Regulation of DNA-damage response by phosphorylation clusters in the p53 signaling network

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The transcription factor p53 coordinates the cellular response to DNA damage. P53 protein level and activity are controlled by a signaling network comprising amongst others the DNA-damage response (DDR) kinases ATM/ATR/DNA-PK (PIKKs), the E3-ubiquitin ligase Mdm2, the phosphatase Wip1 and the kinase Chk2. The activity of the kinases/phosphatases and reciprocal feedbacks generate repeated p53 accumulation pulses, whose duration and number determine p53-mediated transcriptional responses and cell fate. The current view of the system is that i) constitutive expression of p53 and feedback regulation by its E3-ubiquitin ligase Mdm2 maintain low basal protein levels; ii) PIKKs activate p53 and inhibit Mdm2 as long as DNA-damage is present, iii) p53 accumulation triggers Mdm2 and Wip1 expression; iv) Wip1 reverses PIKK-mediated modifications. While these activities largely explain the observed p53 dynamics, recent studies using pharmacological perturbations and biochemical measurements provided evidence for additional regulatory mechanisms.Our groups have focused on unexplored phosphorylation mechanisms in the p53-DDR network: i) proteins show abundant, clustered (de)phosphorylation sites of PIKKs, Chk2, and Wip1, which affect the modification kinetics of key functional phosphosites; ii) sustained p53 pulses depend on Chk2 rather than PIKK activity. Based on these findings, we hypothesize that i) clustered modification sites act as buffers to set thresholds and molecular timers upon DNA-damage, and ii) ATM triggers p53 pulses in response to acute damage, while Chk2 is responsible to maintain p53 activity and enable cellular responses to sustained damage. We propose to validate this model by combining experiments at the biochemical and cellular levels. We will evaluate how the presence of clustered phosphosites shapes the kinetics of the acute and sustained DDR. To this end, we will focus on the modification of Mdm2, Chk2 and Wip1 and how it affects their activity. Specifically, we will i) delineate the competitive activities of PIKKs, Chk2, and Wip1 on p53, Mdm2, Chk2 and Wip1 in a site-specific and quantitative manner using NMR spectroscopy; ii) carry out structural studies to elucidate Mdm2, Chk2 and Wip1 inhibition/destabilization by their phosphorylation; iii) examine the functional role of individual phosphosites using Cas9-mediated genomic engineering and time-resolved live-cell microscopy; iv) evaluate how the balance between PIKKs, Chk2 and Wip1 activities shapes the DDR in cells. This will allow us to build mathematical models accounting for DNA-damage thresholds and long-term dynamics of the p53-driven DDR. Hence, integrating biochemical and structural information, real-time cellular signaling data and a systems biology approach, we will gain a better understanding of the p53-driven DDR and how to manipulate it in the context of cancer therapy.