Oocyte DNA damage quality control requires consecutive interplay of CHK2 and CK1 to activate p63

Abstract

The survival rate of cancer patients is steadily increasing, owing to more efficient therapies. Understanding the molecular mechanisms of chemotherapy-induced premature ovarian insufficiency (POI) could identify targets for prevention of POI. Loss of the primordial follicle reserve is the most important cause of POI, with the p53 family member p63 being responsible for DNA-damage-induced apoptosis of resting oocytes. Here, we provide the first detailed mechanistic insight into the activation of p63, a process that requires phosphorylation by both the priming kinase CHK2 and the executioner kinase CK1 in mouse primordial follicles. We further describe the structural changes induced by phosphorylation that enable p63 to adopt its active tetrameric conformation and demonstrate that previously discussed phosphorylation by c-Abl is not involved in this process. Inhibition of CK1 rescues primary oocytes from doxorubicin and cisplatin-induced apoptosis, thus uncovering a new target for the development of fertoprotective therapies.

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

“…Activation can be suppressed using selective inhibitors of ATM, CHK2 and CK1, suggesting that tetramerization is based on the same pathway as activation following the treatment with cisplatin or doxorubicin (Supplementary Fig. 1a) 13,14,18 . In order to investigate the time dependent induction of apoptosis, we measured the ratio of germ cell nuclear acidic peptidase (GCNA) 19 positive and cleaved poly(ADP-ribose)-polymerase 1 (PARP1) double positive cells in whole mouse ovaries using our 3D staining and light sheet-based fluorescence microscopy combined with semi-automated segmentation method (Fig.…”

“…The first step of the kinase cascade is the activation of checkpoint kinase 2 (CHK2) or checkpoint kinase 1 (CHK1) by ataxia telangiectasia mutant kinase (ATM) 11 . Activated CHK2 phosphorylates TAp63α on S582 12 which renders TAp63α a substrate for casein kinase 1 (CK1) 13 . CK1 requires pre-phosphorylated substrates with the consensus sequence pS/T-x-x-S/T, where pS/T is a phosphorylated serine or threonine.…”

“…CK1 requires pre-phosphorylated substrates with the consensus sequence pS/T-x-x-S/T, where pS/T is a phosphorylated serine or threonine. In TAp63α CK1 adds four phosphate groups that lead to the disruption of the autoinhibitory complex through electrostatic repulsion 13 . Our previous experiments have shown that the inhibited dimeric state of TAp63α constitutes a kinetically trapped high energy state 14 .…”

Frontiers in Molecular Evolutionary Medicine

“…Because controlling the quality of the oocytes that are fertilized, implant, and become fetuses should be a top priority in the evolutionary design of the female reproductive tract, it was suggested that oocytic atresia functions to eliminate genetically defective oocytes (Stearns and Ebert 2001). The mechanisms involved are being discovered: one is DNAdamage-induced apoptosis of resting oocytes mediated by p63, a member of the p53 family (Tuppi et al 2018). Oocyte death can also be triggered by defects in the synapsis and recombination of chromosomes during meiosis that produce double strand breaks, which also induce apoptosis (Qiao et al 2018).…”

Kinase-independent inhibition of cyclophosphamide-induced pathways protects the ovarian reserve and prolongs fertility

“…Another study demonstrated that CHK2 inhibitor II hydrate affected the activities of a broad spectrum of kinases and impacted the global repression of the DNA damage response (DDR) in cultured ovaries 10 , thereby raising some questions about its specificity. Tuppi et al reported that CHK2 and the executioner kinase CK1 are both involved in the TAp63a-mediated oocyte degeneration in cultured ovaries exposed to chemotherapy 11 . As cultured mouse ovaries exposed to chemotherapeutic drugs may not recapitulate the signaling pathways physiologically occurring in the ovary in vivo, we aimed to perform all our experiments under in vivo conditions.…”