Cdc2p controls the forkhead transcription factor Fkh2p by phosphorylation during sexual differentiation in fission yeast
2007; Springer Nature; Volume: 27; Issue: 1 Linguagem: Inglês
10.1038/sj.emboj.7601949
ISSN1460-2075
AutoresMidori Shimada, Chisato Yamada‐Namikawa, Yuko Murakami‐Tonami, Takashi Yoshida, Makoto Nakanishi, Takeshi Urano, Hiroshi Murakami,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle6 December 2007free access Cdc2p controls the forkhead transcription factor Fkh2p by phosphorylation during sexual differentiation in fission yeast Midori Shimada Midori Shimada Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Chisato Yamada-Namikawa Chisato Yamada-Namikawa Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Yuko Murakami-Tonami Yuko Murakami-Tonami Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Takashi Yoshida Takashi Yoshida Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Makoto Nakanishi Makoto Nakanishi Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Takeshi Urano Takeshi Urano Department of Biochemistry II, Graduate School of Medicine, Nagoya University, Showa-ku, Nagoya, Japan Search for more papers by this author Hiroshi Murakami Corresponding Author Hiroshi Murakami Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Midori Shimada Midori Shimada Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Chisato Yamada-Namikawa Chisato Yamada-Namikawa Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Yuko Murakami-Tonami Yuko Murakami-Tonami Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Takashi Yoshida Takashi Yoshida Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Makoto Nakanishi Makoto Nakanishi Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Takeshi Urano Takeshi Urano Department of Biochemistry II, Graduate School of Medicine, Nagoya University, Showa-ku, Nagoya, Japan Search for more papers by this author Hiroshi Murakami Corresponding Author Hiroshi Murakami Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan Search for more papers by this author Author Information Midori Shimada1, Chisato Yamada-Namikawa1, Yuko Murakami-Tonami1, Takashi Yoshida1, Makoto Nakanishi1, Takeshi Urano2 and Hiroshi Murakami 1 1Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, Mizuho-ku, Nagoya, Japan 2Department of Biochemistry II, Graduate School of Medicine, Nagoya University, Showa-ku, Nagoya, Japan *Corresponding author. Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, 1 Kawasumi, Mizuho-cho, Nagoya, Aichi 467-0001, Japan. Tel.: +81 52 853 8145; Fax: +81 52 842 3955; E-mail: [email protected] The EMBO Journal (2008)27:132-142https://doi.org/10.1038/sj.emboj.7601949 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In most eukaryotes, cyclin-dependent kinases (Cdks) play a central role in control of cell-cycle progression. Cdks are inactivated from the end of mitosis to the start of the next cell cycle as well as during sexual differentiation. The forkhead-type transcription factor Fkh2p is required for the periodic expression of many genes and for efficient mating in the fission yeast Schizosaccharomyces pombe. However, the mechanism responsible for coordination of cell-cycle progression with sexual differentiation is still unknown. We now show that Fkh2p is phosphorylated by Cdc2p (Cdk1) and that phosphorylation of Fkh2p on T314 or S462 by this Cdk blocks mating in S. pombe by preventing the induction of ste11+ transcription, which is required for the onset of sexual development. We propose that functional interaction between Cdks and forkhead transcription factors may link the mitotic cell cycle and sexual differentiation. Introduction The mechanism responsible for the switch from growth to sexual development has been studied in many organisms including the fission yeast Schizosaccharomyces pombe. In fission yeast, the onset of sexual development requires both a pheromone signal and the depletion of nutrients, especially that of nitrogen (Yamamoto, 1996; Yamamoto et al, 1997). If cells of the opposite mating type are available, those that have committed to sexual development conjugate to form diploids. These diploid cells then undergo meiosis and complete sexual development. The transcription factor Ste11p plays a central role in commitment to sexual development in fission yeast (Sugimoto et al, 