The p53 tumor suppressor protein is a critical regulator of cell cycle progression that responds to DNA damage and other cellular stresses by arresting cell cycle progression or by inducing apoptosis. The p53 protein is stabilized in response to DNA damage, which also activates p53 as a transcription factor. Both stabilization of the p53 protein and activation of its sequence-specific DNA binding ability are widely believed to be mediated, at least in part, by post-translational modifications to the p53 protein. p53 is phosphorylated on several sites (15,33 and 37) in its N-terminal transactivation domain as well as on several sites in the C-terminal tetramerization/regulatory domain after cells are exposed either to ionizing radiation (IR) or to UV light. The N-terminal segment of p53 also binds Mdm2, a protein that targets p53 for degradation through a ubiquitin-mediated mechanism. Recently, we have shown that the direct association between p53 and Mdm2 is disrupted by phosphorylation of p53 on Thr-18 but not by phosphorylation at other N-terminal sites. Thr-18 was phosphorylated in vitro by casein kinase 1 (CK1); phosphorylation by CK1 required prior phosphorylation of Ser-15. Thr-18 was phosphorylated in vivo in response to DNA damage, and phosphorylation required Ser-15. These results suggest that the initial stabilization of p53 after ionizing radiation may result from inhibiting Mdm2 binding through a DNA damage- activated phosphorylation-phosphorylation cascade. Expression of oncogenic ras is associated with activation of cell cycle arrest and senescence in a p53-dependent manner. We have analyzed various downstream components of the ras signaling pathway for their contribution to the activation of cellular p53 . In collaboration with Drs.D. Bulavin and A. Fornace, our results indicate that p38 kinase had a pronounced effect on p53 activation in comparison to other members of the ras pathway. In vitro, p38 kinase phosphorylated p53 at serines 33 and 46. Mutation of these sites resulted in a significant decrease in the ability of p53 to trigger apoptosis. These results indicate that p38 kinase has a prominent role in the regulation of p53-dependent apoptosis after UV radiation. DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have indicated that four molecules of the DNA binding domain of human p53 (p53DBD) bind a response element with high cooperativity and bend the DNA. However, using A-tract phasing experiments, we find significant differences in the bending and twisting of DNA upon binding by p53DBD and full length human wild type p53 (WT p53). Our data show that four subunits of p53DBD and WT p53 bend the DNA by 30-35o and 50-55o respectively, and that the directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. Phasing analyses also demonstrate that p53DBD and WT p53 overtwist the DNA response element by ~35o and ~70o, respectively and these results are in accord with molecular modeling studies of the tetrameric complex. Overall, the four p53 moieties are associated laterally in a staggered array at the external side of the DNA loop with numerous inter-protein interfaces increasing the stability and cooperativity of binding. This novel architectural organization of the p53 tetrameric complex may have important functional implications including possible p53 interactions with chromatin. The activities of p53 leading to cell cycle arrest or apoptosis are restricted in unstressed cells both by the low level of p53 and by negative regulation through its carboxy terminal domain. The carboxy-terminal domain of p53 binds to non-sequence specific DNA as well as some unusual DNA structures. We have recently observed that p53 binds preferentially to supercoiled DNA via its C-terminal domain. This preferential binding does not result from an unwinding of the DNA by p53 nor a chiral wrapping of the DNA around the p53 and occurs with highly positively as well as highly negatively supercoiled DNA. These observations suggest that p53 preferentially interacts with DNA crossovers and may provide a mechanism for retaining latent p53 near actively transcribed DNA. - DNA binding domain, equilibrium centrifugation, modeling system, p, protein, phosphorylated peptides, - Neither Human Subjects nor Human Tissues
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