Research ArticleCELL BIOLOGY

A methylation-phosphorylation switch determines Plk1 kinase activity and function in DNA damage repair

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Science Advances  06 Mar 2019:
Vol. 5, no. 3, eaau7566
DOI: 10.1126/sciadv.aau7566
  • Fig. 1 Plk1 can be monomethylated on Lys209 by G9a.

    (A) Flag-tagged Plk1 was immunoprecipitated (IP) from HeLa S3 cells, and the methyl status of Plk1 was examined by probing with α-pan mono and dimethyl antibody (Pan me1/me2). IgG, immunoglobulin G. (B) Mass spectrum of Plk1 peptide shows K209 monomethylation. m/z, mass/charge ratio. (C) Alignment of the consensus Plk1 sequences between different species near the K209 methylation site. (D) Characterization of α-Plk1 K209me1-specific antibody. Top: Western blot analysis. Bottom: Dot blot analysis. (E) In vitro methylation assays were performed to examine enzymatic activities of various methyltransferases toward the nonmethylated K209 peptide (me0 pep) of Plk1 using the α-K209me1 antibody. A peptide bearing K209 monomethylation (me1 pep) served as a control. (F) In vitro methylation assays were performed and analyzed by liquid scintillation counting to measure 3H incorporation using recombinant proteins. Three independent reactions with SDs are shown. CPM, count per minute. (G) In vitro methylation of the recombinant N-terminal fragment of Plk1 (GST–N-Plk1) by wild-type (WT) or SET domain–defective G9a (ΔSET). Top: Radioactive graph. Bottom: Coomassie brilliant blue staining (CBB). (H and I) Western blot analysis showing the effect of ectopically expressed mouse G9a (H) or knockout of G9a (I) on Plk1 K209me1. To increase K209me1 signal intensities, cells were synchronized at the G1/S boundary in (I). S.E., short exposure; L.E., long exposure; GAPDH, glyceraldehyde phosphate dehydrogenase.

  • Fig. 2 K209me1 of Plk1 antagonizes pT210.

    (A and B) Dot blot analysis (A) or in vitro methylation assay (B) showing the kinase activity of Aurora A on different K209me methyl status of peptides. (C to E) After preincubated N-terminal fragment of Plk1 (N-Plk1 and K82M) with the SET domain fragment of G9a (G9a-SET), Aurora A–dependent Plk1 phosphorylation was examined. The numbers represent individual reactions. (C) Diagram of the sequential biochemical assays. (D) Liquid scintillation counting showing the methylation status of N-Plk1 by G9a (top); kinase assays showing the phosphorylation status of N-Plk1by Aurora A (middle); the indicated proteins used in the sequential reactions were examined by Coomasie blue staining (bottom). The arrows represent the positions of purified recombinant proteins. (E) Western blot assay showing T210 phosphorylation (pT210) and K209 monomethylation (K209me1) status of Plk1 in the indicated reactions. (F to H) Western blot assay showing the effect of ectopically expressing G9a or G9a inhibitor BIX-01294 on Plk1 pT210 and K209me1 in HeLa S3 (F) or 293T (G and H) cells. The dose-dependent effects of BIX-01294 on the levels of K209me1 and pT210 of Plk1 are shown in (H). The H3K9me2 levels served as a control. The numbers below individual Western strips represent relative intensity of the corresponding band (pT210 or K209me1) normalized to the amount of immunoprecipitated Flag-Plk1 using ImageJ. DMSO, dimethyl sulfoxide.

  • Fig. 3 Aurora A–mediated T210 phosphorylation blocks K209me1 of Plk1.

    (A and B) Dot blot analysis (A) or liquid scintillation counting (B) showing that G9a was able to methylate K209me0 peptide but barely able to methylate the phosphorylated T210 (pT210) and K209me1 peptides. (C and D) Western blot assay showing the effects of knockdown of Aurora A or ectopically expressing Aurora A on the K209me1 and pT210 levels of Plk1 in 293T cells. The numbers represent the relative intensity of corresponding band normalized to the amount of immunoprecipitated Flag-Plk1 using ImageJ.

  • Fig. 4 Plk1 K209me1 peaks at S phase and the methylation attenuates its kinase activity.

    (A) Western blot analysis showing the antagonism of K209me1 and pT210 of Plk1 in synchronized G1/S or mitotic cells. The immunoprecipitated Plk1 proteins were adjusted to similar levels. The kinase activity of immunoprecipitated Plk1 from the indicated cells on casein protein was measured by 32P radioactive labeling assay. (B) Western blot assay showing the opposite changes of K209me1 and pT210 of stably expressed Flag-Plk1 in the double-thymidine (T/T) synchronized and released cells (left). The cell cycle profile was analyzed by FACS. (C) Western blot analysis showing the interactions of stably expressed Flag-Plk1 with G9a or Aurora A at different time points taken from the T/T-synchronized cells. Each number represents the relative binding intensities of G9a with Plk1 or the intensities of Aurora A with Plk1 (left). The cell cycle profile was analyzed by FACS (right). (D and E) The indicated cells taken from various time points after synchronized and released from T/T block were examined by FACS (D) or Western blot (E). (F) T/T-blocked cells were released into a different culture medium as indicated, and Western blot analyses were performed against the indicated antibodies. HU, hydroxyurea.

  • Fig. 5 Plk1 K209M mutant severely delayed metaphase-to-anaphase transition by preventing sister chromatid separation.