1991; Yamamoto, 1996; Yamamoto et al, 1997). It regulates the transcription of many genes required for the initiation and progression of conjugation and meiosis (Mata and Bahler, 2006; Xue-Franzen et al, 2006). Expression of ste11+ itself is regulated by several pathways (Yamamoto, 1996; Yamamoto et al, 1997), including the cyclic AMP (cAMP) pathway. Nutrient exhaustion results in a decrease in the intracellular concentration of cAMP and a consequent inactivation of cAMP-dependent protein kinase (PKA). The transcriptional activator Rst2p, which is negatively regulated by PKA, then binds to the upstream region of ste11+ and induces the production of ste11+ mRNA (Kunitomo et al, 2000; Higuchi et al, 2002). In addition, a stress signal mediated by the mitogen-activated protein kinase pathway is required for the induction of ste11+ mRNA in response to nutrient deprivation (Takeda et al, 1995; Kato et al, 1996; Shiozaki and Russell, 1996). In addition to the regulation of ste11+ transcription, the activity of the encoded protein (Ste11p) is regulated at a post-translational level (Li and McLeod, 1996; Kitamura et al, 2001; Qin et al, 2003; Kjaerulff et al, 2005). In fission yeast, the single cyclin-dependent kinase (Cdk) Cdc2p controls cell-cycle progression in a manner dependent on various internal and external conditions including nutrient availability (MacNeill and Nurse, 1997). Both nitrogen starvation and pheromone induce arrest in G1 phase of the cell cycle by inhibiting the activity of Cdc2p, an effect in turn mediated by cyclin degradation and upregulation of Cdk inhibitors. Pheromone induces degradation of the B-type cyclins Cig2p and Cdc13p as well as upregulation of the Cdk inhibitor Rum1p (Stern and Nurse, 1997, 1998). Nitrogen exhaustion also promotes the degradation of both Cig2p and Cdc13p by activating the anaphase-promoting complex (Yamaguchi et al, 1997; Kitamura et al, 1998; Yamano et al, 2004). Linkage between cell-cycle control and sexual development is likely provided by Cig2p. Loss of Cig2p function promotes mating, whereas overproduction of this cyclin negatively regulates sexual differentiation (Obara-Ishihara and Okayama, 1994). Members of the forkhead-box family of transcription factors are present in almost all eukaryotes (Costa, 2005; Costa et al, 2005; Wang et al, 2005). More than 50 such proteins that share homology in the winged-helix DNA-binding domain have been identified in higher eukaryotes. This family of transcription factors is implicated in the regulation of a variety of cellular processes, including the cell cycle, apoptosis, DNA repair, stress resistance, and metabolism. The Sanger Center database (http://www.sanger.ac.uk/) indicates the existence of four forkhead proteins in fission yeast: Fkh2p, Fhl1p, Sep1p, and Mei4p (Bahler, 2005). Fkh2p is required for efficient G2–M transition, normal septation, and periodic gene expression (Buck et al, 2004; Bulmer et al, 2004; Rustici et al, 2004; Szilagyi et al, 2005). It is also required for efficient mating (Szilagyi et al, 2005). However, the mechanism responsible for the partial sterile phenotype of fkh2 mutant cells has remained unknown. We have now investigated the role of Cdc2p in coordination of cell-cycle control and sexual differentiation in fission yeast. We show here that the forkhead transcription factors, including Fkh2p, are responsible for mediating a signal from the kinase Cdc2p to the transcription factor Ste11p. Results Forkhead transcription factors are required for mating Fkh2p, Fhl1p, Mei4p, and Sep1p are the forkhead transcription factors in fission yeast. Given that fkh2-deleted cells show a partial sterile phenotype (Szilagyi et al, 2005), we investigated the role of forkhead transcription factors in mating in fission yeast. We constructed homothallic strains in which each gene for the forkhead transcription factors was individually deleted. Consistent with previous observations (Ribar et al, 1999), sep1-deleted cells manifested a pronounced septation defect and slow growth. We, therefore, did not further characterize the role of sep1+ in mating. In addition to fkh2-deleted cells, we found that fhl1-deleted and unexpectedly mei4-deleted cells exhibited a partial sterile phenotype, whereas fkh2 fhl1, fkh2 mei4 and fhk2 fhl1 mei4 mutant cells showed a more pronounced