The DNA damage checkpoint and PKA pathways converge on APC substrates and Cdc20 to regulate mitotic progression

Abstract

The conserved checkpoint kinases Chk1 and Rad53-Dun1 block the metaphase to anaphase transition by the phosphorylation and stabilization of securin, and block the mitotic exit network regulated by the Bfa1-Bub2 complex. However, both chk1 and rad53 mutants are able to exit from mitosis and initiate a new cell cycle, suggesting that both pathways have supporting functions in restraining anaphase and in blocking the inactivation of mitotic cyclin-Cdk1 complexes. Here we find that the cyclic-AMP-dependent protein kinase (PKA) pathway supports Chk1 in the regulation of mitosis by targeting the mitotic inducer Cdc20. Cdc20 is phosphorylated on PKA consensus sites after DNA damage, and this phosphorylation requires the Atr orthologue Mec1 and the PKA catalytic subunits Tpk1 and Tpk2. We show that the inactivation of PKA or expression of phosphorylation-defective Cdc20 proteins accelerates securin and Clb2 destruction in chk1 mutants and is sufficient to remove most of the DNA damage-induced delay. Mutation of the Cdc20 phosphorylation sites permitted the interaction of Cdc20 with Clb2 under conditions that should halt cell cycle progression. These data show that PKA pathways regulate mitotic progression through Cdc20 and support the DNA damage checkpoint pathways in regulating the destruction of Clb2 and securin.

New insights into the regulation of anaphase by mitotic cyclins in budding yeast

“…Therefore, complementation by other mitotic cyclins was less efficient when G 2 /M checkpoint was activated. This could possibly be due to deregulated activity of the anaphase-promoting complex/cyclosome-Cdc20 that is regulated by mitotic cyclins 6,48,49 but also by DNA damage checkpoint [50][51][52][53][54][55] and reciprocally controls the stability/turnover of mitotic cyclins. 56 Interestingly, a role for Cdc20 in controlling spindle length and the stability of kinetochore microtubule in response to genotoxic stress has been suggested.…”

ATRMec1 Phosphorylation-independent Activation of Chk1 in Vivo

“…In this case the checkpoint proteins in addition to the kinase Chk1 and the mediator/signal amplifier protein Rad9 inhibit spindle elongation and chromosome segregation in response to DNA damage sensed in late S or G 2 . One effector of Chk1 in this response is the securin Pds1, which is stabilized by phosphorylation in response to DNA damage (3)(4)(5)(6). Although the Chk1/Rad9 DNA damage checkpoint arm is not required during replication blocks in the budding yeast, we have shown that Chk1 is activated when replication is slowed down by the ribonucleotide reductase inhibitor hydroxyurea (HU).…”

The kinomes of apicomplexan parasites

“…In yeast, Cdc5 phosphorylates and activates the Anaphase Promoting Complex (APC). The P. falciparum kinase found to be closest sequence-wise to higher eukaryotic Polo kinases is PfPKA, and yeast/mammalian PKA is also known to phosphorylate APC with the opposite effect of suppressing its activity [92]. Therefore, Polo kinases and PKA regulate mitotic progression by controlling APC activity in opposing ways.…”