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

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.

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).

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.

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.

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.