Abstract
The p53 tumor suppressor is a key mediator of the cellular response to stress. Phosphorylation induced by multiple stress-activated kinases has been proposed to be essential for p53 stabilization, interaction with transcriptional co-activators, and activation of p53 target genes. However, genetic studies suggest that stress-activated phosphorylation may not be essential for p53 activation. We therefore investigated the role of p53 phosphorylation on six key serine residues (Ser(6), Ser(15), Ser(20), Ser(37), Ser(46), and Ser(392)) for p53 activation using nutlin-3, a recently developed small molecule MDM2 antagonist. We show here that nutlin does not induce the phosphorylation of p53. Comparison of the activity of unphosphorylated and phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide in HCT116 and RKO cells revealed no difference in their sequence-specific DNA binding and ability to transactivate p53 target genes and to induce p53-dependent apoptosis. We conclude that p53 phosphorylation on six major serine sites is not required for activation of p53 target genes or biological responses in vivo.
Highlights
The tumor suppressor p53 is a transcription factor that coordinates a complex network of cellular proteins evolved to protect cells from malignant transformation [1, 2]
Comparison of the activity of unphosphorylated and phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide in HCT116 and RKO cells revealed no difference in their sequence-specific DNA binding and ability to transactivate p53 target genes and to induce p53-dependent apoptosis
Despite the lack of detectable phosphorylation, nutlin-induced p53 showed equal or better sequence-specific DNA binding, ability to transactivate p53 target genes, and p53-dependent apoptotic activity than phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide
Summary
The tumor suppressor p53 is a transcription factor that coordinates a complex network of cellular proteins evolved to protect cells from malignant transformation [1, 2]. Comparison of the activity of unphosphorylated and phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide in HCT116 and RKO cells revealed no difference in their sequence-specific DNA binding and ability to transactivate p53 target genes and to induce p53-dependent apoptosis. Despite the lack of detectable phosphorylation, nutlin-induced p53 showed equal or better sequence-specific DNA binding, ability to transactivate p53 target genes, and p53-dependent apoptotic activity than phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide.
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