Abstract
p53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. A phospho-specific monoclonal antibody to the C-terminal Ser(315) phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser(315) can be detected in vivo, whether steady-state Ser(315) phosphorylation increases or decreases in an irradiated cell, and whether this phosphorylation event activates or inhibits p53 in vivo. A native phospho-specific IgG binding assay was developed for quantitating the extent of p53 phosphorylation at Ser(315) where one, two, three, or four phosphates/tetramer could be defined after in vitro phosphorylation by cyclin-dependent protein kinases. Using this assay, near-stoichiometric Ser(315) phosphorylation of endogenous p53 protein was detected in vivo after UV irradiation of MCF7 and A375 cells, coinciding with elevated p53-dependent transcription. Transfection of the p53 gene with an alanine mutation at the Ser(315) site into Saos-2 cells gave rise to a form of p53 protein with a substantially reduced specific activity as a transcription factor. The treatment of cells with the cyclin-dependent protein kinase inhibitor Roscovitine promoted a reduction in the specific activity of endogenous p53 or ectopically expressed p53. These results indicate that the majority of p53 protein has been phosphorylated at Ser(315) after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.
Highlights
P53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression
A variety of biochemical, cellular, and genetic approaches have been developed to indicate that post-translational modification of p53 protein might be central to the control of its transactivation function
The artificial manipulation and activation of p53 in vivo by using an antibody that mimics kinases by targeting the C-terminal negative regulatory domain of p53 [14, 46, 48] or an antibody that mimics N-terminal p53-BOXI domain kinases by disrupting MDM2 binding [66] suggests that the function and stability of endogenous p53 protein in cells is dependent upon post-translational modification
Summary
P53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. P53 is composed of at least four functional domains that regulate its activity as a transcription factor: (i) an N-terminal trans-activation domain that is required for interaction with components of the transcriptional machinery, including p300 [10, 11]; (ii) the central conserved core DNAbinding domain containing most of the inactivating mutations found in human tumors [12]; (iii) a tetramerization domain [13]; and (iv) a C-terminal negative regulatory domain whose phosphorylation, acetylation, or SUMOylation correlates with activation of the latent sequence-specific DNA binding function of p53 (14 –19) Each of these domains on p53 contain multiple sites for modification by both covalent and noncovalent interactions, and it seems likely that it is the combined action of many enzymes that coordinately modulate p53-dependent gene expression in response to cellular stress. These studies identified one key signal transduction cascade that could stimulate p53-dependent transcription via modification of the N-terminal domain of p53
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