Caffeine stabilizes Cdc25 independently of Rad3 in Schizosaccharomyces pombe contributing to checkpoint override

Abstract

Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both S chizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S . pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S . pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25.

Caffeine stabilises fission yeast Wee1 in a Rad24-dependent manner but attenuates its expression in response to DNA damage identifying a putative role for TORC1 in mediating its effects on cell cycle progression

“…We previously demonstrated that caffeine stabilises both wild type (wt) Cdc25-GFP int and the Cdc25 (9A) -GFP int mutant that lacks 9 major inhibitory phosphorylation sites and is normally degraded following exposure to genotoxic agents (Frazer & Young, 2011, Frazer & Young, 2012, Alao et al, 2014. In this study, exposure to 10 mM caffeine also stabilised the Cdc25 (12A) mutant protein that lacks all 12 inhibitory phosphorylation sites ( Fig.…”

“…Mik1 is required for maintenance of the replication damage checkpoint signalling in S. pombe mutants expressing Cdc25 (12A) -GFP int (Frazer & Young, 2011, Frazer & Young, 2012. As observed for rad3Δ and cds1Δ mutants (Alao et al, 2014), Cdc25 appeared more stable in a mik1Δ genetic background ( Fig. 1 A).…”

“…3 C). We previously demonstrated that caffeine stabilises Cdc25 in the nucleus of S. pombe cells exposed to genotoxic agents (Alao et al, 2014). Caffeine thus both stabilises Cdc25 and suppresses Wee1 expression under genotoxic conditions.…”

“…3 A). Caffeine activates both the DNA damage response and environmental stress response pathways (Calvo et al, 2009, Alao et al, 2014. The effect of caffeine on Wee1 stability may thus be context dependent.…”

“…The inhibition of TORC1 activity suppresses Wee1 expression, results in increased Cdc25 activation and drives cells into mitosis. In addition, the effect of caffeine on cell cycle progression resembles that of TORC1 inhibition (Petersen, 2009, Alao et al, 2014, Atkin et al, 2014. We previously demonstrated that caffeine overrides checkpoint signalling in part, by stabilising Cdc25 expression.…”