p63 sets the threshold for induction of apoptosis using a kinetically encoded ‘doorbell-like’ mechanism

Abstract

27 quality control, ovarian reserve, premature ovarian insufficiency, light sheet microscopy 28 29 Abbreviations: DSBs, DNA double strand breaks; POI, premature ovarian insufficiency; TAD, 30 transactivation domain; DBD, DNA binding domain; TD, tetramerization domain; SAM, sterile 31 alpha motif; PAD, phosphorylation activation domain; TID, transcriptional inhibitory domain; 32 NMR, nuclear magnetic resonance; Dox, doxorubicin; CHK2, checkpoint kinase 2; CK1, 33 casein kinase 1; Cs, Cisplatin; PARP 1, poly(ADP-ribose)-polymerase; DSBs, double strand 34 breaks; 35 36 2 Abstract 37Cell fate decisions such as apoptosis require cells to translate signaling input into a binary 38 yes/no response. A tight control of the process is required to avoid loss of cells by accidental 39 activation of cell death pathways. One particularly critical situation exists in primary oocytes 40 because their finite number determines the reproductive capacity of females. On the one hand 41 a stringent genetic quality control is necessary to maintain the genetic integrity of the entire 42 species; on the other hand an overly stringent mechanism that kills oocytes with even minor 43 DNA damage can deplete the whole primary oocyte pool leading to infertility. The p53 homolog 44 TAp63α is the key regulator of genome integrity in oocytes. After DNA damage TAp63α is 45 activated by multistep phosphorylation involving multiple phosphorylation events by the kinase 46 CK1, which triggers the transition from a dimeric and inactive conformation to an open and 47 active tetramer. By measuring activation kinetics in ovaries and single site phosphorylation 48 kinetics in vitro with peptides and full length protein we show that TAp63α phosphorylation 49 follows a biphasic behavior. While the first two CK1 phosphorylation events are fast, the third 50 one that constitutes the decisive step to form the active conformation is slow. We reveal the 51 structural mechanism for the difference in the kinetic behavior based on an unusual 52 CK1/TAp63α substrate interaction and demonstrate by quantitative simulation that the slow 53 phosphorylation phase determines the threshold of DNA damage required for induction of 54 apoptosis. 55activation mechanism has to be adjusted to a certain level of damage that on the one hand 93 must be sufficiently low to protect the integrity of the genetic pool of a species but on the other 94 hand tolerant enough not to endanger reproductive capacity. This situation requires a 95 mechanism that ideally works similar to a doorbell: an input signal (mechanical pressure) below 96 a certain threshold has no effect. If, however, this threshold is surpassed the output signal is 97 independent of the actual input signal strength (pressing harder does not make the doorbell 98 louder). Indeed, a tight dose-response curve has been measured in four-day old mice: while 99 most oocytes survive irradiation with 0.1 Gy (~three DSBs per cell), virtually all primary oocytes 100 were eliminated by 0.45 Gy irradiation (~ten DSBs per cell) 4 .101 Sw...

Chek2 Signaling Is the Key Regulator of Oocyte Survival After Chemotherapy

“…Both genetic and biochemical evidence indicate that phosphorylation by CHEK2, followed by CK1δ, are needed to fully activate TAp63 (26,32). Hyperphosphorylated TAp63 can then tetramerize and function as a transcription factor (32,58,59,94). However, the role of CHEK2 in the response to alkylating agents has been unclear due to discrepancies in TAp63 hyperphosphorylation status and its requirement for oocyte elimination.…”