Ca2+-Stimulated AMPK-Dependent Phosphorylation of Exo1 Protects Stressed Replication Forks from Aberrant Resection

Abstract

Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca²⁺ concentration ([Ca²⁺]_i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca²⁺]_i and the replication stress response as well as a function of the Ca²⁺-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.

Keywords: 14-3-3; AMPK; Ca(2+); CaMKK2; Exo1; calcium signaling; genome maintenance; protein phosphorylation; replication fork resection; replication stress response.

Figure 1.. Upon replication stress Exo1 undergoes phosphorylation at S746, resulting in 14-3-3s binding, which in turn suppresses chromatin recruitment of Exo1, thereby preventing aberrant fork processing, chromosomal instability and cell death.

(a) Co-IP of Exo1(WT)-Flag or Exo1(S746A)-Flag with 14-3-3s in cells treated with HU (2 mM) or H₂O for 3 h. (b) S4/8-phosphorylation of RPA in wild-type or Exo1-KO HeLa cells transfected with empty vector or mCherry-tagged Difopein after treatment with HU (2 mM) or H₂O for 4 h. (c) Result of DNA fiber analysis in the transfected cells described in (b). Top panel: Experimental scheme (see Methods). Bottom panel: dot plot of the ratio of CIdU/IdU track length from two independent experiments. Red bars represent the median. 150 tracts were scored for each sample. ****, p⩾0.0001 (unpaired t-test). (d) S746-phosphorylation of endogenous Exo1 in HeLa cells upon replication stress. Cells were treated with HU (2 mM) or H₂O for 3 h and S746-phosphorylation of immunoprecipitated Exo1 was detected by western blotting with an anti-pS746 antibody. (e) S746-phosphorylation of ectopically expressed Exo1 in HeLa cells treated with HU (2 mM) or APH (5 μg/ml) for 3 h. (f) Association of Exo1(WT)-GFP and Exo1(S746A)-GFP with chromatin in HU-treated or H₂O-treated U2OS cells after extraction. (g) S4/8-phosphorylation of RPA in Exo1-KO HeLa cells transfected with empty vector or FLAG-tagged Exo1(WT), Exo1(S746A), Exo1(D173A) or Exo1 (DM) after treatment with HU (2 mM) or H₂O for 4 h. (h) Quantified BrdU signal in ssDNA for the Exo1-replacement cells described in (g). Representative BrdU IF images are shown in Figure S1g. Cells with a BrdU signal above that in the majority (98%) of HU-untreated control cells (black dots) were taken as BrdU-positive (green dots). Red bars represent the median BrdU intensity of BrdU-positive cells. 1,000 cells were analyzed for each sample. n=3, ***, p≤0.001, ****, p≤0.0001 (unpaired t-test). (i) Result of DNA fiber analysis in the Exo1-replacement cells described in (g). Top panel: Experimental scheme (see Methods). Bottom panel: dot plot of the ratio of CIdU/IdU track length from two independent experiments. Red bars represent the median. 150 tracts were scored for each sample. ****, p≤0.0001 (unpaired t-test). (j) Left panel: Representative images of metaphase chromosome spreads of Exo1-KO cells transfected with empty vector, Exo1(WT)-Flag or Exo1(S746A)-Flag after HU treatment. Chromosomal aberrations are marked by arrows. Right panel: Quantified results of the samples depicted in the left panel. 150 metaphases from three independent experiments were examined for each sample. (k) Result of clonogenic analysis of the survival of Exo1-KO cells transfected with empty-vector, Exo1(WT)-Flag or Exo1(S746A)-Flag after treatment with indicated concentrations of HU. Data represent mean ± S.D. from triplicates.

Figure 2.. Both AMPK and Chk1 phosphorylate Exo1 at S746 upon replication stress.

