild-type p53 and a p53 mutant lacking the CK2 phosphorylation site (p53 Δ4).
- In vivo analysis of p53 forms produced under different cellular conditions.
Main Results:
- Three distinct in vivo biochemical forms of p53 were identified: one latent and two activated states.
- The two activated p53 forms are differentially regulated by constitutive and UV-inducible signaling pathways.
- p53 Δ4 lacking the CK2 site still produced two activated forms, indicating CK2-independent activation pathways.
Conclusions:
- p53 activation in vivo is a complex process involving multiple biochemical states.
- Both constitutive and stress-induced signaling pathways contribute to p53 activation.
- p53 activation can occur independently of CK2 phosphorylation, suggesting the involvement of other regulatory factors.
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