Phosphorylation of p53 is a mechanism for responding to signaling events initialized by DNA damage or other stresses. To characterize the sites in human p53 that become phosphorylated in response to DNA damage, we have developed polyclonal antibodies that recognize p53 only when it is phosphorylated at specific sites. Several attempts to generate an antibody to p53 phosphorylated at Ser 6 using a phosphoserine-containing peptide as an immunogen were unsuccessful; however, phosphorylation-specific antibodies were produced by using the phosphoserine mimetic, l-2-amino-4-phosphono-4, 4-difluorobutanoic acid (F(2)Pab), in place of phosphoserine. Affinity-purified antibodies elicited by immunizing rabbits with an F(2)Pab peptide coupled to keyhole limpet hemocyanin recognized a p53(1-39) peptide phosphorylated only at Ser 6. Untreated A549 cells exhibited a background of constitutive phosphorylation at Ser 6 that increased approximately 10-fold upon exposure to either ionizing radiation or UV light. Similar results were obtained for Ser 9 using antibodies raised against a conventional phosphopeptide. Ser 9 was phosphorylated by casein kinase 1 in vitro in a phospho Ser 6-dependent manner. Our data identify two additional DNA damage-induced phosphorylations in human p53 and show that F(2)Pab-derivatized peptides can be used to develop phosphorylation site-specific polyclonal antibodies. Ser15 in human p53 (corresponding to Ser18 of mouse p53) is phosphorylated by ATM family kinases in response to ionizing radiation (IR) and UV light. To determine the effects of phosphorylation of endogenous murine p53 at Ser18 on biological responses to DNA damage, we introduced a missense mutation (Ser18 to Ala) by homologous recombination into both alleles of the endogenous p53 gene in mouse embryonic stem (ES) cells. Our analyses showed that phosphorylation of murine p53 at Ser18 in response to IR or UV radiation was required for a full p53-mediated response to these DNA damage-inducing agents. In contrast, phosphorylation of p53 at Ser18 was not required for ATM-dependent cellular resistance following exposure to IR. Additionally, efficient acetylation of the C-terminus of p53 in response to DNA damage did not require phosphorylation of murine p53 at Ser18. Activation of p53-mediated transcription is essential for cell cycle arrest, but its importance for apoptosis remains controversial. To address this question, we employed homologous recombination and LoxP/Cre-mediated deletion to produce mutant murine embryonic stem cells that express p53 with Gln and Ser in place of Leu 25 and Trp 26, respectively. p53(Gln25Ser26) was stable and nuclear and did not accumulate after DNA damage; p21/Waf1 expression was not induced. After exposure of mutant ES cells to UV light, p53(Gln25Ser26) was not acetylated and its DNA binding activity did not increase. More importantly, p53(Gln25Ser26) ES cells, like p53-/- cells, did not undergo DNA damage-induced apoptosis. The tumor suppressor p53 induces cellular senescence in response to oncogenic signals. p53 activity is modulated by protein stability and post-translational modifications, including phosphorylation and acetylation. The mechanism of p53 activation by oncogenes remains largely unknown. Recently, we have reported that the tumor suppressor PML regulates the p53 response to oncogenic signals. We found that oncogenic Ras upregulates PML expression, and overexpression of PML induces senescence in a p53-dependent manner. p53 is acetylated at Lysine 382 upon Ras expression, an event that is essential for its biological function. Ras induces re-localization of p53 and the CBP acetyltransferase within the PML nuclear bodies and induces the formation of a trimeric p53-PML-CBP complex. Lastly, Ras-induced p53 acetylation, p53-CBP complex stabilization and senescence are lost in PML-/- fibroblasts. These data establish a link between PML and p53 and indicate that integrity of the PML bodies is required for p53 acetylation and senescence upon oncogene expression.
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