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DNA damage triggers DRB-resistant phosphorylation of human p53 at the CK2 site
1Department of Molecular and Cellular Pathology, Ninewells Medical School, The University of Dundee, Scotland, UK.
Abstract:
The sequence-specific DNA binding activity of p53 is negatively regulated by a C-terminal domain whose phosphorylation in vitro can activate the latent DNA binding function of the protein. The DNA binding activity of p53 is a core component of its stress-activated transcription function, yet it is not yet clear whether phosphorylation within the C-terminal domain plays a role in the p53 damage response in vivo. As the casein kinase 2 (CK2) site at serine 392 is the C-terminal phosphorylation motif that exhibits the most pronounced conservation at the primary amino acid level, we have focused on determining whether the CK2 site is modified in vivo and whether radiation effects the extent of that phosphorylation. Using antibodies that can detect serine 392-phosphorylation of p53, we demonstrate that UV radiation can trigger extensive phosphorylation at the CK2 site. The CK2 inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), can partially inhibit the UV-induced phosphorylation at serine 392, suggesting that CK2 is one of the major serine 392-kinases. However, a striking increase in UV-induced serine 392 phosphorylation and p53 transactivation function at higher levels of DRB suggests that a DRB-resistant/stress-activated pathway may target serine 392 in vivo. These data demonstrate that radiation-induced phosphorylation of p53 can occur in vivo at serine 392 and implicate a CK2-independent signal cascade that can function to modulate serine 392 phosphorylation in cells.
Insights
Radiation exposure triggers p53 protein phosphorylation at serine 392 in vivo. This stress-activated pathway involves casein kinase 2 (CK2) and a distinct DRB-resistant signaling cascade, impacting p53 DNA binding activity.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Cancer Research
Background:
- The tumor suppressor protein p53 regulates transcription in response to cellular stress.
- p53's DNA binding activity is crucial for its function but is negatively regulated by its C-terminal domain.
- Phosphorylation of the C-terminal domain can modulate p53's DNA binding and transcriptional activity in vitro, but its in vivo role in stress responses remains unclear.
Purpose of the Study:
- To investigate whether the conserved casein kinase 2 (CK2) phosphorylation site at serine 392 in p53 is modified in vivo.
- To determine if radiation exposure affects the extent of serine 392 phosphorylation.
- To explore the kinases involved in p53 serine 392 phosphorylation during cellular stress.
Main Methods:
- Utilized antibodies specific for serine 392-phosphorylated p53.
- Exposed cells to UV radiation and treated with the CK2 inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole).
- Assessed p53 phosphorylation levels and transactivation function.
Main Results:
- UV radiation induced significant phosphorylation of p53 at serine 392 in vivo.
- The CK2 inhibitor DRB partially reduced UV-induced serine 392 phosphorylation, indicating CK2's role.
- Higher DRB concentrations led to increased UV-induced serine 392 phosphorylation and p53 transactivation, suggesting a DRB-resistant pathway.
Conclusions:
- Radiation-induced phosphorylation of p53 at serine 392 occurs in vivo.
- A CK2-independent signaling pathway contributes to serine 392 phosphorylation during stress.
- These findings highlight a novel stress-activated mechanism modulating p53 function.