Dar, A. | bioRxiv Science

bioRxiv2025

Mdm2 restrains the activity of the CRL4Cdt2 E3 ubiquitin ligase to promote cell cycle progression through the G2/M phase

The canonical function of the Mdm2 oncoprotein is widely recognized as the negative regulation of the p53 tumor suppressor. However, growing evidence indicates that its physiological activities extend far beyond p53. Here, we show that Mdm2 promotes cell cycle progression at the G2/M phase through the ubiquitin-mediated degradation of the substrate recognition adaptor Cdt2 of the CRL4Cdt2 E3 ubiquitin ligase complex, independently of p53. The attenuation of CRL4Cdt2 activity by Mdm2 stabilizes its cell cycle-specific substrates including p21, Set8, and Cdt1, at the G2/M phase following their proteasomal degradation in the S phase. Furthermore, the delay in cell cycle progression at the G2/M phase and the decreased cell proliferation observed in the absence of Mdm2 are largely caused by an increase in Cdt2 and a subsequent decrease in p21. Collectively, our data illustrate a previously unexplored mechanism by which Mdm2 regulates the cell cycle and promotes cellular proliferation by neutralising the CR4Cdt2 E3 ubiquitin ligase activity and subsequently stabilizing p21.

molecular biology↗

bioRxiv2022

A small molecule screen reveals the essentiality of p38 kinase for choanoflagellate cell proliferation

Animal kinases regulate cellular responses to environmental stimuli, including cell differentiation, migration, survival, and response to stress, but the ancestry of these functions is poorly understood. Choanoflagellates, the closest living relatives of animals, encode homologs of diverse animal kinases and have emerged as model organisms for reconstructing animal origins. However, efforts to identify key kinase regulators in choanoflagellates have been constrained by the limitations of currently available genetic tools. Here, we report on a framework that combines small molecule-driven kinase discovery with targeted genetics to reveal kinase function in choanoflagellates. To study the physiological roles of choanoflagellate kinases, we established two high-throughput platforms to screen the model choanoflagellate Salpingoeca rosetta with a curated library of human kinase inhibitors. We identified 95 diverse kinase inhibitors that disrupt S. rosetta cell proliferation. By focusing on one inhibitor, sorafenib, we identified a p38 kinase as a regulator of the heat shock response in S. rosetta. This finding reveals a conserved p38 function between choanoflagellates, animals, and fungi. Moreover, this study demonstrates that existing kinase inhibitors can serve as powerful tools to examine the ancestral roles of kinases that regulate modern animal development.

biochemistry↗