Suppression of CHK1 protein levels in TS cells induces expression of p57 and p21. (A) TS cells were cultured in the absence of FGF4 and conditioned medium for the indicated times, and total cell extracts were then analyzed by Western immunoblotting for the indicated proteins. (B) Proliferating TS cells and TG cells (2 days of FGF4 deprivation) were infected with lentivirus expressing either control shRNA (ctrl) or shRNA targeted against Chk1 mRNA (CHK1) and selected for puromycin resistance. Total extracts of surviving cells were analyzed as described for panel A. (C) Total RNA was isolated 3 days after proliferating TS cells were infected with lentivirus as described for panel B, and the presence of mRNAs characteristic of either TS or TG cells was assayed by RT-PCR, as previously described (48). Genes expressed in TS cells but suppressed in TG cells included Chk1, Fgfr2 (the fibroblast growth factor receptor for FGF4), and Esrrb/Err2 (estrogen-related receptor-β). Genes expressed in both TS and TG cells included Chk2 and Gapdh (glyceraldehyde-3-phosphate dehydrogenase). Genes upregulated in TG cells included Prl3d1/Pl1 (prolactin family 3, subfamily d, member 1) and Prl3b1/Pl2 (prolactin family 3, subfamily b, member 1). (D) TS cells infected with lentivirus as described for panel B were stained with 4′,6′-diamidino-2-phenylindole (DAPI) to visualize DNA (blue) and antibodies to visualize p57 protein (red). Photos are at a ×60 magnification. (E) The fraction of nuclei expressing p57 protein was quantified under the various conditions described above and in the legend of Fig. 2. Error bars indicated standard errors of the means. AZD, AZD7762.

Suppression of CHK1 kinase activity in TS cells induces giant cell morphology, blocks cell proliferation, and inhibits induction of endocycles. (A) TS cells were cultured for 3 days either in the absence of FGF4 (−FGF4) or in the presence of 50 nM AZD7762 (+AZD). Cells were photographed in phase contrast (×10 magnification). Similar results were obtained with TS cells treated with UCN01 or transfected with shRNA^CHK1 (data not shown). TS cells grow in tightly packed colonies in which individual cells are difficult to distinguish. The difference in sizes between TS and TG cells is clearest when cells are dissociated (48). (B) TS cells were cultured either in the presence (•, ▪, ▴) or absence (○) of UCN01, AZD7762, and shRNA^CHK1, respectively. The number of cells per T75 flask was scored at the indicated time. The extent to which cells underwent endoreduplication was determined by FACS analysis (48). (C) TS cells cultured for 3 days either in the presence or absence of 200 nM UCN01 were assayed for CDK1 and CDK2 protein kinase activities by immunoprecipitating each CDK protein and then assaying it for its ability to phosphorylate histone H1. (D) TS cells cultured for 3 days either in the presence of FGF4 alone (top panels) or in the presence of FGF4 and either shRNA^CHK1 (+shRNA), 50 nM AZD7762 (+AZD), or 200 nM UCN01 (+UCN01) (bottom panels). (E) TS cells deprived of FGF4 for 3 days (top panel) were then cultured for an additional 3 days in the presence or absence of 200 nM UCN01 (bottom panel).

Expression of an active CHK1 protein in TG cells suppresses p57 expression, whereas expression of CHK2 does not. TS cells were deprived of FGF4 for 3 days to induce differentiation into TG cells. The cells were then transfected with a plasmid expressing either YFP, a CHK1 protein with a mutation of K38 to R38 that abolishes kinase activity (CHK1*-YFP) (15, 30), or wild-type CHK1 protein linked to YFP (CHK1-YFP) or wild-type CHK2 linked to YFP (CHK2-YFP), as well as a neomycin resistance gene. (A) At 24 h posttransfection, cells were treated with neomycin, and the surviving cells were sonicated 48 h later and subjected to Western immunoblotting. (B) YFP was then immunoprecipitated from total cell extract and assayed for protein kinase activity on CHKtide. (C) TG cells transfected with the indicated plasmid were fixed 2 days posttransfection and analyzed by fluorescence microscopy. Cells were stained with 4′,6′-diamidino-2-phenylindole (DAPI) to visualize DNA (blue) and antibodies to visualize p57 (red). YFP and YFP-tagged proteins (green) were visualized directly by using a green filter. (D) The fraction of nuclei that were p57 positive (red), YFP positive (green), or both p57 and YFP positive (yellow) was determined as a percentage of transfected cells. In each case, at least 200 transfected cells were counted in three independent experiments. Error bars are standard errors of the means. Notably, none of the cells transfected with active CHK1-YFP (green) also expressed p57 (red).

