Role of calcium calmodulin kinases in modification of the p53 signalling pathway

Abstract

P53 is a tetrameric transcription factor which exhibits DNA binding activity through its core domain which encompasses the conserved domains (known as Box II, III, IV and V). The N-terminal domain of p53 provides a scaffold for binding of components of the transcriptional machinery. Phosphorylation at residues within the N-terminal transactivation domain of p53 such as Serine 20 is a crucial event in the activation of p53. It stabilises the binding of the co-activator p300, reduces the binding of the inhibitory partner Mdm2 and enhances activation of p53 target genes. The identification of enzymes that phosphorylate p53 transactivation domain is an important development in the ongoing mapping of signaling pathways that control p53-dependent transcription and resultant tumour suppression. Environmental and physiological stresses activate p53 which has led to the creation of several hypothetical models in which tumour suppressor kinases mediate p53 activation by phosphorylation at Serine 20. Although much researched the identity of the main Serine 20 kinase in cells remains undefined. In this study we have identified Calcium Calmodulin kinase superfamily (CAMK) members as potent Serine 20 kinases in cells and show that the co-transfection of p53 peptides derived from the conserved domains can modify this response. Moreover, we show that the multi-protein docking site, p53 Box V domain, is required for Serine 20 phosphorylation and ubiquitination of p53. To further define the domains required for the interaction of p53 with CAMK superfamily members, mutagenesis of p53 was performed. Using transcriptional and binding based assays we were able to establish that p53 does indeed form an interaction with Chk1 and DAPK1. Development of cell models and gene expression studies demonstrated that depletion of Chk1 and DAPK1 results in activation of the p53 signalling pathway. There may therefore be a role for kinases as negative regulators of p53 and a potential for the development of kinases as drug targets for reactivation of the p53 pathway.