Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism

Abstract

Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and thereby promote cell cycle progression. In vertebrates, Chk1 and Chk2 phosphorylate Cdc25A at multiple N-terminal sites and target it for rapid degradation in response to genotoxic stress. Here we show that Chk1, but not Chk2, phosphorylates Xenopus Cdc25A at a novel C-terminal site (Thr504) and inhibits it from C-terminally interacting with various Cdk-cyclin complexes, including Cdk1-cyclin A, Cdk1-cyclin B, and Cdk2-cyclin E. Strikingly, this inhibition, rather than degradation itself, of Cdc25A is essential for the Chk1-induced cell cycle arrest and the DNA replication checkpoint in early embryos. 14-3-3 proteins bind to Chk1-phosphorylated Thr504, but this binding is not required for the inhibitory effect of Thr504 phosphorylation. A C-terminal site presumably equivalent to Thr504 exists in all known Cdc25 family members from yeast to humans, and its phosphorylation by Chk1 (but not Chk2) can also inhibit all examined Cdc25 family members from C-terminally interacting with their Cdk-cyclin substrates. Thus, Chk1 but not Chk2 seems to inhibit virtually all Cdc25 phosphatases by a novel common mechanism.

Figure 1

Chk1 phosphorylation sites required for Cdc25A degradation. (A) A schematic representation of Xenopus Cdc25A protein. Seven serine residues that lie in the consensus Chk1/Chk2 phosphorylation motif (Arg-X-X-Ser) are shown. RD, regulatory domain; CD, catalytic domain; CT, C-terminal tail. (B) GST-Cdc25A peptide fusion proteins (GST-S120, GST-A120, etc., each named after the relevant Ser or substituted Ala residue numbers) or GST-Cdc25C peptide fusion proteins (GST-S287 and GST-A287), 2 μg each, were incubated with [γ-³²P]ATP and either Δ60-Chk1 protein, wild-type Chk2 protein or their kinase-dead forms, subjected to SDS–PAGE, stained with Coomassie blue (CB), and then autoradiographed (³²P/Chk1 and ³²P/Chk2). Because of the very low activity of Chk2, ³²P/Chk2 was exposed substantially longer than ³²P/Chk1. The arrows indicate autophosphorylated Δ60-Chk1 or Chk2. (C) Activated eggs were injected with 1 ng of mRNA encoding Myc-tagged wild-type Cdc25A (WT) or indicated Myc-tagged Cdc25A mutants (see text for the naming of the mutants), reinjected 2.5 h later with 2 ng of Δ60-Chk1 mRNA, and analyzed at 20 min intervals by immunoblotting using anti-Myc antibody. For WT Cdc25A, eggs expressing no Δ60-Chk1 (−Chk1) were also analyzed. (D) Fertilized eggs were injected with 1 ng of mRNA encoding Myc-tagged wild-type or 4A Cdc25A and, at 30 min intervals after the early blastula stage 8, analyzed as in (C). The MBT occurred 1 h after stage 8.

Figure 2

Phosphorylation of Cdc25A Thr504 by Chk1 but not Chk2. (A) Alignment of the C-terminal regions of Xenopus (X.l.), human (Hu), and mouse (Mu) Cdc25A proteins. The asterisks show conserved Ser/Thr residues. (B) GST-Cdc25A C-terminal peptide fusion proteins (each with a Ser/Thr → Ala substitution) were phosphorylated by Δ60-Chk1 or Chk2 in vitro and analyzed as in Figure 1B. (C) Extracts from the eggs expressing Myc-tagged S73A Cdc25A together with or without Δ60-Chk1 were mock-treated or treated with bacterial alkaline phosphatase (BAP) and analyzed by immunoblotting (IB) using anti-Myc or anti-phospho-Thr504 antibodies. (D) One-cell embryos were injected with 1 ng of mRNA encoding Myc-tagged S73A Cdc25A. At the early blastula stage 7, the embryos were mock-treated or treated with aphidicolin (APH) together with or without caffeine (CAF) and, at 20 min intervals, analyzed by immunoblotting using anti-Myc antibody, anti-phospho-Thr504 antibody, or anti-human Chk1 phospho-Ser345 antibody (pChk1, which can recognize well the activating phosphorylation of Xenopus Chk1; Shimuta et al, 2002). (E) One-cell embryos were coinjected with 1 ng of mRNA encoding Myc-tagged S73A Cdc25A and 15 ng of mRNA encoding either control GST (left panel) or a dominant-negative (or kinase-dead) form of Chk1 (DN-Chk1), and then analyzed as in (D).

