Study of the cytolethal distending toxin (CDT)-activated cell cycle checkpoint. Involvement of the CHK2 kinase.
AbstractThe bacterial cytolethal distending toxin (CDT) triggers a G2/M cell cycle arrest in eukaryotic cells by inhibiting the CDC25C phosphatase-dependent CDK1 dephosphorylation and activation. We report that upon CDT treatment CDC25C is fully sequestered in the cytoplasmic compartment, an effect that is reminiscent of DNA damage-dependent checkpoint activation. We show that the checkpoint kinase CHK2, an upstream regulator of CDC25C, is phosphorylated and activated after CDT treatment. In contrast to what is observed with other DNA damaging agents, we demonstrate that the activation of CHK2 can only take place during S-phase. Use of wortmannin and caffeine suggests that this effect is no…
Escherichia coli cytolethal distending toxin blocks the HeLa cell cycle at the G2/M transition by preventing cdc2 protein kinase dephosphorylation and activation
Cytolethal distending toxins (CDT) constitute an emerging heterogeneous family of bacterial toxins whose common biological property is to inhibit the proliferation of cells in culture by blocking their cycle at G2/M phase. In this study, we investigated the molecular mechanisms underlying the block caused by CDT from Escherichia coli on synchronized HeLa cell cultures. To this end, we studied specifically the behavior of the two subunits of the complex that determines entry into mitosis, i.e., cyclin B1, the regulatory unit, and cdc2 protein kinase, the catalytic unit. We thus demonstrate that CDT causes cell accumulation in G2 and not in M, that it does not slow the progression of cells th…
Use of CDC2 from etoposide-treated cells as substrate to assay CDC25 phosphatase activity
International audience; Cyclin-dependent kinases (CDKs) regulate the key transition of the cell cycle in all organisms. In response to Etoposide (VP-16) induced DNA damage, cells undergo a G2-phase arrest resulting in the accumulation of inactive CDK1 (CDC2) kinase complexes. Here we report that upon Etoposide treatment CDC2 is phosphorylated on tyrosine 15 and is dephosphorylated and activated in vitro by recombinant CDC25 phosphatase. We also show that inactive CDC2 kinase from Etoposide-treated cells can be used as a substrate in a sensitive two-step assay of CDC25 phosphatase. This assay, which is very simple to set-up, is based on the monitoring of CDC2 kinase activity after CDC25-depe…