(a) Sequence alignment between S746 or the corresponding site in human, mouse and Xenopus Exo1 and the consensus phosphorylation sites of AMPK and Chk1. X, any amino acid. (b) Coomassie blue staining of purified recombinant His-Chk1(WT), His-Chk1(KD) (D130A, kinase-dead mutant) and Exo1-His proteins expressed in insect cells. (c) S746-phosphorylation of Exo1 by an equal amount of recombinant His-Chk1(WT) or His-Chk1(KD) proteins in vitro. Exo1 phosphorylation was detected by immunoblotting using anti-pS746 antibody. Exo1 and Chk1 proteins were detected by using anti-His antibody. The gel mobility changes and apparent decrease in western blot signal of Chk1(WT) likely resulted from its autophosphorylation. (d) Coomassie blue staining of purified recombinant AMPKα1/β1/γ1(WT) and AMPKα1/β1/γ1(KD) (D159A, kinase-dead mutant) complexes expressed in insect cells. (e) S746-phosphorylation of Exo1 by recombinant AMPKα1/β1/γ1 complexes in vitro. Exo1 phosphorylation was detected by immunoblotting using anti-pS746 antibody. Exo1 and AMPKα1 proteins were detected by using anti-His antibody. The gel mobility changes and apparent decrease in western blot signal of AMPKα1(WT) likely resulted from its autophosphorylation. (f) S746-phosphorylation of Exo1 and S345-phosphorylation of Chk1 in wild-type HeLa cells and in two independent AMPKα-KO cell clones treated with HU (2 mM) or H₂O for 4 h. (g) S746-phosphorylation of Exo1 and S345-phosphorylation of Chk1 in AMPKα-KO cells transfected with control siRNA or Chk1 siRNA in response to HU (2 mM) or H₂O treatment for 4 h. (h) S746-phosphorylation of Exo1 and S345-phosphorylation of Chk1 induced by HU (2 mM, 4 h) in cells pretreated with Compound C (20 μM), AZD7762 (150 nM), or both.

Figure 3.. AMPK prevents aberrant fork processing and promotes chromosomal stability and cell survival after replication stress.

(a) S4/8-phosphorylation of RPA and S345-phosphorylation of Chk1 in WT or AMPKα-KO HeLa cells treated with HU (2 mM) or H₂O for 4 h. (b) Quantified BrdU signal in ssDNA in WT or AMPKα-KO HeLa cells treated with HU (2 mM) or H₂O for 4 h. Representative BrdU IF images are shown in Figure S2b. Red bars represent the median BrdU intensity of BrdU-positive cells. 1,000 cells were analyzed for each sample. n=3, ****, p≤0.0001 (unpaired t-test). (c) Result of DNA fiber analysis in WT or AMPKα-KO HeLa cells treated with HU (4 mM) or H₂O. Red bars represent the median. 150 tracts were scored for each sample. n=3, ****, p≤0.0001 (unpaired t-test). (d) Effects of CC (20 μM) on BrdU signal in ssDNA in HeLa cells after treatment of HU (2 mM) or H₂O for 4 h. Representative IF images are shown in Figure S2c. Red bars represent the median BrdU intensity of BrdU-positive cells. 1,000 cells were analyzed for each sample. n=3, ****, p≤0.0001 (unpaired t-test). (e) Quantified BrdU signal in ssDNA in WT or AMPKα-KO MEFs treated with HU (0.5 mM) or H₂O for 4 h. Representative IF images are shown in Figure S2h. Red bars represent the median BrdU intensity of BrdU-positive cells. 1,000 cells were analyzed for each sample. n=3, ****, p≤0.0001 (unpaired t-test). (f) Upper panel: Representative images of metaphase chromosome spreads of WT or AMPKα-KO HeLa cell clones after HU treatment. Chromosomal aberrations are marked by arrows. Lower panel: Quantified results for the samples depicted in the upper panel. 150 metaphases from three independent experiments were examined for each sample. (g) Result of clonogenic analysis of WT or AMPKα-KO HeLa cells after HU treatment. Data represent mean ±S.D. from triplicates. (h) Result of clonogenic analysis of WT or AMPKα-KO MEFs after HU treatment. Data represent mean ± S.D. from triplicates.

Figure 4.. Exo1 is the primary target of AMPK in fork protection after replication stress.

(a) Effects of siRNA-mediated knockdown of Exo1 on S4/8-phosphorylation of RPA in WT or AMPKα-KO HeLa cells after treatment with HU (2 mM) or H₂O for 4 h. (b) Quantified BrdU signal in ssDNA in WT or AMPKα-KO HeLa cells transfected with control or Exo1 siRNAs after HU (2 mM) or H₂O treatment for 4 h. Representative IF images are shown in Figure S3a. Red bars represent the median BrdU intensity of BrdU-positive cells. 1,000 cells were analyzed for each sample. n=3, **, p≤0.01; ***, p≤0.001 (unpaired t-test). (c) Result of DNA fiber analysis in WT or AMPKα-KO HeLa cells transfected with control siRNAs or Exo1 siRNAs after HU or H₂O treatment. Red bars represent the median. 150 tracts were scored for each sample. n=3, ****, p≤0.0001 (unpaired t-test). (d) Result of clonogenic analysis of WT or AMPKα-KO HeLa cells transfected with control siRNAs or Exo1 siRNAs after HU treatment. Data represent mean ± S.D. from triplicates.

Figure 5.. CaMKK2 promotes AMPK activation to prevent aberrant fork resection upon replication stress.

