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Phosphorylation of the tumor suppressor protein p53 by mitogen-activated protein kinases
D M Milne1, D G Campbell, F B Caudwell
1Department of Biochemistry, University of Dundee, United Kingdom.
Abstract:
The p53 tumor suppressor protein is tightly regulated in the cell and is phosphorylated at multiple sites by several different protein kinases. We have investigated the phosphorylation of p53 by mitogen-activated protein (MAP) kinase, a protein kinase that plays a central role in mediating many mitogenic and differentiation signals. Recombinant wild-type mouse p53 was phosphorylated in vitro by activated recombinant p42-MAP kinase but not by inactive MAP kinase or by the activating protein, MAP kinase kinase. Phosphorylation of p53 by MAP kinase occurred at two N-terminal sites, threonine residues 73 and 83. Tryptic phosphopeptides of recombinant p53 phosphorylated in vitro by MAP kinase comigrated on two-dimensional maps with p53 from SV3T3 cells labeled in vivo with [32P]orthophosphate, suggesting that MAP kinase targets a site in p53 that is phosphorylated in the cell. Following serum stimulation of quiescent C57MG cells, two p53 kinases, which were resolved by chromatography on Mono Q, were stimulated 15-20-fold within 5 min. Each of these kinase activities co-eluted with myelin basic protein kinase activity and could be inactivated following treatment with protein phosphatase 2A, a serine/threonine phosphatase, or leukocyte antigen receptor, a protein tyrosine phosphatase, suggesting that these activities were members of the MAP kinase family. The two kinase activities from the lysates targeted the same phosphorylation sites on p53 as the purified recombinant MAP kinase. These protein kinase activities were also stimulated following exposure of the cells to ultraviolet radiation, but with slightly delayed kinetics. Phorbol ester treatment of SV3T3 cells led to increased phosphorylation of the peptide containing the residues targeted by MAP kinase. The data suggest that p53 may be phosphorylated by MAP kinase physiologically and that this interaction may be involved in the cell's response to UV exposure, growth factor stimulation, or transformation by oncogenes.
Insights
Mitogen-activated protein (MAP) kinase phosphorylates the p53 tumor suppressor protein at specific sites. This interaction is physiologically relevant and may play a role in cellular responses to growth factors and UV radiation.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The p53 tumor suppressor protein is crucial for cellular regulation and is modified by phosphorylation.
- Mitogen-activated protein (MAP) kinase is a key signaling molecule involved in cell growth and differentiation.
Purpose of the Study:
- To investigate the direct phosphorylation of p53 by MAP kinase.
- To identify the specific sites of p53 phosphorylation by MAP kinase.
- To determine the physiological relevance of p53 phosphorylation by MAP kinase in cellular responses.
Main Methods:
- In vitro phosphorylation assays using recombinant wild-type mouse p53 and activated p42-MAP kinase.
- Two-dimensional phosphopeptide mapping to compare in vitro and in vivo phosphorylation sites.
- Chromatographic separation (Mono Q) of cellular kinases and characterization of their activity.
- Stimulation assays using serum, UV radiation, and phorbol ester.
Main Results:
- MAP kinase directly phosphorylates p53 at N-terminal threonine residues 73 and 83 in vitro.
- In vivo and in vitro phosphorylation sites on p53 were found to be identical.
- Two distinct MAP kinase family members in C57MG cells were stimulated by serum and UV radiation, phosphorylating p53 at the same sites.
- Phorbol ester treatment also increased p53 phosphorylation at these sites.
Conclusions:
- MAP kinase physiologically phosphorylates p53 at specific N-terminal sites.
- p53 phosphorylation by MAP kinase is likely involved in cellular responses to growth factors, UV exposure, and oncogenic transformation.