Figure 3

Requirement of Cdc25A phosphorylation on Thr504 for the DNA replication checkpoint. (A, B) Activated eggs were injected with 1 ng of mRNA encoding Myc-tagged wild-type Cdc25A or indicated Myc-tagged Cdc25A mutants, reinjected 2.5 h later with 2 ng of Δ60-Chk1 mRNA, and then analyzed by immunoblotting using anti-Myc or anti-Cdk1 phospho-Tyr15 antibodies. (C–E) One-cell embryos were uninjected (Cont.) or injected with 1 ng of mRNA encoding either wild-type Cdc25A or T504A Cdc25A, cultured, and analyzed for immunoblotting (after stage 8 with anti-Cdc25A or anti-Cdk1 phospho-Tyr15 antibodies; C), for the external morphology (D) and for the percentage embryonic death at stage 11 (E).

Figure 4

Inhibition of Cdc25A/Cdk–cyclin interactions by Thr504 phosphorylation. (A) GST-Cdc25A fusion proteins (wild type (WT), T504A, or C428S) were first incubated with GST-Δ60-Chk1 (Δ60) or its kinase-dead form (DA) and then with Tyr15-phosphorylated Cdk1–cyclin A1 complexes. The reaction mixture was then analyzed by immunoblotting using anti-Cdc25A, anti-phospho-Thr504, anti-cyclin A1, or anti-Cdk1 phospho-Tyr15 antibodies. (For exact experimental conditions, see Supplementary data.) (B) Activated eggs were injected with 2 ng of mRNA encoding GST (Cont.) or GST-Cdc25A (WT or T504A), incubated for 2.5 h, reinjected or not with 2 ng of Δ60-Chk1 mRNA, and then incubated further for 1.5 h. Egg extracts (Input; equivalent to half an egg) and GST-pulled down proteins (GST-PD; equivalent to 10 eggs) were then immunoblotted for GST-Cdc25A or endogenous cyclins A1, B1, or E1 (left panel). (The blot of GST-Cdc25A in GST-PD is short-exposed.) The levels of cyclins (in both Input and GST-PD) were quantified using the NIH Image program from four independent experiments, and the levels of cyclins bound to GST-Cdc25A proteins were normalized to the input of cyclins; values obtained for WT Cdc25A (−Chk1) were set at 1.0 (right panel). (C) Extracts from the eggs expressing GST-Cdc25A proteins (together with or without Δ60-Chk1) as in (B) were mixed with extracts from the eggs expressing Myc-tagged cyclins A1 or B1 (together with Xe-Wee1B (Okamoto et al, 2002) to induce Cdk1 Tyr15 phosphorylation), incubated for 1 h at 4°C, and analyzed for GST-Cdc25A or ectopic cyclins A1 or B1 as in (B, left). (D, E) Activated eggs were injected with 2 ng of mRNA encoding wild-type GST-Cdc25A, GST-Cdc25A-ΔC23 (D) or GST-Cdc25A-3A (E), incubated for 2.5 h, and analyzed as in (B). In (B–E), all the GST-Cdc25A constructs had both S73A and C428S mutations (see text).

