Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation

Abstract

Cyclin-dependent kinases (Cdks) coordinate cell division, and their activities are tightly controlled. Phosphorylation of threonine 14 (T14) and tyrosine 15 (Y15) inhibits Cdks and regulates their activities in numerous physiologic contexts. Although the roles of Cdk1 inhibitory phosphorylation during mitosis are well described, studies of Cdk2 inhibitory phosphorylation during S phrase have largely been indirect. To specifically study the functions of Cdk2 inhibitory phosphorylation, we used gene targeting to make an endogenous Cdk2 knockin allele in human cells, termed Cdk2AF, which prevents Cdk2 T14 and Y15 phosphorylation. Cdk2AF caused premature S-phase entry, rapid cyclin E degradation, abnormal DNA replication, and genome instability. Cdk2AF cells also exhibited strikingly abnormal responses to replication stress, accumulated irreparable DNA damage, and permanently exited the cell cycle after transient exposure to S-phase inhibitors. Our results reveal the specific and essential roles of Cdk2 inhibitory phosphorylation in the successful execution of the replication stress checkpoint response and in maintaining genome integrity.Wee1 | cyclin A C yclin-dependent kinases (Cdks) are regulated by positive and negative phosphorylations (1). T14/Y15 phosphorylation by the Wee1 and Myt1 kinases inhibits Cdks by preventing ATP binding, and these conserved phosphorylations are highly regulated during the cell cycle and by signaling pathways (1, 2). Wee1 and Myt1 are opposed by cell division cycle 25 (Cdc25) phosphatases, which, in mammals, comprise three related enzymes that dephosphorylate T14/Y15 to promote Cdk activity (2-4).Wee1 and Cdc25 functions in mammals are best understood in G 2 /M, where Cdk1 inhibitory phosphorylation is required for mitotic progression and DNA damage responses. Cdk1 regulates Wee1 and Cdc25C by phosphorylation, which results in feedback loops that produce switch-like mitotic transitions (5). DNA damage pathways also use Cdk1 T14/Y15 phosphorylation to prevent mitotic entry by inhibiting Cdk1 (6, 7).Cdk2 is activated by cyclin E and cyclin A; cyclin E-Cdk2 regulates G 1 progression and S-phase entry, whereas cyclin A-Cdk2 acts later in S phase and mitosis (8, 9). Cdk2 is also regulated by Wee1 and Cdc25A, but the consequences of Cdk2 inhibitory phosphorylation are less well understood than for Cdk1 (10-13). Studies of Wee1 and Cdc25A have implicated Cdk2 inhibition in DNA replication and S-phase DNA damage checkpoints (14-17). However, the contributions of Cdk1 vs. Cdk2 to these phenotypes have been difficult to disentangle because manipulations such as Wee1 inhibition concurrently impact multiple Cdks. Indeed, a recent mouse model unexpectedly found that Cdk2 T14/Y15 phosphorylation is not required for DNA damage-induced cell cycle arrest, but instead has roles in S-phase entry and centrosome duplication (18).Cyclin E-Cdk2 activity peaks at the G 1 -S transition, and its periodicity results from regulated transcription and cyclin E degradation by the SCF (Skp1/Cul1/F-box protein)-Fbw7...

The same, only different – DNA damage checkpoints and their reversal throughout the cell cycle

“…With the continuous degradation of p21, the intra-S checkpoint instead relies on Wee1 kinase, which becomes expressed in Sphase and phosphorylates Cdk2 to inhibit its activity (Chow et al, 2003;Watanabe et al, 1995). Chk1 and Chk2 deposit activating phosphates on Wee1 kinase and target the counteracting phosphatase Cdc25A for degradation to impose an immediate break on further Cdk activation (Beck et al, 2010;Hughes et al, 2013; O'Connell et al, 1997).…”

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

“…Cdk2 appears to be under similar checkpoint control and is also phosphorylated by Wee1 on Tyr-15, which prevents unscheduled S-phase entry. Conversely, Cdk2 must be dephosphorylated by Cdc25 phosphatases to become active, a process which is also controlled by the DDR (14,15). In addition to this fast-acting kinase-driven DDR network, a transcriptional program is activated through p53 stabilization (16).…”

The PP2A-B56 Phosphatase Opposes Cyclin E Autocatalytic Degradation via Site-Specific Dephosphorylation

“…Reactions were quenched by the addition of sample buffer, boiled, resolved on polyacrylamide gels, and exposed to film. Endogenous cyclin E half-life was measured by [ 35 S]Met pulse-chase as previously described (61).…”