(a) T172-phosphorylation of AMPKα in HeLa cells treated with HU (4 mM) or APH (5 μg/ml) for 4 h. (b) Representative IF images of T172-phosphorylated AMPKα in HeLa cells treated with HU (4 mM) or APH (5 μg/ml) for 4 h. γH₂AX signal marks cells with replication stress. Quantified result is shown in Figure S4c. (c) Upper panel: Representative FRET images of pan-ABKAR in U2OS cells treated with HU (4 mM) or APH (5 μg/ml) for 4 h. Lower Panel: Quantified FRET signals of the samples depicted in the upper panel. Red bars represent the median FRET ratio. 500 cells were analyzed for each sample. n=3, ****, p≤0.0001 (unpaired t-test). (d) T172-phosphorylation of AMPKα and S4/8-phosphorylation of RPA in WT HeLa cells and two independent clones of CaMKK2-KO knockout cells that were treated with HU (2 mM) or H₂O for 4 h. (e) Effects of the CaMKK2 inhibitor STO609 (25 μM) on AMPKα T172-phosphorylation and RPA S4/8-phosphorylation in HeLa cells in response to HU treatment (2 mM, 4 h). (f) S746-phosphorylation of Exo1-GFP in WT and CaMKK2-KO HeLa cells treated with HU (2 mM) or H₂O for 4 h. (g) Quantified BrdU signal in ssDNA in WT or CaMKK2-KO HeLa cells after treatment of HU (2 mM) or H₂O for 4 h. Representative BrdU IF images are shown in Figure S4h. Red bars represent the median BrdU intensity of BrdU-positive cells. 1,000 cells were analyzed for each sample. n=3, ****, p≤0.0001 (unpaired t-test). (h) Effects of STO609-pretreatment (25 μM, 30 min) on BrdU signal in ssDNA in HeLa cells after treatment with HU (2 mM) or H₂O for 4 h. Representative BrdU IF images are shown in Figure S4k. Red bars represent the median BrdU intensity of BrdU-positive cells. 1,000 cells were analyzed for each sample. n=3, ****, p≤0.0001 (unpaired t-test). (i) Result of clonogenic analysis of WT or CaMKK2-KO HeLa cells after HU treatment. Data represent mean ± S.D. from triplicates.

Figure 6.. Replication stress induces [Ca²⁺]_i elevation, which prevents aberrant fork resection by eliciting AMPK activation and Exo1 phosphorylation.

(a) Upper panel: Representative images of GCaMP6s reporter signal in S phase-synchronized U2OS cells treated with HU (4 mM) or APH (5 μg/ml) for 4 h. Lower panel: Quantified results for the samples depicted in the upper panel. Red bars represent the median. 100 cells were scored for each sample. n=3, ****, p≤0.0001 (unpaired t-test). (b) Effects of BAPTA-AM pretreatment (50 μM, 30 min) on AMPKα T172-phosphorylation in U2OS cells induced by HU (4 mM) or APH (5 μg/ml) treatment for 4 h. γH₂AX signal marks the cells with replication stress. Quantified result is shown in Figure S5g. (c) Effects of BAPTA-AM (50 μM) on S746-phosphorylation of transfected Exo1-GFP in HeLa cells induced by HU (2 mM) or H₂O treatment for 4 h. (d) Effects of BAPTA-AM on BrdU signal in ssDNA in HeLa cells after HU (2 mM) or H₂O treatment for 4 h. Representative BrdU IF images are shown in Figure S5h. Red bars represent the median BrdU intensity of BrdU-positive cells. 1,000 cells were analyzed for each sample. n=3, ****, p≤0.0001 (unpaired t-test). (e) S746-phosphorylation of Exo1 in cells cultured in regular medium or in Ca²⁺-free medium after HU (2 mM) or H₂O treatment for 4 h. Pre-treatment with BAPTA-AM (membrane permeable), but not BAPTA (membrane impermeable), inhibited HU-induced Exo1 phosphorylation in cells cultured in Ca²⁺-free medium. (f) T172-phosphorylation of AMPKα and S746-phosphorylation of transfected Exo1-GFP in HeLa cells after treatment with Thapsigargin (1 μM) or A23187 (2 μM) for 1 h. (g) T172-phosphorylation of AMPKα and S746-phosphorylation of transfected Exo1-GFP in WT or AMPKα-KO HeLa cells after treatment with A23187 (2 μM) for 1 h. (h) A model for the role of the Ca²⁺-CaMKK2-AMPK-Exo1 signaling pathway in protecting replication fork structure to promote genomic stability and cell survival after replication stress. See text for details.