To ensure genomic integrity, dividing cells implement multiple checkpoint pathways during the course of the cell cycle. In response to DNA damage, cells may halt the progression of the cycle, termed a cell cycle arrest, or undergo apoptosis depending on the extent of damage and the cell's capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks relies on the activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors (e.g., Chk2 and p53) to inhibit cell cycle progression. ATM is recruited to double-stranded DNA breaks by a complex of sensor proteins resulting in its monomerization, autophosphorylation, and activation. In characterizing the protein Aven, a reported apoptotic inhibitor, we have found that Aven can function as an ATM activator to inhibit the G2/M transition. Binding experiments revealed an interaction between Aven and ATM, and Aven overexpression in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knock-down of Aven in human cells prevented autophosphorylation of ATM in response to DNA damage. We also demonstrated that Aven is a substrate of the ATM kinase and that the phosphorylation of Aven by ATM is required for full ATM activation, possibly forming a positive feedback loop during ATM activation. Thus, these findings place Aven as a critical transducer of the DNA damage signal to regulate mitotic entry.

Exit from mitosis is controlled by the degradation of Cyclin B, the loss of Cdc2 kinase activity, and the dephosphorylation of Cdc2 phosphorylated proteins. When Aven was depleted from cycling Xenopus egg extracts, we found that the extracts arrested at mitosis, despite Cyclin B degradation. This observation suggested that Aven might play a role in the dephosphorylation of mitotic phosphoproteins. However, the identity and mechanisms of phosphatases that modulate Cdc2 substrate dephosphorylation at mitotic exit remained relatively unknown. Our studies have revealed that protein phosphatase-1 (PP1) is the major catalyst of mitotic phosphoprotein dephosphorylation and that PP1 activity is tightly controlled during mitosis and mitotic exit. We demonstrate that suppression of PP1 during early mitosis is maintained through the dual inhibition of PP1 by Cdc2 phosphorylation and the binding of Inhibitor-1. As Cyclin B degrades and Cdc2 levels decrease, autodephosphorylation of PP1 at the site of Cdc2 phosphorylation allows partial PP1 activation. This permits PP1-mediated dephosphorylation of I-1 at its activating site, dissociation of the I-1-PP1 complex, and full PP1 activation to promote mitotic exit. By elucidating the mechanism(s) of PP1 regulation, we have laid the groundwork to further our understanding of the regulatory functions of Aven during mitotic exit. Moreover, these studies raise the interesting possibility that Aven function at mitotic entry also reflects a role for Aven in controlling phosphatases upstream of ATM activation.