The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery
- Department of Oral Biology, University of Nebraska Medical Center, United States
- Department of Cell Biology and Physiology, School of Medicine, Washington University in St. Louis, United States
Figures
Figure 1 with 2 supplements
The protein level of MASTL is upregulated after DNA damage.
(A) HEK293 cells were treated with 0.5 µM doxorubicin (DOX) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32, and α-tubulin. (B) HEK293 cells were treated with 10 mM hydroxyurea (HU) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32, and α-tubulin. (C) HEK293 cells were treated with 10 nM camptothecin (CPT) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32, and α-tubulin. (D) HeLa cells were treated with 10 mM HU, and incubated as indicated. Cell lysates were collected and analyzed by immunoblotting for MASTL, phospho-H2AX Ser-139, RPA32, phospho-ATM/ATR substrates, and α-tubulin. (E) HeLa cells were treated with or without 0.5 µM DOX, 10 nM CPT, 10 mM HU, and 20 Gy ionizing radiation (IR) for 4 hr. Cell lysates were collected and analyzed by immunoblotting for MASTL, phospho-H2AX Ser-139, phospho-ATM/ATR substrates, and α-tubulin. (F) HeLa cells were treated with 0.5 μM DOX, and analyzed by immunoblotting for MASTL, α-tubulin, Aurora A, Aurora B, CDK1, cyclin B1, and phospho-ATM/ATR substrates. (G) SCC38 cells were incubated with or without HU and caffeine, as indicated, and analyzed by immunoblotting for MASTL and β-actin.
Figure 1—figure supplement 1
MASTL upregulation after DNA damage.
(A, B) SCC38 cells were treated with 10 mM hydroxyurea (HU) (A) or 10 Gy ionizing radiation (IR) (B), and incubated for 0–10 hr, as indicated. The expression levels of MASTL, RPA, and α-tubulin were analyzed by immunoblotting. (C, D) HeLa cells were treated with 0.5 µM doxorubicin (DOX) (C) or 10 mM HU (D) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL and α-tubulin. (E) Quantification of MASTL expression, measured by immunoblotting, and normalized to a loading control (α-tubulin or β-actin) in HeLa cells treated without or with IR, DOX, or HU. Standard derivations were calculated from three independent experiments.
Figure 1—figure supplement 2
The expression levels of cell cycle kinases after DNA damage.
HeLa (A) or SCC38 (B) cells were treated without or with 2 mM of hydroxyurea (HU) for 8 hr (A), or 3–6 hr (B). Cell lysates were harvested for immunoblotting.
Figure 2 with 1 supplement
MASTL upregulation after DNA damage is mediated by protein stabilization.
(A) HeLa cells were treated with 10 mM hydroxyurea (HU) for 2 hr, and harvested for gene expression analysis. Quantitative RT-PCR was performed to detect the RNA levels of MASTL and GAPDH. The ratio of MASTL to GAPDH expression is shown. The mean values were calculated from three experiments, and statistical significance was evaluated using an unpaired two-tailed Student’s t test. A p-value more than 0.05 was considered non-significant (ns). (B) CFP-tagged MASTL was expressed in SCC38 cells. Cells were treated with or without 10 mM HU for 3 hr and analyzed by immunoblotting for CFP-MASTL and H2B. (C) HeLa cells were treated with or without 10 nM CPT for 1 hr. These cells were then treated with cycloheximide (CHX, 20 μg/ml) at time 0 to block protein synthesis, and analyzed by immunoblotting for the protein stability of MASTL and α-tubulin. (D–F) SCC38 cells were treated without (D) or with (E) 10 mM HU for 2 hr. These cells were then treated with CHX (20 μg/ml) at time 0 to block protein synthesis, and analyzed by immunoblotting for the protein stability of MASTL and β-actin. In panel F, the band signals were quantified using ImageJ, and the mean values and standard deviations of MASTL/β-actin were calculated based on results of three experiments.
Figure 2—figure supplement 1
Increased protein stability of MASTL after DNA damage.
