YAP expression promotes inactivation of the checkpoint regulators ATM and Chk2
Entry into mitosis is marked by activation of the cyclin dependent kinase (Cdk)1: cyclin B1 complex. During G2, Cdk1 is maintained in an inactive state by phosphorylation on Thr 14 and Tyr 15 (Castedo et al 2002); de-phosphorylation by cdc25 disinhibits Cdk1. To confirm that YAP-expressing cells continued to enter mitosis after irradiation, indicating inactivation of the G2/M checkpoint, we carried out western blotting for Tyr 15-phosphorylated Cdk1. As shown in Figure 4A, irradiation results in increased Tyr15-phosphorylated Cdk1 in both GFP- and YAP-infected CGNPs. However, after three hours, YAP-infected CGNPs show reduced levels of Tyr15-phosphorylated Cdk1, indicating re-activation of Cdk1 at this early timepoint, when DNA breaks still persist. The reduction of inactive Cdk1 was associated with an increase in levels of cyclin B1, which is synthesized during interphase. Following metaphase, cyclin B1 is degraded; this degradation is required for completion of mitosis. Taken together, these results confirm that YAP-expressing irradiated CGNPs enter mitosis despite the presence of double-stranded DNA breaks, and they suggest that the G2/M arrest checkpoint is compromised by YAP expression.
These observations prompted us to examine whether YAP expression blocked activation of the G2/M checkpoint by affecting the activity of proteins involved in its regulation. Upon irradiation, the kinase ATM is activated by autophosphorylation and subsequently phosphorylates and activates Chk2 kinase. Chk2, in turn, phosphorylates cdc25c, resulting in its inactivation and preventing entry into mitosis (Reinhardt and Yaffe 2009). Irradiation resulted in increased levels of phosphorylated ATM and its substrate Chk2 in both GFP- and YAP-infected cells. However, in the presence of ectopic YAP, ATM and Chk2 were more rapidly dephosphorylated (Figure 4B, western blot with quantification below). We observed a similar trend towards Chk2 inactivation in medulloblastoma cells (MBC) obtained from SmoA1 medulloblastomas, cultured in vitro and transduced with either GFP or YAP. Our observation of ATM and Chk2 inactivation in YAP-infected CGNPs and MBC is consistent with relief from the G2/M checkpoint and consequently, re-entry into mitosis despite the presence of unrepaired DNA breaks. We also observed reduced phosphorylation of Histone H2AX in YAP-transduced irradiated CGNPs, further indicative of impaired DNA damage response pathway signaling (Supplementary Figure 4A). Interestingly, we did not observe differential effects of YAP expression on regulation of Chk1 or p53 activity after exposure to radiation in CGNPs or MBC, indicating that YAP’s downstream effectors preferentially target the ATM/Chk2 DNA damage response pathway (Figure 4B).