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p53 Antiproliferative function is enhanced by aspartate substitution at threonine 18 and serine 20
1Department of Pathology, Cancer Genomics Laboratory, Graduate Program in Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Abstract:
Previous studies have demonstrated the irradiation-induced phosphorylation of p53 at Thrl8 and Ser20, residues integral within an a-helical segment of the transactivation domain. Importantly, phosphorylation at either site has been correlated with decreased binding to the inhibitory partner Mdm-2 and enhanced transactivation of p53 target genes. In this study, we investigated the impact of Asp substitution at Thrl8 and Ser20 (p53Tl8D/S20D) on the functional regulation of p53. Asp substitution is commonly accepted as a means of mimicking phosphorylation due to the introduction of negative charge within the functional group. p53T18D/S20D was refractory to in vitro digestion by calpain, a protease recognizing a-helical structure within the transactivation domain. In addition, transfected p53T18D/S20D poorly bound GST-Mdm-2 in vitro, enhanced the endogenous expression of the p53 transactivation targets p21(Waf1/Cip1) and fas/APO-1, and significantly curtailed cell proliferation relative to wild-type p53 transfected cells. Thus, Asp substitution at Thr18 and Ser20 within the a-helical segment of the transactivation domain reduced Mdm-2 interaction, upregulating transactivation of cell-cycle and apoptotic regulatory targets, curtailing cellular proliferation.
Insights
Mimicking phosphorylation of p53 at Thr18 and Ser20 with Asp substitution enhanced its tumor suppressor activity. This modification reduced Mdm-2 binding, boosting target gene expression and inhibiting cell proliferation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- p53 phosphorylation at Thr18 and Ser20 is induced by irradiation.
- Phosphorylation at these sites decreases Mdm-2 binding and enhances p53 target gene transactivation.
Purpose of the Study:
- To investigate the functional impact of Asp substitution at Thr18 and Ser20 (p53Tl8D/S20D) on p53 regulation.
- To mimic phosphorylation using Asp substitution to introduce a negative charge.
Main Methods:
- Investigated p53Tl8D/S20D resistance to calpain digestion.
- Assessed GST-Mdm-2 binding of p53Tl8D/S20D in vitro.
- Measured endogenous expression of p53 targets p21(Waf1/Cip1) and fas/APO-1.
- Compared cell proliferation rates between p53Tl8D/S20D and wild-type p53 transfected cells.
Main Results:
- p53Tl8D/S20D was resistant to calpain digestion.
- p53Tl8D/S20D exhibited reduced binding to GST-Mdm-2.
- Transfected p53Tl8D/S20D enhanced p21(Waf1/Cip1) and fas/APO-1 expression.
- p53Tl8D/S20D significantly curtailed cell proliferation compared to wild-type p53.
Conclusions:
- Asp substitution at Thr18 and Ser20 mimics phosphorylation, reducing Mdm-2 interaction.
- This modification upregulates cell-cycle and apoptotic regulatory targets.
- p53Tl8D/S20D effectively curtails cellular proliferation, highlighting its role in tumor suppression.