Antagonism of Chk1 signaling in the G2 DNA damage checkpoint by dominant alleles of Cdr1

Abstract

Activation of the Chk1 protein kinase by DNA damage enforces a checkpoint that maintains Cdc2 in its inactive, tyrosine-15 (Y15) phosphorylated state. Chk1 downregulates the Cdc25 phosphatases and concomitantly upregulates the Wee1 kinases that control the phosphorylation of Cdc2. Overproduction of Chk1 causes G(2) arrest/delay independently of DNA damage and upstream checkpoint genes. We utilized this to screen fission yeast for mutations that alter sensitivity to Chk1 signaling. We describe three dominant-negative alleles of cdr1, which render cells supersensitive to Chk1 levels, and suppress the checkpoint defects of chk1Delta cells. Cdr1 encodes a protein kinase previously identified as a negative regulator of Wee1 activity in response to limited nutrition, but Cdr1 has not previously been linked to checkpoint signaling. Overproduction of Cdr1 promotes checkpoint defects and exacerbates the defective response to DNA damage of cells lacking Chk1. We conclude that regulation of Wee1 by Cdr1 and possibly by related kinases is an important antagonist of Chk1 signaling and represents a novel negative regulation of cell cycle arrest promoted by this checkpoint.

Figure 1.—

Isolation of mutants supersensitive to Chk1 overexpression. (A) The indicated strains were streaked onto minimal media containing phloxin B together with thiamine (nmt1 promoter repressed) or without thiamine (nmt1 promoter derepressed), and the plates were incubated at 30° for 4 days. Note that the residual growth of mutants on plates lacking thiamine is from a heavy inoculum from a plate containing thiamine and also that the nmt1 promoter takes about five cell cycles to fully derepress. These inviable cells are stained with phloxin B and cannot be propagated on a fresh plate. (B) Cells were grown to midlogarithmic phase in the presence of thiamine, washed three times in thiamine-free medium, and then grown in the absence of thiamine for 18 hr at 30°. Cells were fixed with formaldehyde and stained with DAPI. Cytoplasmic staining is from mitochondrial genomes. Bar, 10 μm. (C) The same cultures as in B were harvested and snap frozen. Extracts were prepared and analyzed by Western blotting using anti-Chk1 polyclonal antibodies, which can detect only overexpressed Chk1. The filter was stripped and reprobed with antitubulin antibodies.

Figure 2.—

Normal cell cycle progression of the supersensitive mutants in the absence of Chk1 overexpression. (A) B3 and C47 were separated from sup3-5 nmt1∷chk1 by tetrad dissection and transformed with pREP41X∷chk1 (medium strength nmt1) together with vector and wild-type controls. Sensitivity to Chk1 overexpression in the presence and absence of thiamine was assayed as described in Figure 1. (B) Midlogarithmic cultures of the indicated strains were fixed with formaldehyde and stained with DAPI, and the mean length at division (±standard deviation, n = 100) was measured with an eyepiece micrometer. (C) Wild type (□), B3 (•), C47 (▪), and D8 sup3-5 (○) were grown to midlogarithmic phase, and cell cycle progression was assayed by triplicate cell number determination using a Coulter counter for 10 hr.

Figure 3.—

The Chk1 supersensitive mutants are alleles of cdr1. (A) A plasmid shuffling screen (see materials and methods) was used to identify a termination codon in cdc5 (CGA to TGA) in D8 cells, but cdc5 mutations were not present in B3 or C47. The cdr1 locus is located 13.2 kb to the left of cdc5 on chromosome I. (B) The mutants exhibit a changed division response (Cdr) phenotype. Cultures were started at 2 × 10⁶ cells/ml and grown for 48 hr to saturation in the presence or absence of NH₄Cl, fixed with 70% ethanol, and processed for DNA content determination by FACS. Note the absence of a G₁ peak in cdr1 mutants and the cdr1Δ control. (C) Mutations in the Cdr1 catalytic domain (subdomains I, VIII, and IX; substituted amino acid is underlined) were present in our cdr1 mutants.

Figure 4.—

Dominant-negative cdr1 mutations partially suppress the MMS and UV-C hypersensitivity of chk1Δ. (A) Ten-fold serial dilutions of the indicated strains were spotted onto YES agar with a range of concentrations of MMS, and colonies were allowed to form over 4 days at 30°. (B) UV-C survival assays of the same series of strains shown in A. (C) Plate assays of sensitivity to Chk1 overexpression were carried out as described in Figure 1. Note that cdr1Δ shows an intermediate sensitivity to Chk1 compared to wild-type and cdr1-D8 controls. (D) MMS sensitivity and (E) UV-C survival was assayed as in A and B. Note that cdr1Δ does not partially suppress chk1Δ.

Figure 5.—

Regulation of Wee1 by Cdr1. (A) Each cdr1 allele was tagged with His₆Flag₃ at the 3′-end of its endogenous locus. Proteins were immunoprecipitated with anti-Flag M2 beads and detected with M2 antibodies. (B) GST-tagged wild-type and mutant Cdr1 were expressed from the nmt1 promoter and purified on glutathione sepharose, and autophosphorylation was assayed in vitro from 50% of the recovered material. “Activity” is the arbitrary units determined by a Bio-Rad FX phosphorimager of the ³²P signal, and levels of GST-Cdr1 in the assays were determined by Western blotting. (C) GST and GST-Cdr1 fusion proteins were expressed from the nmt1 promoter for 15 hr at 30° in cells expressing Wee1-His₆HA₃ from the endogenous locus. Anti-GST Western blots show GST and GST-Cdr1 levels, and Wee1 is detected with anti-HA antibodies. Identical effects on Wee1 were obtained with untagged Cdr1 constructs. Total Cdc2 and Y15 Cdc2 were detected from the same extracts. (D) Cell extracts were prepared as in C, and Wee1-His₆HA₃ was recovered on Ni-NTA agarose. Samples were split and treated with calf intestinal phosphatase (CIP) or mock treated. (E) Cdr1 and Cdr1-D8 were expressed from the nmt1 promoter in wild-type cells for 18 hr at 30°, fixed, and stained with DAPI. Bar, 10 μm. Overexpressed Cdr1-B3 and -C47 behave identically to Cdr1-D8 (data not shown).

Figure 6.—

Cdr1 overexpression results in a partially defective checkpoint response. (A) MMS sensitivity and (B) UV-C survival assays were performed as in Figure 4. Note that nmt1∷cdr1 cells exhibit a sensitivity similar to that of wee1Δ cells and that Cdr1 overexpression, while somewhat toxic, substantially enhances the MMS and UV-C hypersensitivity of chk1Δ cells. (C) The indicated strains were mock irradiated (0 J/m²) or UV-C irradiated (100 J/m²) and grown at 30°. At 30-min intervals for 3 hr, samples were taken and fixed with formaldehyde. Fixed samples were stained with DAPI, and the percentage of cells that were binucleate (□) or exhibited an aberrant mitosis (cut phenotype, ▪) were counted in triplicate (±standard deviation, n = 100). A drop in binucleates indicates a cell cycle delay, and aberrant mitoses (arrowed) are indicative of checkpoint failure. Bar, 10 μm.