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Articles

The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

Takahiro Negishi, Jiri Veis, David Hollenstein, Mizuho Sekiya, Gustav Ammerer, Yoshikazu Ohya
Takahiro Negishi
aDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan
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Jiri Veis
bDepartment of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
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David Hollenstein
bDepartment of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
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Mizuho Sekiya
aDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan
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Gustav Ammerer
bDepartment of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
cChristian Doppler Laboratory for Proteome Analysis, University of Vienna, Vienna, Austria
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Yoshikazu Ohya
aDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan
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DOI: 10.1128/MCB.00952-15
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  • FIG 1
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    FIG 1

    Overexpression of Hcm1 induces a defective cell wall integrity checkpoint. (A) Hcm1 overexpression induces a defective cell wall integrity checkpoint. Cells of the indicated strains (YOC1087 and YOC4462) were synchronized at G1 phase by elutriation and released in YPD or YPGS medium at 37°C. The inhibition of bud growth (left) and bipolar spindle formation (right) was quantified at the indicated time points (n > 200 for each time point). (B) Representative images of data from panel A 240 min after release. Arrows indicate the bipolar spindle.

  • FIG 2
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    FIG 2

    Late-S-phase transcription factor Hcm1 is downregulated by the cell wall integrity checkpoint. (A) Hcm1 protein level was examined during the activation of the cell wall integrity checkpoint. HA-tagged Hcm1 from FKS1 (YOC4878) and fks1-1154 (YOC4879) cells were synchronized to G1 phase by elutriation, grown at 37°C, and subjected to Western blotting in a time course manner together with the asynchronized untagged controls (YOC1001 and YOC1087). (B) Quantification of Hcm1 level normalized to that of the Cdc28 loading control from panel A. AU, arbitrary units. (C) Hcm1 protein level was regulated similarly in FKS1 (YOC4878) and fks1-1154 (YOC4879) strains at the permissive temperature of 25°C. HA-tagged Hcm1 from FKS1 (YOC4878) and fks1-1154 (YOC4879) cells were prepared as described for panel A and were cultured at 25°C, followed by Western blotting in a time course manner together with untagged controls (YOC1001 and YOC1087). (D) Quantification of Hcm1 level normalized to that of the Cdc28 loading control from panel C. (E) HCM1 transcription was examined during functions of the active cell wall integrity checkpoint. Cells of the indicated strains (YOC1001 and YOC1087) were synchronized to G1 phase by elutriation and grown at 37°C, followed by Northern blotting in a time course manner. The ACT1 transcript level is shown as the loading control. (F) Hcm1-regulated gene expression, including the key transcription factors NDD1 and FKH2, was delayed or downregulated by activation of the cell wall integrity checkpoint. Cells were prepared as described for panel E. The expression of genes reported to be regulated by Hcm1 in the genome-wide transcription study of Pramila et al. (14) was examined while the cell wall integrity checkpoint was active.

  • FIG 3
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    FIG 3

    Hcm1 is a phosphoprotein. (A) Cells harboring HA-tagged (YOC4878) or untagged Hcm1 (YOC1001) were subjected to protein extraction following phosphatase treatment with or without inhibitors. The protein extracts were subjected to SDS-PAGE supplemented with the Phos-tag or without supplement. Western blotting was performed using anti-HA (11MO) antibody for Hcm1-HA and anti-Cdc2/p32 (PSTAIRE) antibody for Cdc28/Pho85 as loading controls. (B) The 39 in vivo serine, threonine, and tyrosine phosphorylation sites (in red) identified in this study in reference to the Hcm1 primary amino acid sequence (see Table S2 in the supplemental material). Red boldface S and T (serine and threonine) show proline-directed amino acid residues. Underlining shows the DNA-binding domain (from SGD or Superfamily 1.73, HMM server). (C) The phosphorylation sites identified in this study were compared to those reported previously from systematic studies and the ISPB PhosphoPep database (see Table S2). Numbers in parentheses indicate the number of sites identified in each study. (D) A schematic model is shown for Hcm1 with the DNA-binding domain and the phosphorylation sites investigated in this study. In addition to the sites identified in panel B, the in vivo phosphorylation sites identified in the ISPB phosphopep database and in Soufi et al. are listed, and the color keys indicate the characteristics of each site (41). Some of the phosphorylation sites are preferred by CDK and MAPK (44).

  • FIG 4
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    FIG 4

    Hcm1 S61, S65, and S66 are required for the cell wall integrity checkpoint. (A) Hcm1 phosphorylation site amino acid replacement mutants were constructed to produce nonphosphorylatable alleles. (B) Cells of the indicated strains (YOC4879, YOC4839, YOC5084, YOC4896, YOC5085, and YOC4841) harboring alleles depicted in panel A were tested for their function in the cell wall integrity checkpoint. Cells were synchronized to the G1 phase by centrifugal elutriation and budding morphology (top; med-large bud indicates a bud that is approximately one-third larger than the mother) was investigated at each time point after release of the cells at the permissive growth temperature of 25°C, and bipolar spindle morphology (bottom) was investigated by visualizing the spindle using indirect immunofluorescence microscopy (n > 200 for each time point). The percentage of med-large budded cells or cells with a bipolar spindle in the population after release is shown for each time point. Strains are as described in the graph. (C) Cells depicted in panel B also were investigated at the restrictive temperature of 37°C in terms of budding (top) and bipolar spindle morphology (bottom). (D) Representative images of the cells described in panel C at the indicated time points are shown. Arrows indicate bipolar spindles.