sterile phenotype than did cells lacking either gene alone (Figure 1A). These results suggested that, among the forkhead transcription factors, Fkh2p plays the predominant role in mating, with Fhl1p and Mei4p having minor roles that partially overlap with that of Fkh2p. Figure 1.Forkhead transcription factors are required for the induction of ste11+ mRNA and efficient mating. (A) wt (HM6), fkh2 (HM5657), fhl1 (HM4837), mei4 (HM50), fkh2 fhl1 (HM4887), fkh2 mei4 (HM5515), or fkh2 fhl1 mei4 (HM5544) cells were grown in EMM2 medium to a density of 1 × 107 cells/ml, washed, and resuspended at a density of 2 × 107 cells/ml in EMM2 medium lacking nitrogen. They were then cultured at 30°C and samples were collected at the indicated times for determination of mating frequency. Data are from representative experiments. (B) Total RNA was extracted from cells treated as in (A), and the abundance of ste11+ mRNA was examined by northern blot analysis. Ethidium bromide staining of rRNA is shown as a loading control. The ratios of intensities of ste11+ to rRNA signals were used to calculate the relative fold enrichment, shown below the rRNA. The samples from wt to fkh2, from fhl1 to mei4, from fkh2 mei4 to fkh2 fhl1 mei4 were from the same gel. All the samples were treated equally and the exposure time was the same. (C) Cells were transformed with pcL-ste11+ (ste11+), pAL-fkh2+ (fkh2+), or the empty vector pcL-X (Vec) and were cultured as in (A) for the determination of mating efficiency at 24 h after transfer to EMM2 medium without nitrogen. Data are from representative experiments. Download figure Download PowerPoint Given that the induction of ste11+ mRNA plays a central role in mating, we monitored the abundance of this mRNA in the various mutant strains (Figure 1B). In contrast to wt cells, the induction of ste11+ mRNA was greatly delayed or virtually abolished in fkh2, fkh2 fhl1, fkh2 mei4, or fkh2 fhl1 mei4 mutant cells. In fhl1-deleted cells, the increase in ste11+ mRNA was apparent, but slightly reduced. In mei4-deleted cells, the increase in ste11+ mRNA was apparent but delayed. Ectopic expression of ste11+ indeed restored fertility not only to the fkh2-deleted cells but also to fkh2 fhl1, fkh2 mei4, and fkh2 fhl1 mei4 mutant cells to an extent similar to that observed with ectopic expression of fkh2+ (Figure 1C). We thus concluded that the sterility of the forkhead mutant cells was caused largely by poor induction of ste11+, not by slow growth. In addition, the sterility was not attributable to a defect in the induction of G1 arrest by nitrogen starvation, given that the forkhead mutant cells arrested in G1 phase in a manner similar to that of wt cells (Supplementary Figure 1). Fkh2p binds to a FLEX element upstream of ste11+ both in vivo and in vitro Given that the forkhead family of transcription factors recognizes the core sequence GTAAAYA (Pierrou et al, 1994), we searched for this sequence in the vicinity of the genomic locus of ste11+. One such sequence, designated FLEX1, was detected in the putative 5′ regulatory region of ste11+ (Figure 2A). If one mismatched base is allowed, three FLEX-like sequences—designated FLEXL1, FLEXL2, and FLEXL3—were also apparent in this region. Figure 2.Fkh2 binds to FLEX and FLEXL sequences in the putative promoter region of ste11+ and thereby induces ste11+ mRNA. (A) Schematic representation of the region upstream of the open reading frame (ORF) of ste11+ showing FLEX and FLEXL sequences. The major transcription initiation site of ste11+ is indicated by the arrow, and the regions targeted by primer sets in ChIP analysis are also shown. (B) An EMSA was performed with recombinant GST–Fkh2p(216–330) (or GST alone) and with FLEX1, FLEXL1, FLEXL2, FLEXL3, or TR (negative control) probes labeled with 32P. Competition was evaluated with excess amounts of unlabeled FLEX1, TR, or FLEXL1 oligonucleotides, and supershift analysis was performed with antibodies to GST, as indicated. The positions of shifted and supershifted bands are shown. (C) No tagged cells (no tag, HM6) and cells expressing GFP-tagged Fkh2p (Tag, HM5719) were grown to late log-phase, washed, and resuspended in medium without nitrogen. After incubation for 2 h at 30°C, cells were collected and analyzed by ChIP with antibodies to GFP and with the primer sets indicated