(A–C) HeLa cells expressing CFP-MASTL were treated without or with ionizing radiation (IR). Cells were analyzed by direct fluorescence for CFP-MASTL expression, and by immunofluorescence for the phosphorylation of ATM/ATR substrates. Quantifications are shown in panels B and C (>10 cells/time point). (D, E) HEK293 cells were treated without (D) or with (E) 10 mM hydroxyurea (HU) for 2 hr. These cells were then treated with cycloheximide (CHX, 20 μg/ml) at time 0 to block protein synthesis, and analyzed by immunoblotting for the protein stability of MASTL and α-tubulin.
Figure 3 with 1 supplement
E6AP associates with MASTL.
(A) Immunoprecipitation (IP) was performed in HeLa cell lysates, as described in Materials and methods. The lysate input, MASTL IP, and control (ctr) IP samples were analyzed by immunoblotting for E6AP and MASTL. (B) A pulldown assay was performed in HeLa cell lysates using MBP-tagged E6AP, as described in Materials and methods. The pulldown product, cell lysis input, and a control (-) pulldown (using empty beads) were analyzed by immunoblotting for E6AP and MASTL. (C) A pulldown assay was performed using MBP-tagged full-length E6AP in Xenopus egg extract. Purified segments of MASTL, including N (aa 1–340), M (aa 335–660), and C (aa 656–887), were supplemented in the extracts. The pulldown products, egg extract inputs, and a control (-) pulldown (using empty beads) were analyzed by immunoblotting for GST and MBP. (D) Segments of E6AP, including N (aa 1–280), M (aa 280–497), and C (aa 497–770), were tagged with GFP, and transfected into HeLa cells for expression. 24 hr after transfection, cell lysates were harvested for GFP IP. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for MASTL and GFP.
Figure 3—figure supplement 1
E6AP and MASTL associate via their N-terminal motifs.
(A) MBP-E6AP and GST-MASTL were expressed and purified, as described in Materials and methods. GST-MASTL on glutathione beads, or control glutathione beads, were incubated with MBP-E6AP, followed by pulldown, as described in Materials and methods. The input, GST-MASTL pulldown, and control pull down samples were analyzed by immunoblotting for MASTL and E6AP. (B) GFP-MASTL, FL or N-terminus (aa 1–340), was expressed in HeLa cells. Immunoprecipitation (IP) was performed using a GFP antibody, and the presence of E6AP in the IP products was examined by immunoblotting. (C) IP was performed using GFP-tagged segments of E6AP expressed in HeLa cells. The IP product, cell lysate input, and a control IP (using empty beads) were analyzed by immunoblotting for MASTL and GFP. N: aa 1–280; N1: aa 1–99; N2: aa 100–207; N3: aa 108–280.
Figure 4 with 1 supplement
E6AP mediates MASTL degradation.
(A) HeLa cells were transfected with control or E6AP-targeting siRNA E6AP for 24 hr. Cells were analyzed by immunoblotting for E6AP, MASTL, and β-actin. (B) HeLa cells were transfected with HA-tagged E6AP. 24 hr after transfection, cells were analyzed by immunoblotting for E6AP, MASLT, and α-tubulin. (C) As in panel B, HeLa cells were transfected with HA-E6AP, and analyzed by immunofluorescence (IF) for HA (green) and MASTL (red). Cells with HA-E6AP expression, as denoted by arrowheads, exhibited lower MASTL expression. (D) E6AP gene knockout (KO) was performed in HeLa cells, as described in Materials and methods. HA-E6AP was expressed in E6AP KO cells, as indicated. Cells were analyzed by immunoblotting for MASTL, E6AP, and α-tubulin. (E) HeLa cells transfected with control or E6AP siRNA were treated with 20 μg/ml cycloheximide (CHX), as indicated. Cells were harvested and analyzed by immunoblotting for MASTL and β-actin. (F) HeLa cells were treated as in panel E, MASTL and β-actin protein levels were quantified, and the ratio is shown for the indicated time points after CHX treatment, after normalized to that of time 0. The mean values and standard deviations were calculated from three experiments. (G) WT or E6AP KO HeLa cells were transfected with HA-tagged ubiquitin for 12 hr, followed 50 μM MG132 treatment for 4 hr. Cell lysates were harvested for HA immunoprecipitation (IP) or ctr IP using blank beads. The input and IP products were analyzed by immunoblotting for MASTL and HA. (H) In vitro ubiquitination assay was performed using E6AP as E3 ligase, and MASTL as substrate, as described in Materials and methods. S5a was added as a control substrate. The reactions were incubated as indicated, as analyzed by immunoblotting for MASTL, ubiquitination, and S5a.