    (A and B) Mitotic cell populations in cells transfected with indicated Plk1 constructs were determined by FACS analysis staining with an α-MPM2 antibody, which specifically recognizes mitotic phosphoproteins (A), or using Western blot analysis (B). (C and D) Kinetics of mitotic progression in the indicated HeLa/RFP-H2B cells was analyzed by time-lapse microscopy. Cell images was captured every 5 min, and still frames from movies of representative cells are shown in (C). Scale bar, 10 μm. About 20 cell images from each cell line were captured, and the durations of mitotic progression were quantified in (D). (E to G) Arm cohesion of chromosomes in the wild-type or the K209M cells was analyzed by spreading and Giemsa staining. (E) Representative images from the indicated cells are shown, and insets show whether arm cohesion is absent (arm open) or present (arm closed). The ratio of open arms versus closed arms from more than 50 indicated cells each was quantified in (F). Scale bar, 100 μm. (G) The distance of sister chromatids at the arm ends from 50 indicated cells each was quantified. The error bars represent three independent experiments. (H) The mitotic cells stably expressing wild type, K209A, or K209M mutant of Flag-Plk1 were subjected to immunoprecipitation by α-Flag resins, the enriched Plk1 proteins were incubated with or without recombinant casein, and the Plk1 activities were measured by autoradiography (left). The mitotic cell cycle regulators were examined by immunoblotting (right). Data are presented as means ± SD in (A), (F), and (G). P values were determined by unpaired t test. ns, not significant.

  • Fig. 6 Plk1 K209me1 is required for DNA damage repair.

    (A) Asynchronous cells stably expressing Flag-Plk1 were treated with VP-16 (40 μM) or CPT (2 μM) for 1 hour. Plk1 were immunoprecipitated using an α-Flag antibody, and samples were probed with the indicated antibodies. (B) Inhibition of ATR pathway by using the ATR inhibitor VE-821 (10 μM) resulted in premature mitotic entry. Synchronized G2 cells were treated with VP-16 (40 μM) or CPT (2 μM) for 1 hour and an addition with or without VE-821 treatment for 2 hours. The changes of K209me1 and pT210 levels were examined by Western blot. (C, F, and H) The indicated cells were treated with VP-16 (40 μM for 1 hour) and allowed to release into normal medium for the indicated times and analyzed using immunofluorescent staining with an α-γH2A.X, α-RPA2, or α-RAD51 antibody, respectively. (D) Quantification of γH2A.X-positive cells in (C) using ImageJ. The data represent means ± SD (n > 100 each) from three independent experiments. (E) The indicated cells that were treated as described in (C) were analyzed using Western blotting. (G and I) Quantification of RPA2 or RAD51 foci numbers in individual cells described in (F) or (H) using ImageJ. The boxes designate cells with more than 10 foci, whose percentage is indicated above each box. ***P < 0.001. (J) The indicated cells were treated as described in (C), the chromatin fractions were collected, and chromatin-bound RPA2 and RAD51 levels were examined using Western blotting.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaau7566/DC1

    Fig. S1. Identification of the methyltransferase responsible for methylating Plk1.

    Fig. S2. Validation of K209me1 or pT210 antibody recognition of various peptides and mutant cells.

    Fig. S3. K209me1 of Plk1 antagonizes pT210.

    Fig. S4. Generation of K209A and K209M knock-in Plk1 mutant cell lines.

    Fig. S5. Plk1 K209M mutant slightly delays G2/M transition.

    Fig. S6. Plk1 K209me1 does not affect the kinetochore tension on the metaphase onset.

    Fig. S7. Plk1 K209me1 is required for DNA damage repair.

    Fig. S8. Plk1 K209me1 is not required for the recruitment of DNA damage factors to DNA damage sites.

    Movie S1. A representative HeLa/RFP-H2B cell bearing wild-type Plk1 was recorded from nuclear breakdown to cytokinesis by time-lapse microscopy.

    Movie S2. A representative HeLa/RFP-H2B cell bearing K209A mutant Plk1 was recorded from nuclear breakdown to cytokinesis by time-lapse microscopy.

    Movie S3. A representative HeLa/RFP-H2B cell bearing K209M mutant Plk1 was recorded from nuclear breakdown to cytokinesis by time-lapse microscopy.

    Movie S4. A population of HeLa/RFP-H2B cells bearing K209M mutant Plk1 was recorded from nuclear breakdown to cytokinesis by time-lapse microscopy.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Identification of the methyltransferase responsible for methylating Plk1.
    • Fig. S2. Validation of K209me1 or pT210 antibody recognition of various peptides and mutant cells.
    • Fig. S3. K209me1 of Plk1 antagonizes pT210.
    • Fig. S4. Generation of K209A and K209M knock-in Plk1 mutant cell lines.
    • Fig. S5. Plk1 K209M mutant slightly delays G2/M transition.
    • Fig. S6. Plk1 K209me1 does not affect the kinetochore tension on the metaphase onset.
    • Fig. S7. Plk1 K209me1 is required for DNA damage repair.
    • Fig. S8. Plk1 K209me1 is not required for the recruitment of DNA damage factors to DNA damage sites.
    • Legends for movies S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). A representative HeLa/RFP-H2B cell bearing wild-type Plk1 was recorded from nuclear breakdown to cytokinesis by time-lapse microscopy.
    • Movie S2 (.avi format). A representative HeLa/RFP-H2B cell bearing K209A mutant Plk1 was recorded from nuclear breakdown to cytokinesis by time-lapse microscopy.
    • Movie S3 (.avi format). A representative HeLa/RFP-H2B cell bearing K209M mutant Plk1 was recorded from nuclear breakdown to cytokinesis by time-lapse microscopy.
    • Movie S4 (.avi format). A population of HeLa/RFP-H2B cells bearing K209M mutant Plk1 was recorded from nuclear breakdown to cytokinesis by time-lapse microscopy.

    Files in this Data Supplement:

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