Polo-like kinase 1 inhibits DNA damage response during mitosis

Abstract

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.

Keywords: 53BP1; 53BP1, p53 binding protein 1; ATM, ataxia telangiectasia mutated kinase; BRCA1, breast cancer type 1 susceptibility protein; Cdk, cyclin dependent kinase; DDR, DNA damage response; DNA damage response; H2AX, histone variant H2AX; IR – ionizing radiation; MDC1, mediator of DNA damage checkpoint protein 1; NCS – neocarzinostatin; NZ – nocodazole; PTIP, PAX transactivation activation domain-interacting protein; Plk1, Polo-like kinase 1; Polo like kinase 1; RIF1, Rap1-interacting factor 1 homolog; RNAi, RNA interference; RNF168, RING finger protein 168; RNF8, RING finger protein 8; mitosis; phosphorylation.

Figure 1.

53BP1 is phosphorylated during mitosis by Cdk1 and Plk1. (A) U2OS cells were irradiated with 3 Gy, fixed after 1 h and probed for γH2AX, 53BP1 and DAPI and analyzed by confocal microscopy. Single focal plane is shown. Bar indicates 10 μm. (B) Endogenous 53BP1 was immunoprecipitated from exponentially grown cells (Asynch.) or from cells arrested in mitosis by NZ, separated on 3–8% Tris-Acetate gel and analyzed by immunoblotting. Where indicated immunopurified 53BP1 was incubated with λ-phosphatase for 15 min at 30°C. (C) U2OS cells were grown exponentially (Asynch.), treated for 12 h with RO-3306 to arrest them in G2, or with BI2536 or NZ to arrest them in mitosis. Mitotic cells treated with NZ were collected by shake-off and incubated for additional 60 min with DMSO or with RO-3306 or SB202190. Whole cell lysates were separated on 3–8% Tris-Acetate or 4–12% Bis-Tris gels and probed with indicated antibodies.

Figure 2

(See previous page). Plk1 phosphorylates 53BP1 in the UDR domain. (A) Purified GST or GST-53BP1-C-term were incubated with His-Plk1 in the presence of ³²P-γ-ATP and then separated on SDS-PAGE. Phosphorylation was detected by autoradiography or by immunoblotting with pS1618–53BP1 antibody. (B) Purified GST, GST-53BP1-C-term-WT or -S1618A were incubated with His-Plk1 and Phosphorylation was detected by autoradiography or by immunoblotting. (C) Unsynchronized cells (Asynch.) or cells arrested in mitosis by nocodazole or by Plk1 inhibitor (BI2536) were lyzed and probed with indicated antibodies. (D) U2OS cells were transfected with GAPDH or 53BP1 siRNA and grown asynchronically or arrested in mitosis by nocodazole. Arrowhead indicates the same position on the gel (E) U2OS cells were transfected by siRNA targeting GAPDH or Plk1. Nocodazole was added to cells transfected with GAPDH siRNA. Cells depleted of Plk1 spontaneously arrested in mitosis. Mitotic cells were collected by mitotic shake-off and analyzed by immunoblotting. (F) HeLa or U2OS cells were synchronized at G1/S transition by a double thymidine block, released to fresh media with nocodazole and collected in 2 h intervals. Media without nocodazole was used as control for cells that progressed to the following G1. (G) hRPE-TERT cells were grown exponentially or arrested in mitosis by nocodazole or BI2536 for 16 h and collected by mitotic shake-off. (H) Mitotic U2OS cells (NZ) were released to the fresh media and collected in 1 h intervals.

Figure 3.

53BP1 phosphorylated at S1618 colocalize with Plk1. (A) U2OS cells were transfected with GAPDH or 53BP1 siRNA, fixed after 48 h and probed for endogenous 53BP1 and CREST (marker of centromeres) using confocal microscopy. Images represent single focal planes. Insets show magnified regions of the same image. Bar indicates 10 μm or 1 μm in the insets. (B) Mitotic cells expressing EGFP-Plk1 were probed with 53BP1 or pS1618–53BP1 and CREST and analyzed by confocal microscopy as in A. (C) U2OS cells transfected with control, 53BP1 or Plk1 siRNA were analyzed after 48 h by confocal microscopy. Alternatively cells were treated with BI2536 (10 nM, 3 h). Images represent single focal plane and bar indicates 10 μm. (D) Exponentially growing U2OS cells were fixed and stained with pS1618–53BP1 antibody. Bar indicates 10 μm. Total cell fluorescence was quantified in interphase and mitotic cells. Each dot represents one cell. Error bars indicate mean and SD. (E) U2OS cells were transfected with GAPDH or 53BP1 siRNA, fixed and probed for tubulin and DAPI. Morphology of mitotic spindles was scored as bipolar or aberrant (monopolar and multipolar; n=3, error bars indicate SD). (F) U2OS stably expressing EGFP-53BP1 were grown exponentially (Asynch.), synchronized in G2 by RO-3306 or in mitosis by nocodazole and 53BP1 was immunoprecipitated by GFP-Trap. Bound proteins were analyzed by immunoblotting.