Figure 4—figure supplement 1
E6AP mediates MASTL ubiquitination and degradation.
(A) HeLa cells were treated without or with E6AP siRNA. The RNA levels of E6AP and MASTL were quantified by real-time PCR. The mean values were calculated from three experiments. Statistical significance was determined using an unpaired two-tailed Student’s t test. A p-value more than 0.05 was considered non-significant (ns). (B) Control or E6AP knockout (KO) HeLa cells were treated without or with MG132, as indicated. The protein levels of MASTL and α-tubulin are shown by immunoblotting. (C) The E6AP in vitro ubiquitination assay was performed using MASTL as substrates, as in Figure 4H. S5a was added as a control substrate. E1/2 and E3 (E6AP) enzymes were added in the reactions, as indicated. Samples were analyzed by immunoblotting for MASTL and S5a after 90 min incubation. (D) In vitro ubiquitination assay was performed using E6AP as E3 ligase, and ΔN MASTL (aa 335–887) as substrate. S5a was added as a control substrate. The reactions were incubated as indicated, as analyzed by immunoblotting for MASTL and S5a.
Figure 5 with 1 supplement
E6AP depletion promotes DNA damage checkpoint recovery via MASTL.
(A) WT or E6AP knockout (KO) HeLa cells were treated with or without MASTL siRNA. The cells were incubated in 0.1 μM etoposide (ETO) for 18 hr, and released in fresh medium for recovery. Cells were harvested at the indicated time points (after the removal of ETO) for immunofluorescence (IF) using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired two-tailed Student’s t test was used to determine the statistical significance (*p<0.05, **p<0.01, n>500 cell number/measurement). MASTL knockdown by siRNA was shown by immunoblotting in the panel E. (B) WT or E6AP KO HeLa cells with or without MASTL siRNA, as in panel A, were treated with 2 mM hydroxyurea (HU) for 18 hr. Cells were then released in fresh medium, and incubated as indicated, for recovery. The cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS), as described in Materials and methods. (C) WT or E6AP KO HeLa cells were treated with or without 0.5 μM doxorubicin (DOX) for 4 hr. Cells were then analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, phospho-CHK1 Ser-345, phospho-CHK2 Thr-68, γ-H2AX, and α-tubulin. (D) WT, E6AP KO, or E6AP KO with expression of HA-E6AP HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, and α-tubulin. (E) WT, E6AP KO, or E6AP KO with transfection of MASTL siRNA HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for MASTL, phospho-ATM/ATR substrates, and α-tubulin.
Figure 5—figure supplement 1
Impaired DNA damage checkpoint signaling in E6AP-null cells.
(A) Control or MASTL knockdown HeLa cells were maintained in cell culture, and cell viability was determined by counting cell numbers. The cell numbers in days 2 and 3 were normalized to those in day 1. The mean values and standard derivations, calculated from three experiments, are shown. (B) Control or E6AP knockout (KO) HeLa cells were treated with 0.1 μM etoposide (ETO) for the indicated hours. Cells were analyzed by immunoblotting for phospho-ATM/ATR substrates and α-tubulin.
Figure 6
The E6AP and MASTL association is disrupted by DNA damage-induced ATM/ATR signaling.
(A) HeLa cells expressing CFP-MASTL were treated without or with 10 mM hydroxyurea (HU) for 2 hr. CFP-MASTL immunoprecipitation (IP) was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, and β-actin. (B) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU and 4 mM caffeine, as indicated, for 2 hr. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, phospho-CHK1 Ser-345, and α-tubulin.