  • FIG 5
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    FIG 5

    Downregulation of Hcm1 during functions of the cell wall integrity checkpoint is lost in cells harboring Hcm1 S61A, S65A, or S66A. (A) Cells of Hcm1 phosphorylation site amino acid replacement mutants of the indicated strains (YOC4879, YOC4896, YOC5085, and YOC4841) were examined for their protein levels during the functioning of the cell wall integrity checkpoint. Cells were synchronized to G1 phase by elutriation, cultured at 37°C, and subjected to Western blot analysis in a time course manner along with the asynchronized untagged control (YOC1087, fks1-1154 Asyn.). Protein extracts were subjected to SDS-PAGE, followed by anti-HA Western blotting along with the loading controls (PSTAIRE). (B) Quantification of Hcm1 level to that of the Cdc28 loading control from panel A.

  • FIG 6
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    FIG 6

    S61, S65, and S66 Hcm1 phosphorylation sites are required to regulate the protein level of the cell wall integrity checkpoint. (A) The loss of Hcm1 phosphorylation sites inhibits Hcm1 protein degradation. Cells of the indicated strains (YOC4878 and YOC5057) were subjected to cycloheximide chase assay, and protein extracts were collected at different time points, followed by Western blotting together with the untagged control (YOC1001). (B) Quantification of images shown in panel A. (C) Cells harboring hcm1 S61A_S65A_S66A have a defective cell wall integrity checkpoint. Cells of the indicated strains (YOC4879 and YOC5058) were synchronized at the G1 phase by elutriation and released into YPD media at 37°C. The inhibition of bud growth (left) and bipolar spindle formation (right) were quantified at the indicated time points (n > 200 for each time point). (D) Representative images of data shown in panel C 0 and 180 min after release. Arrows indicate bipolar spindles. (E) Downregulation of Hcm1 during the functioning of the cell wall integrity checkpoint is lost in cells harboring hcm1 S61A_S65A_S66A. Cells were prepared as described for panel C and subjected to SDS-PAGE, followed by anti-HA Western blotting after Phos-tag gel electrophoresis, along with control electrophoresis and loading controls (PSTAIRE). (F) Quantification of Hcm1 level to that of the Cdc28 loading control from panel E. Proportions of phospho- and dephospho-Hcm1 are indicated by gray and black bars, respectively, at each level of Hcm1 detected. (G) Cells prepared in a manner similar to that for panel C were examined for HCM1, FKH2, and ACT1 transcription during functions of the active cell wall integrity checkpoint by Northern blotting in a time course manner. (H) Inhibition of Clb2 expression by the cell wall integrity checkpoint is partially suppressed by the loss of Hcm1 phosphorylation sites. Cells of the indicated strains (YOC5096 and YOC5097) were prepared as described for panel E, and their Myc-tagged Clb2 protein levels were determined by Western blotting. (I) Quantification of Clb2 level normalized to that of the Cdc28 loading control from panel F. (J) Cells of the indicated strains (YOC5096 and YOC5097) were prepared as described for panel H, except they were cultured at the permissive temperature of 25°C. (K) Quantification of Clb2 level normalized to that of the Cdc28 loading control from panel H.

  • FIG 7
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    FIG 7

    CWI MAPK signaling pathway regulates Hcm1 protein level. (A) The loss of Hcm1 phosphorylation sites also inhibits Hcm1 degradation under 37°C heat stress. Cells of the indicated strains (YOC4879 and YOC5058) were shifted to 37°C, and protein extracts were collected in a time course manner followed by Western blotting together with the untagged control (YOC1087). (B) Quantification of Hcm1 level normalized to that of the Cdc28 loading control from panel A. (C) The CWI MAPK pathway was inactivated by deleting SLT2 MAPK (indicated strain, YOC5090; YOC4878 was used as the control), and the Hcm1 protein level during 37°C heat stress was determined in a time course manner by Western blotting. (D) Quantification of images shown in panel C. (E) Cells after heat stress prepared in a manner similar to that for panel C were examined for HCM1 and ACT1 transcription by Northern blotting in a time course manner. (F) The loss of Slt2 inhibits Hcm1 protein degradation at 37°C. Cells of the indicated strains (YOC4878 and YOC5090) were subjected to cycloheximide chase assay at 37°C, and protein extracts were collected at different time points, followed by Western blotting together with the untagged control. (G) Quantification of images shown in panel F. (H) Activation of the CWI MAPK pathway using hyperactive Bck1 (Bck1-20; MAPKKK of the CWI MAPK pathway) was introduced to the indicated strains (YOC5086, YOC5087, YOC5088, and YOC5089) via a high-copy-number plasmid, and the protein extract was subjected to Western blotting. (I) Quantification of the Hcm1 level for images shown in panel H.

Additional Files

  • Figures
  • Supplemental material

    • Supplemental file 1 -

      Table S1 (Mass spectrometry data)

      XLSX, 107K

    • Supplemental file 2 -

      Tables S2 (Phosphorylation sites of Hcm1), S3 (Strains), S4 (Plasmids), and S5 (Oligonucleotides)

      PDF, 1.6M

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The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint
Takahiro Negishi, Jiri Veis, David Hollenstein, Mizuho Sekiya, Gustav Ammerer, Yoshikazu Ohya
Molecular and Cellular Biology Mar 2016, 36 (6) 941-953; DOI: 10.1128/MCB.00952-15

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The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint
Takahiro Negishi, Jiri Veis, David Hollenstein, Mizuho Sekiya, Gustav Ammerer, Yoshikazu Ohya
Molecular and Cellular Biology Mar 2016, 36 (6) 941-953; DOI: 10.1128/MCB.00952-15
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