Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency.DOI: http://dx.doi.org/10.7554/eLife.16270.001
SUMMARY Vertebrate centrioles lose their geometric scaffold, the cartwheel, during mitosis, concurrently with gaining the ability to recruit the pericentriolar material (PCM) and thereby function as the centrosome. Cartwheel removal has recently been implicated in centriole duplication, but whether “cartwheel-less” centrioles are intrinsically stable, or must be maintained through other modifications remains unclear. Here, we identify a newborn centriole-enriched protein, KIAA1731/CEP295, specifically mediating centriole-to-centrosome conversion but dispensable for cartwheel removal. In the absence of CEP295, centrioles form in S/G2 phase, and lose their associated cartwheel in mitosis, but cannot be converted to centrosomes, uncoupling the two events. Strikingly, centrioles devoid of both the PCM and cartwheel progressively lose centriolar components, whereas centrioles associating with either the cartwheel or PCM alone can exist stably. Thus, cartwheel removal can have grave repercussions to centriole stability, and centriole-to-centrosome conversion mediated by CEP295 must occur in parallel to maintain cartwheel-less centrioles for duplication.
Background: Postnatal expansion of the pancreatic β-cell mass is required to maintain glucose homeostasis immediately after birth. This β-cell expansion is regulated by multiple growth factors, including glucose, insulin, insulin-like growth factor (IGF-1) and epidermal growth factor (EGF). These mitogens signal through several downstream pathways (AKT, ERK, STAT3, and JNK) to regulate the survival and proliferation of β-cells. Survivin, an oncofetal protein with both pro-proliferative and anti-apoptotic properties, is a known transcriptional target of both IGF-1 and EGF in cancer cells. Here, we analyzed the effects of the β-cell mitogens IGF-1 and EGF on survivin regulation in the established pancreatic β-cell model cell lines, MIN6 and INS-1 and in primary mouse islets. Results: In pancreatic β-cells, treatment with glucose, insulin, or EGF increased survivin protein levels at early time points. By contrast, no significant effects on survivin were observed following IGF-1 treatment. EGF-stimulated increases in survivin protein were abrogated in the presence of downstream inhibitors of the Raf-1/MEK/ERK pathway. EGF had no significant effect on survivin transcription however it prolonged the half-life of the survivin protein and stabilized survivin protein levels by inhibiting surviving ubiquitination. Conclusions: This study defines a novel mechanism of survivin regulation by EGF through the Raf-1/MEK/ERK pathway in pancreatic β-cells, via prolongation of survivin protein half-life and inhibition of the ubiquitin-mediated proteasomal degradation pathway. This mechanism may be important for regulating β-cell expansion after birth.
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