Figure 7 with 1 supplement
ATM/ATR mediates E6AP S218 phosphorylation to disrupt MASTL association and proteolysis.
(A) The sequence alignment of the conserved E6AP Ser-218 motif in human, mouse, and Xenopus. (B) A phospho-specific antibody recognizing E6AP Ser-218 was generated, as described in Materials and methods. WT or E6AP knockout (KO) HEK293 cells were treated without or with 10 mM hydroxyurea (HU) for 4 hr, and analyzed by immunoblotting for phospho-E6AP Ser-218, E6AP, and α-tubulin. (C) HeLa cells were treated without or with 0.5 μM doxorubicin (DOX) and 5 μM KU55933 (ATMi), as indicated, for 1 hr, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-E6AP Ser-218, and α-tubulin. (D) HeLa cells were transfected with HA-tagged WT, S218A, or S218D E6AP. Cell lysates were harvested for immunoprecipitation (IP) assays. The input, MASTL IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL and HA. (E) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX for 3 hr, and harvested for IP assays. The input, HA IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL, phospho-E6AP Ser-218, and HA. (F) E6AP KO HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX, incubated as indicated, and harvested for immunoblotting for MASTL and α-tubulin.
Figure 7—figure supplement 1
DNA damage-induced E6AP Ser-218 phosphorylation is mediated by ATM.
(A) WT or E6AP knockout HEK293 cells were treated without or with doxorubicin (DOX, 0.5 μM) for 4 hr. Cells were harvested and analyzed by immunoblotting for phospho-E6AP Ser-218 and α-tubulin. (B) HEK293 cells were treated without or with DOX (0.5 μM), or ATM inhibitor (KU55933, 10 μM), for 4 hr, and analyzed by immunoblotting. (C) HeLa cells were treated with hydroxyurea (HU, 10 mM) combined with ATM/ATR inhibitor (caffeine, 4 mM) or ATM inhibitor (KU55933, 10 μM), for 12 hr, as indicated. Cells were harvested and analyzed by immunoblotting.
Figure 8
E6AP S218 phosphorylation is required for DNA damage recovery.
(A) E6AP knockout (KO) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in Figure 7. Cells were treated with 0.1 μM etoposide (ETO) for 18 hr, and released in fresh medium for recovery. Cells were then harvested at the indicated time points (after the removal of ETO) for immunofluorescence (IF) using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired two-tailed Student’s t test was used to determine the statistical significance (**p<0.01, n>500 cell numbers/measurement). (B) E6AP KO HeLa cells expressing HA-tagged WT or S218A E6AP, as in panel A, were treated with 2 mM hydroxyurea (HU) for 18 hr. Cells were then released in fresh medium, and incubated as indicated, for recovery. Cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS). (C) WT or S218A E6AP was expressed in E6AP KO HEK293 cells. Cells were treated without or with 0.1 μM ETO for 18 hr, released in fresh medium for recovery, and incubated as indicated. Cells were analyzed by immunoblotting for phospho-cyclin-dependent kinase (CDK) substrates and histone H3. (D) WT or S218A E6AP was expressed in E6AP KO HEK293 cells, as in panel C. Cells were treated without or with 1 μM CPT for 90 min, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, and α-tubulin.
Figure 9
A ‘timer’ model for the role of the ATM-E6AP-MASTL axis in cell cycle arrest and recovery after DNA damage.
DNA damage induces ATM/ATR activation and checkpoint signaling. At this stage (the initial DNA damage sensing/signaling), MASTL expression is normal (not yet upregulated). Activated ATM/ATR also phosphorylates E6AP Ser-218, leading to the dissociation of E6AP from MASTL and reduced MASTL degradation. The subsequent accumulation of MASTL, at upregulated levels several hours after DNA damage, promotes de-activation of the DNA damage checkpoint and initiates cell cycle resumption by inhibiting dephosphorylation of cyclin-dependent kinase (CDK) substrates.
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Source data 1
Original gel blots, as presented in the figures and figure supplements, are provided as source data.
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The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery
eLife 12:RP86976.
https://doi.org/10.7554/eLife.86976.3