in (A). Data are means±s.e. *P<0.006 (Student's t-test). (D) No tagged cells (no tag, HM6) and cells expressing GFP-tagged Fkh2p (wt; HM5719 and ste11-dFLEX1: HM6124) were treated and analyzed as in (C) with primer set A. Samples were collected at 0 and 2 h after nitrogen withdrawal. Data are means±s.e. of values from three separate experiments. *P<0.007 (Student's t-test). (E) wt (HM6) or ste11-dFLEX1 (HM5832) cells were treated and analyzed for mating efficiency as in Figure 1A. (F) Total RNA was extracted from cells treated as in (E) and was subjected to northern blot analysis of ste11+ mRNA. Download figure Download PowerPoint To examine whether Fkh2p binds to these FLEX or FLEX-like elements, we prepared a fusion protein consisting of glutathione-S-transferase (GST) and the forkhead DNA-binding domain of Fkh2p (amino acids 216–330) and performed an electrophoretic mobility-shift assay (EMSA) with this protein and radioactive oligonucleotides containing the FLEX1 or FLEXL sequences as probes (Figure 2B). Shifted bands were observed with FLEX1 and, to a lesser extent, with FLEXL1, but they were not detected with FLEXL2, FLEXL3, or an unrelated (TR) probe. The shifted bands were specific for Fkh2p and for FLEX1 or FLEXL1, given that they were not observed with GST in place of the fusion protein and that the corresponding unlabeled oligonucleotides, but not an unrelated oligonucleotide (TR), inhibited the binding of the GST–Fkh2 fusion protein to the labeled probes. The shifted band observed with the FLEX1 probe was also supershifted in the presence of antibodies to GST. These results thus indicated that the GST–Fkh2 fusion protein directly binds to FLEX1 and, to a lesser extent, to FLEXL1 in vitro. To examine whether Fkh2p binds to the FLEX or FLEX-like sequences upstream of ste11+ in vivo, we performed a chromatin immunoprecipitation (ChIP) assay with cells expressing green fluorescent protein (GFP)-tagged Fkh2p by nmt41 promoter (Figure 2C). In cells expressing GFP-tagged Fkh2p, the mating efficiency and the induction of ste11+ mRNA were comparable to those in wt cells, suggesting that GFP-tagged Fkh2p functions like wt protein (Supplementary Figure 2). Immunoprecipitation with antibodies to GFP revealed that GFP-Fkh2p associates with genomic DNA containing both FLEX1 and FLEXL1 (primer set A), whereas association with genomic DNA containing both FLEXL2 and FLEXL3 (primer set B) is little. The amount of either region of genomic DNA immunoprecipitated with the antibodies to GFP was greatly reduced for cells not expressing GFP-Fkh2p. These results showed that Fkh2p binds in vivo to the genomic locus containing the FLEX1 and FLEXL1 elements upstream of ste11+. To test whether such binding depends on nutrient conditions, we measured the binding activity of Fkh2p in cells subjected to nitrogen deprivation. The ChIP assay revealed that nitrogen withdrawal resulted in an increase in the binding of GFP-Fkh2p to genomic DNA containing FLEX1 and FLEXL1 (Figure 2D), but not to the region upstream of cdc15+(Supplementary Figure 3). The amount of genomic DNA containing FLEX1 and FLEXL1 immunoprecipitated with the antibodies to GFP was low for cells not expressing GFP-Fkh2p upon nitrogen starvation. These results suggest that Fkh2p associates with the upstream region of ste11+ in vivo when cells are able to mate. To examine the role of FLEX1 in mating, we deleted the 7-bp core sequence of this site from its chromosome locus. The mating efficiency of the resulting mutant strain (ste11-dFLEX1) was greatly reduced compared with that of wt cells (Figure 2E; Supplementary Figure 2). This sterility was not attributable to a defect in induction of G1 arrest (Supplementary Figure 4). These observations suggested that the core sequence of FLEX1 is required for efficient mating. In addition, induction of ste11+ mRNA by nitrogen withdrawal was largely abolished in ste11-dFLEX1 cells (Figure 2F; Supplementary Figure 2), suggesting that the core sequence of FLEX1 is also required for activation of ste11+ expression. The ChIP assay revealed that the core FLEX deletion resulted in a decrease in the binding of GFP-Fkh2p to genomic DNA around FLEX1 (Figure 2D), suggesting that the core FLEX1 is required for Fkh2p to associate with the upstream region of ste11+ in vivo. Effects of phosphorylation