Figure 4.

53BP1 is phosphorylated by Cdk1/cyclin B. (A) Purified GST or GST-53BP1-C-term was phosphorylated in vitro by active Cdk1/cyclin B or p38a and phosphorylation was detected by autoradiography for 30 min or 5 h. (B) Various alanine mutants of GST-53BP1-C-term were phosphorylated in vitro by Cdk1/cyclin B.

Figure 5.

Phosphorylation of 53BP1 by Plk1 and Cdk1 inhibits its binding to ubiquitinated histones. (A) Purified GST, GST-53BP1-C-WT, GST-53BP1-C-S1618D or GST-53BP1-C-S1618D-S1609D were incubated with extract from U2OS-FLAG-ubiqiuitin cells treated with etoposide. Pull down was done by glutathione sepharose and bound proteins were analyzed by immunoblotting. (B) Purified GST-53BP1-C-WT was phosphorylated in vitro by Plk1 or Plk1 and Cdk1/cyclin B or mock phosphorylated and incubated with extract from U2OS-FLAG-ubiqiuitin cells treated with etoposide as in A. (C) U2OS cells transfected with EGFP-53BP1-WT, -S1618D or –S1609D-S1618D were pre-treated with BrdU, laser micro-irradiated and recruitment of EGFP-tagged proteins to irradiated area was assayed by life imaging. (D) Cells from (C) were fixed 3 h after exposure to ionizing radiation (3 Gy) and DNA damage foci were analyzed by automated high-content microscopy and spot detection module. Percentage of cells with more than 5 53BP1 foci is shown. (n=3, error bars indicate SD) (E) U2OS cells transfected with EGFP-53BP1-WT, -S1618D or -S1609D-S1618D were grown exponentially and analyzed 48 h after transfection by automated high-content microscopy. Average number of 53BP1 foci was quantified in G1 cells gated by the intensity of the DAPI. (n=3, error bars indicate SD) (F) Exponentially growing U2OS cells were fixed 3 h after irradiation with 3 Gy and probed with antibodies against 53BP1 and γ-tubulin. Note no difference in 53BP1 foci formation in late G2 cell with separated centrosomes.

Figure 6.

Inhibition of Plk1 increases DNA repair capacity in mitotic cells. (A) U2OS cells were synchronized in mitosis by NZ (16 h), incubated for additional 2 h with DMSO or BI2536 (50 nM) and treated with NCS (2 nM) for indicated times. DNA lesions were quantified by neutral comet assay. Plotted is average amount of DNA in tails, error bars indicate SD. Circles and triangles indicate individual cells. (B) Cells were treated as in (A), fixed at indicated times and γH2AX levels were measured by FACS (at least 10⁴ cells per condition, n=4, error bars indicate SD). (C) U2OS cells were transfected with siRNA targeting coding region or 3-UTR region of 53BP1 and knock down was evaluated by immunoblotting. (D) U2OS-TR cells stably transfected with EGFP-53BP1-WT, -S1618D or –S1609D-S1618D were transfected with siRNA targeting 3-UTR region of 53BP1. After 48h expression of EGFP-53BP1 was induced by tetracycline for 12h, cells were irradiated with 3 Gy and fixed 8h afterwards. BRCA1 foci were analyzed by automated high-content microscopy. Average number of 53BP1 foci was quantified in G1 cells gated by the intensity of the DAPI and negative Cyclin A signal (n=3, error bars indicate SD). (E) RPE cells stably expressing EGFP-53BP1-WT or –S1609D-S1618D were treated and analyzed as in (D). (n=4, error bars indicate SD). (F) RPE cells stably expressing EGFP-53BP1-WT or –S1609D-S1618D were treated as in (D) and γH2AX-positive foci were analyzed by automated high-content microscopy. Average number of γH2AX- foci was quantified in G1 cells gated by the intensity of the DAPI and negative Cyclin A signal (n=3, error bars indicate SD).

Figure 7.

Model of 53BP1 inhibition by Plk1 and Cdk1 phosphorylation in mitosis. Following exposure of interphase cells to genotoxic stress, activation of ATM eventually leads to monoubiquitination of H2A by RNF168. Together with constitutive H4K20-me2 modification this allows recruitment of 53BP1 to DNA damage foci and its function in NHEJ repair. In mitosis, Cdk1 phosphorylates 53BP1 in the N-terminal part to generate a docking site for Plk1. In turn, Plk1 phosphorylates 53BP1 at S1618 within the UDR domain and disables binding of 53BP1 to H2A-Ub. In addition, Cdk1 phosphorylates S1609 and S1678 further inhibiting the ability of 53BP1 to bind to H2A-Ub. Mitotic 53BP1 is not phosphorylated by ATM in mitosis and its role in NHEJ is blocked.