The p53 tumour suppressor protein is a labile transcription factor that is activated and stabilized in response to a wide range of cellular stresses, through a mechanism involving disruption of its interaction with MDM2, a negative regulatory partner. Induction of p53 by DNA damage additionally involves a series of phosphorylation and acetylation modifications, some of which are thought to regulate MDM2 binding. Here we report the effects of introducing mutations at several known or putative N-terminal phosphorylation sites on the transactivation function of p53. These studies highlight phosphorylation of Ser15, a key phosphorylation target during the p53 activation process, as being critical for p53-dependent transactivation. Biochemical data indicate that the mechanism by which phosphorylation of Ser15 stimulates p53-dependent transactivation occurs through increased binding to the p300 coactivator protein. The data also indicate that Ser15-dependent regulation of transactivation is independent of any involvement in modulating MDM2 binding, and that Ser15 phosphorylation alone is not sufficient to block the p53-MDM2 interaction.
Melanocytes require the RAS/RAF/MEK/ERK and the cyclic AMP (cAMP) signaling pathways to maintain the fine balance between proliferation and differentiation. We have investigated how cross-talk between these pathways affects melanoma progression. We show that cAMP suppresses CRAF activity in melanocytes and that this is essential to suppress the oncogenic potential of CRAF in these cells. As a consequence, BRAF alone is responsible for signaling to MEK. However, when RAS is mutated in melanoma, the cells switch their signaling from BRAF to CRAF. This switch is accompanied by dysregulated cAMP signaling, a step that is necessary to allow CRAF to signal to MEK. Thus, a fundamental switch in RAF isoform usage occurs when RAS is mutated in melanoma, and this occurs in the context of disrupted cAMP signaling. These data have important implications for the development of therapeutic strategies to treat this life-threatening disease.
We also show that Raf-1 and protein kinase A (PKA) form a complex in vivo that is disrupted by cAMP and that ablation of PKA by use of small interfering RNA blocks phosphorylation by cAMP. The ability of PKA to block Raf-1 activation is ablated by the PKA inhibitor H89. These studies suggest that Raf-1 and cAMP form a signaling complex in cells. Upon activation of PKA, Raf-1 is phosphorylated and 14-3-3 binds, blocking Raf-1 recruitment to the plasma membrane and preventing its activation.In mammalian cells, growth factors, mitogens and hormones stimulate the activity of the extracellular signalregulated protein kinases ERK1 and ERK2.
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