of Fkh2p by Cdc2p Cdks regulate forkhead transcription factors in various organisms, and Fkh2p has been shown to be a phosphoprotein in fission yeast (Buck et al, 2004; Bulmer et al, 2004). A search of the Fkh2p sequence for consensus phosphorylation sites for Cdc2p, (pS/pT)-P-X-(R/K) (Nigg, 1993), revealed three such sites at residues T314, S462, and S481 (Figure 3A). The sequence surrounding T314 in the DNA-binding domain of Fkh2p is conserved among members of the forkhead-box family of other species, especially those of the FoxN subfamily (Mazet et al, 2003), although the consensus phosphorylation site sequence is not fully conserved. S462 is conserved, but not as a Cdc2p phosphorylation site, among members of the FoxJ subfamily of transcription factors (Mazet et al, 2003). To assess the potential function of these putative Cdc2p phosphorylation sites in Fkh2p, we changed the serine or threonine residues to glutamic acid by site-directed mutagenesis of the chromosome to mimic the effect of Cdc2p phosphorylation in vivo. Cells in which T314 (fkh2-T314E) or S462 (fkh2-S462E) of Fkh2p was replaced with glutamic acid exhibited a reduced mating efficiency (Figure 3B). In contrast, similar mutation of S481 of Fkh2p (fkh2-S481E) did not substantially affect mating efficiency (data not shown). In addition, the mating efficiency of fkh2-T314E S462E S481E mutant cells was similar to that of fkh2-T314E or fkh2-S462E cells (Figure 3B). These results suggested that dephosphorylation of Fkh2p at T314 and S462 is required for efficient mating. However, unphosphorylated form of Fkh2p (fkh2-T314A S462A S481A) failed to enhance mating efficiency, suggesting that an additional mechanism is required for ectopic mating (Figure 3B). The induction of ste11+ mRNA in response to nitrogen deprivation was greatly reduced in fkh2-T314E or fkh2-S462E cells compared with that apparent in wt cells (Figure 3C). These observations thus suggested that dephosphorylation of Fkh2p on T314 and S462 is required for efficient induction of ste11+ mRNA. Figure 3.Phosphorylation of Fkh2p by Cdc2p negatively regulates mating. (A) A schematic representation of Fkh2p indicating consensus phosphorylation sites (T314, S462, S481) for Cdc2p as well as the FHA and FKH domains is shown in the upper panel. Multiple alignment of Fkh2, FoxN3, and FoxJ2 proteins of S. pombe (Sp), Xenopus laevis (Xl), Mus musculus (Mm), Rattus norvegicus (Rn), Homo sapiens (Hs), and Ciona intestinalis (Ci) is shown in the lower panels. Identical (shaded black) and similar (shaded gray) amino acids as well as two Cdc2p consensus phosphorylation sites (T314 and S462) of Fkh2p are indicated. Dashes represent gaps introduced to optimize alignment. (B) Cells expressing wild type (wt, HM5145) or T314E (fkh2-T314E, HM5910), S462E (fkh2-S462E, HM5911), T314E S462E S481E (fkh2-T314E S462E S481E, HM5827) or T314A S462A S481A (fkh2-T314A S462A S481A, HM5722) mutant forms of Fkh2p were treated and analyzed for mating efficiency as in Figure 1A. (C) Total RNA was extracted from cells treated as in (B) and was subjected to northern blot analysis of ste11+ mRNA. Download figure Download PowerPoint Other defects of fkh2-deleted cells, such as abnormal morphology or septation defect, were less than 1% in wt, fkh2-T314E, or fkh2-S462E cells. In addition, cell length is similar in wt, fkh2-T314E, or fkh2-S462E cells (Supplementary Figure 5). These facts suggest that these point mutations specifically affect mating. To confirm that these point mutations do not affect cell-cycle progression and transcriptional activity during the normal mitotic cell cycle, we measured the timing of mitotic entry and the mRNA levels of cdc15+, spo12+, and slp1+, as Fkh2p is required for periodic expression of these mRNAs (Buck et al, 2004; Bulmer et al, 2004). Cells were transiently arrested in late G2 by the inactivation of cdc25+ and released to the permissive temperature to enter a synchronous cell cycle. In contrast to fkh2-deleted cells that showed the severe delay in entry into mitosis (Buck et al, 2004), wt, fkh2-T314E, or fkh2-S462E cells entered mitosis almost with the same timing as indicated by the coincidence of the peak of septa (Supplementary Figure 6). Additionally, the periodic expressions of cdc15+, spo12+, and slp1+ mRNAs were observed in