Suppression of endogenous p57 in TG cells induces mitosis in the absence of cytokinesis. TS cells were cultured for 2 days in the absence of FGF4 to produce TG cells. Cells were then transfected with a plasmid expressing either recombinant YFP, CHK1*-YFP, CHK1-YFP (shown in A), CHK2-YFP, or CDK1 (shown in B), as described in the legend of Fig. 4. Some cells were stained with propidium iodide to visualize nuclear DNA (B, red in inset). Cells were examined at 4 days posttransfection. (C) For comparison, shRNA against p57 was expressed in TG cells, as described in the legend of Fig. 1. Cells were stained with propidium iodide to label the nuclei and with Troma-1 antibody to label the cytoplasm (48). (D) The fraction of cells containing two or more nuclei was recorded for each condition. In addition, TS cells nullizygous for p57 (p57^−/−) were cultured for 10 days in the absence of FGF4 (48). Error bars indicate the standard deviations for 3 experiments.

CHK1 phosphorylates p57 specifically at Ser19 within the CHK1/CHK2 consensus substrate motif. (A) Aliquots of a total cell extract from TS cells cultured in the presence of either 50 nM AZD7762 or 2 mM MG132 were fractionated by gel electrophoresis for 4 h rather than 1 h to separate the different forms of p57. The indicated aliquots were pretreated with lambda protein phosphatase (λPPase) in the presence and absence of its inhibitor NaF. (B) Synthetic peptide from amino acids 1 to 25 of mouse p57 contains a putative CHK1 phosphorylation site [RXX(T/S)] with two potential phosphorylation sites, Thr12 and Ser19. (C) Recombinant CHK1 and CHK2 proteins were assayed for their ability to phosphorylate CHKtide, p57 peptide (p57tide), and recombinant p57 protein (expressed in E. coli). Samples were fractionated by gel electrophoresis and transferred to a polyvinylidene difluoride membrane. ³²P-labeled proteins of the correct molecular weights were detected by autoradiography. p57 protein was detected by Western immunoblotting. (D, E, and F) Various exposures of the data in panel C and similar results were quantified by densitometry and plotted as percentages of the maximum phosphorylation observed for CHK1. (G) CHK1 phosphorylation of p57 protein immunoprecipitated from TG cells was assayed in the presence or absence of 200 nM UCN01 (p57-P). (H) The activity of CHK1 kinase was assayed with recombinant p57 wild-type protein, p57 with a mutation of threonine 12 to alanine (T12A), and p57 with a mutation of serine 19 to alanine (S19A) (p57-P). The amount of p57 protein in each assay was determined by immunoblotting (p57). (I) Time course for CHK1 phosphorylation of equivalent amounts of wild-type and S19A p57 proteins in vitro. Error bars indicate standard deviations for three to five experiments.

Site-specific phosphorylation of p21 by CHK1. (A) Mouse p21 protein contains two overlapping CHK consensus phosphorylation sites (bold), RRQT-140 and RQTS-141 (NCBI reference NP001104569). (B) Both recombinant CHK1 and CHK2 phosphorylated a CDC25C peptide (CHKtide), but only CHK1 phosphorylated recombinant p21 in vitro under the same conditions used for p57 phosphorylation (Fig. 7). Equivalent amounts of p21(wt), the p21 mutants T140V and S141A, and p21 the double mutant T140V S142A (TV+SA) (C) were tested in parallel for their ability to be phosphorylated by CHK1 (D). (E) These and other data were quantified relative to p21(wt). Error bars indicate standard errors of the means for four experiments.

