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.

Keywords: Wee1; cyclin A.

Fig. 1.

Cdk2^AF/AF cells display early S-phase entry and accelerated cyclin E turnover. (A) Lysates were harvested from Cdk2^+/+ and Cdk2^AF/AF cells. Phospho-Y15 Cdk2 and total Cdk2 abundance were measured by Western blotting. (B) Cdk2^+/+ and Cdk2^AF/AF cells were arrested in G₀/G₁ by serum/leucine starvation and released into media containing nocodazole. Samples were harvested as indicated, and DNA content was analyzed by flow cytometry. (C) Cyclin E-Cdk2 abundance and kinase activity were assayed in the samples harvested in B. (D) Cyclin E half-life was determined by pulse–chase analysis beginning 13 h after serum/leucine release. (E) The indicated cell lines were treated with HU as indicated and assayed for cyclin E and Cdk2 levels. (C and E) PP2A-loading control. (F) Cyclin E turnover was measured in HU-arrested Cdk2^+/+ and Cdk2^AF/AF cells by pulse–chase analysis.

Fig. 2.

Cdk2 inhibitory phosphorylation is required for recovery from replication stress. (A) Cdk2^+/+ or Cdk2^AF/AF cells were arrested in HU and released into media containing nocodazole. Samples were harvested as indicated, and DNA content was analyzed by flow cytometry. (B) Same as in A, but cells were arrested in Aphidicolin. (C) Experimental setup for D and E: On day 0 cells were seeded; on day 1, HU (D) or Aphidicolin (E) were added and were removed on day 2. Proliferation was assayed 3 d later by using Alamar Blue. Percent proliferation is calculated as percentage of proliferation relative to untreated cells. (F) Annexin V staining of Cdk2^+/+ and Cdk2^AF/AF cells treated with HU or staurosporine for 18 h. (STS, positive control). (G) Cdk2^+/+ and Cdk2^AF/AF cells were treated as in C and assayed for senescence-associated β-galactosidase activity 3 d after HU removal. Error bars indicate SD from three independent data points.

Fig. 3.

Loss of Cdk2 inhibitory phosphorylation causes DNA damage during replication stress. (A) Cdk2^+/+ or Cdk2^AF/AF cells were treated with HU (7 h) as indicated and stained with anti-γH2AX antibody and DAPI (to visualize nuclei). (B) Cdk2^+/+ and Cdk2^AF/AF cells were treated with HU (7 h) as indicated, and cells were harvested and analyzed by flow cytometry. The bar graph shows γH2AX staining that exceeded basal γH2AX induction in HU. (see Fig. S5A for γH2AX gating). (C) Cdk2^+/+ and Cdk2^AF/AF cells were treated with HU for increasing times and γH2AX-positive cells were identified by flow cytometry. (D) Cdk2^+/+ and Cdk2^AF/AF cells were treated with HU as indicated and γH2AX abundance and total H2AX abundance were analyzed by immunoblotting. (E) PFGE of Cdk2^+/+ or Cdk2^AF/AF cells that were untreated, treated with ionizing radiation (12 Gy), or HU. The migration of DNA containing DSBs is indicated. (F, Left) U2OS cells were transfected with vectors expressing Cdk2, Cdk2AF, or empty vector and treated with HU as shown. γH2AX abundance was assayed by flow cytometry. (F, Right) Ectopic Cdk2 or Cdk2AF protein expression. Error bars indicate SD from three independent data points.

Fig. 4.

Constitutive Cyclin A-Cdk2 activity drives aberrant S-phase responses to replication stress. (A) Cdk2^+/+ and Cdk2^AF/AF cells were treated with HU for 6 h and Roscovitine as indicated, and γH2AX staining was analyzed by flow cytometry. (B, Upper) Cdk2^AF/AF cells transfected with either control or two Cdk2 siRNAs were treated with 0.5 mM HU for 24 h, and percent proliferation relative to untreated controls was measured by Alamar Blue 3 d after HU release. (B, Lower) Cdk2 abundance after siRNA transfection. (C) Cdk2^AF/AF cells transduced with control or lentiviruses expressing cyclin E or cyclin A shRNAs were grown asynchronously or treated with HU, and γH2AX abundance was assayed by flow cytometry. (D) Cyclin E and cyclin A abundance in samples from C.

Fig. 5.

Cdk2^AF/AF cells display aberrant replication dynamics. (A) Diagram of replication analysis by sequential labeling with EdU and IdU. Asterisk indicates time of harvest. Representative forks are shown. (B) Bar graph shows that origin firing during the labeling period is increased in Cdk2^AF/AF cells (P = 0.042; Materials and Methods) (C) Ongoing forks progress slower in Cdk2^AF/AF cells. Bar graphs show the percentages of replication forks with indicated EdU and IdU segment lengths. (D) Labeling scheme for HU arrest/release experiments. HU (2mM) was added at the end of a 30-min EdU labeling interval. After 3 h, HU and EdU were removed, and IdU was added. After 30 min, IdU was removed, and samples were harvested 30 min later. (E) Ongoing forks (EdU-IdU tracks) and terminated or inactivated forks (EdU only) were counted to determine percentage of ongoing forks. Cdk2^AF/AF cells have significantly reduced fork restart rate after HU treatment (P = 0.0002). (F) Bar graphs show the percentages of replication forks with indicated EdU segment lengths in either untreated or HU-treated cells. (G) Graphic summary showing that Cdk2^AF/AF cells display increased replication in HU. Brackets indicate the increased replication in HU in Cdk2^AF/AF cells compared with Cdk2^+/+ cells. Data in B, C, E, and F represent averages (with SD) from three experiments.