Figure 5

No requirement of 14-3-3 binding for the inhibitory effect of Thr504 phosphorylation. (A) Activated eggs expressing GST-Cdc25A proteins (together with or without Δ60-Chk1) as in Figure 4B were subjected to GST pulldown assays and analyzed by immunoblotting using anti-GST antibody or anti-human 14-3-3β antibody (which can recognize all 14-3-3 isoforms). (B) Chk1-phosphorylated GST-Cdc25A protein (wild type) was GST-pulled down from activated eggs, washed with or without 0.4% Empigen, and analyzed by immunoblotting as in (A) (left). Extracts from the eggs expressing GST-cyclin A1 (together with Xe-Wee1B; see Figure 4C legend) were treated with the indicated peptide-coupled beads and immunoblotted for cyclin A1 or 14-3-3 protein (right). See Materials and methods for details. (C) GST-Cdc25A proteins (phosphorylated or not by Chk1) and egg extracts (equivalent to 10 eggs) were both prepared as in (B) (GST-Cdc25A proteins being eluted from glutathione beads), mixed together, and incubated for 1 h at 4°C. GST-Cdc25A proteins were then immunoprecipitated with anti-Cdc25A antibody (IP) and analyzed by immunoblotting using anti-GST or anti-cyclin A1 antibodies. As control of GST-Cdc25A proteins (wild type or T504A), GST alone was used (Cont.). (D) GST-Cdc25A proteins prepared as in (C) were first incubated or not with 0.1 μg of recombinant Xenopus 14-3-3ɛ protein in 30 μl of an egg extraction buffer for 15 min at 4°C and then with (GST-)Cdk1–cyclin A1 complexes (purified from 10 eggs) for 1 h. (Xenopus 14-3-3ɛ protein can bind to phosphorylated Thr504 of Cdc25A; see Materials and methods.) The mixture was then analyzed as in (C). In (A–D), all the GST-Cdc25A constructs had both S73A and C428S mutations.

Figure 6

Chk1 regulation of other Cdc25 family members by C-terminal phosphorylation. (A) Alignment of the C-terminal tails of Cdc25 family members. A conserved consensus Chk1/Chk2 phosphorylation motif (R/K-X-X-S/T) and three other conserved basic residues are dotted and asterisked, respectively. (B) GST-Cdc25 C-terminal peptide fusion proteins (wild-type and Ser/Thr → Ala mutant forms of Cdc25A, 25B, 25C, or String) were phosphorylated by Δ60-Chk1 or Chk2 in vitro and analyzed as in Figure 2B. (C) Activated eggs were injected with 1 ng of mRNA encoding indicated forms of (Myc-tagged) Cdc25 phosphatases, reinjected 2.5 h later with 2 ng of Δ60-Chk1 mRNA, and then analyzed as in Figure 3A.

Figure 7

Regulation of various Cdc25/Cdk–cyclin interactions by C-terminal phosphorylation. (A, C) Activated eggs were injected with 2 ng of mRNA encoding indicated forms of GST-Cdc25 phosphatases, cultured for 2.5 h, and then subjected to GST pulldown assays as in Figure 4B. (B) Activated eggs were injected with 2 ng of mRNA encoding indicated forms of GST-Cdc25 phosphatases, incubated for 2.5 h, reinjected or not with 2 ng of Δ60-Chk1 mRNA, and then 1.5 h later subjected to GST pulldown assays. In (A–C), all the GST-Cdc25 constructs were phosphatase-dead forms (like the C428S mutant of Cdc25A).

Figure 8

Model for the regulation of Cdc25 phosphatases by Chk1 and Chk2. In response to genotoxic stress, both Chk1 and Chk2 phosphorylate various Cdc25 phosphatases on multiple N-terminal sites (N-℗) and inhibit them by various mechanisms; however, only Chk1 phosphorylates the various phosphatases on a C-terminal site (C-℗) and inhibits them from C-terminally interacting with their Cdk–cyclin substrates. Chk1 also functions during an unperturbed cell cycle. See text for details.