Phosphorylation at Ser19 targets p57 for degradation by the 26S proteasome. (A) 3T3 cells were cotransfected with a plasmid expressing the Tet repressor and a plasmid expressing either LacZ, p57(wt), p57(S19D), or p57(S19A). Tetracycline (1 μg/ml) was added to the medium 30 h after transfection to induce p57 expression. At 18 h after tetracycline induction (48 h posttransfection), the indicated proteins were assayed by Western immunoblotting. p57(S19D) was not detected even with longer exposures of the immunoblot. Total RNA was extracted from one portion of the cells and analyzed by RT-PCR for the indicated gene. (B) The protocol in panel A was applied to TS cells where transfection efficiency was poor. (C) After tetracycline induction, 3T3 cells were cultured in the presence of 2 μM MG132 for 5 h before subjecting them to Western immunoblotting for the indicated proteins. (D) 3T3 fibroblasts were cotransfected with a plasmid expressing Tet repressor and a plasmid expressing either LacZ, p57(wt), p57(S19D), or p57(S19A). At 30 h posttransfection, tetracycline was added to the cells to induce gene expression. Tetracycline was removed from the medium 18 h later in order to terminate expression of the recombinant genes, and total cell extracts were subjected to immunoblotting for the indicated protein. (E) Data collected from immunoblots of various exposures of panel C were quantified by densitometry relative to the amount of actin present. Error bars indicate standard errors of the means for three experiments. (F) 3T3 cells were transfected with a plasmid expressing p57(wt), p57(S19D), or p57(S19A) in the absence of Tet repressor. At 24 h posttransfection, the cells were treated with 50 μg/ml cycloheximide to block further protein synthesis. At the indicated times, total cell extracts were subjected to immunoblotting for the indicated protein, and the data were quantified as described in panel E.

CHK1 is the effector kinase for two checkpoints. The DNA replication checkpoint prevents premature mitosis in response to DNA damage and stalled replication forks. These events activate (+) the ATR kinase that activates the CHK1 kinase that inhibits (⊥) the CDC25 phosphatase through site-specific phosphorylation. Since CDC25 is required to activate CDK1, inhibition of CDC25 prevents entrance into mitosis. The G₂ restriction point prevents expression of p57 and p21 in response to mitogen stimulation. In the case of TS cells, the mitogen is FGF4. In the presence of FGF4, TS cells express the p21 and p57 genes, but CHK1 phosphorylates the p21 and p57 proteins, thereby targeting them for degradation (Ø) by the 26S proteasome. This prevents TS cells from exiting the mitotic cell cycle. In the absence of FGF4 (dashed line), CHK1 protein is degraded, thereby upregulating p57 and p21. p21 may constitute a feedback loop that further suppresses CHK1 expression. Inhibition of CDK1 by p57 triggers endoreduplication and differentiation into TG cells. In TG cells, p57 protein levels oscillate to allow endocycles. p57 (as well as p27) prevents Cdk2 from initiating S phase until increased CcnE levels result in sufficient Cdk2-CcnE activity to phosphorylate p57 (as well as p27) at a CDK-specific site, thereby targeting it for degradation. p21 may be involved in sustaining suppression of CHK1.

Inhibition of CHK1 activity in TS cells induces p57 expression. (A) ES, 3T3, TS, and TG cells were cultured in the presence of 200 nM UCN01, a CHK1 inhibitor, for the indicated times before total cell extracts were analyzed for the indicated proteins by immunoblotting. TG cells were produced by FGF4 deprivation for 3 days before the addition of UCN01. (B) TS cells were cultured for 3 days either in the absence of FGF4 and conditioned medium to produce TG cells or in the presence of FGF4 and either 200 nM UCN01 or 50 nM AZD7762. Cells were stained as described in the legend of Fig. 1D. Since the results with the two CHK1 kinase inhibitors were indistinguishable, only UCN01 data are shown.

CHK1 activity in TS cells targets p57 and p21 proteins for degradation. (A) Total RNA was isolated from proliferating cultures of ES, 3T3, TS, and TG cells and then analyzed by RT-PCR for the indicated transcripts. (B) ES, 3T3, TS, and TG cells were cultured for 5 h in the presence of the indicated concentration of MG132 before total cell extracts were analyzed by Western immunoblotting. (C and D) To compare directly p21 and p57 gene expression, TS cells were cultured in the presence of either 50 nM AZD7762 (C) or 2 μM MG132 (D) for the indicated times and then assayed for p21 and p57 protein by Western immunoblotting. Tubulin was included as a loading control.