wt, fkh2-T314E, or fkh2-S462E cells (Supplementary Figure 6). These results suggest that these point mutations specifically affect ste11+ mRNA expression but not other mitotic genes expression. The poor mating efficiency of the fkh2-T314E or fkh2-S462E mutants was not due to a defect in induction of cell-cycle arrest in G1 phase (Supplementary Figure 7). In addition, the abundance of the mutant proteins was similar to that of the wild-type protein (Supplementary Figure 8), suggesting that the poor mating efficiency of the mutant cells was not attributable to a reduced protein level. Phosphorylation of Fkh2p by Cdc2p in vitro and in vivo We next tested whether Cdc2p directly phosphorylates GST fusion proteins containing various fragments of Fkh2p (residues 305–492, 216–330, or 317–479) in vitro (Figure 4A; Supplementary Figure 9). Cdc2p precipitated from cell extracts with anti-hemagglutinin epitope (HA) antibody or Suc1p-coated beads phosphorylated each of the GST–Fkh2p fusion proteins but not GST alone. We found that mutation to alanine of the consensus phosphorylation sites for Cdc2p in each of the Fkh2p fragments (T314 in Fkh2p (216–330), S462 in Fkh2p (317–479), or T314, S462, and S481 in Fkh2p (305–492)) reduced the extent of phosphorylation by Cdc2p. These results thus suggested that Cdc2p phosphorylates at least T314 and S462 residues of Fkh2p in vitro. In addition, recombinant human Cdc2p complex, but not the kinase inactive complex, phosphorylated Fkh2p, suggesting that Cdc2p directly phosphorylates Fkh2p (Supplementary Figure 9). Figure 4.Phosphorylation of Fkh2p on T314 and S462 by Cdc2p in vitro and in vivo. (A) Kinase assays were performed with Cdc2p precipitates prepared from protein extracts of exponentially growing cells expressing hemagglutinin epitope (HA)-tagged forms of Cdc2p (HM6118; lanes 1–3) or not expressing HA (HM6; lanes 4–6) with anti-HA antibody. Substrates (lanes 1–3, respectively) included GST–Fkh2p(305–492), GST–Fkh2p(305–492) containing T314A, S462A, and S481A mutations, or GST alone. Reaction mixtures were separated by SDS–polyacrylamide gel electrophoresis, and proteins were detected by staining with Coomassie brilliant blue (CBB) and autoradiography (32P). Arrows indicate GST and the GST–Fkh2p fusion proteins. The Cdc2p input into each reaction mixture was also examined separately by Western blotting. (B) Various amounts (10, 2, or 0.4 ng) of Fkh2p peptides containing phosphorylated or nonphosphorylated T314 or S462 were spotted onto a nitrocellulose membrane and subjected to immunodetection with affinity-purified antibodies (anti-pT314 and anti-pS462) generated in response to the corresponding phosphorylated peptides. (C) Cells expressing HA-tagged forms of wild-type Fkh2p (HM5145) or the Fkh2p(T314A,S462A) mutant (HM5722) were grown to mid-log phase at 30°C. Cell lysates were then subjected to immunoprecipitation with antibodies to HA, and the resulting precipitates were subjected to immunoblot analysis with anti-pT314, anti-pS462, and anti-HA, as indicated. (D) Cells expressing HA-tagged Fkh2p were either grown to mid-log phase in EMM2 at 24°C and then incubated at 36.5°C for 7 h (wt, HM5145; cdc2-ts, HM5444) or grown as in (C) (cig2, HM5530). Cell lysates were subjected to immunoprecipitation and immunoblot analysis as in (C). (E) Cells expressing HA-tagged Fkh2p (wt, HM5146; cdc13 off, HM5554) were grown to mid-log phase in EMM2 at 30°C, after which thiamine was added to the culture medium to switch off cdc13+ expression and the cells were incubated for an additional 5 h. Cell lysates were subjected to immunoprecipitation and immunoblot analysis as in (C). (F) Cells expressing HA-tagged Fkh2p (HM6107) were synchronized in G1 by transient temperature arrest and samples taken every 1 h upon release to the permissive temperature. Cell lysates were subjected to immunoprecipitation and immunoblot analysis as in (C). (G) DNA content of the cells in (F) was determined by flow cytometric analysis. Download figure Download PowerPoint To test whether Fkh2p is phosphorylated on T314 or S462 in vivo, we prepared antibodies to Fkh2p peptides containing phosphorylated (p) T314 or pS462. The antibodies (anti-pT314, anti-pS462) specifically recognized the respective Fkh2p peptides containing pT314 or pS462 but not the corresponding nonphosphorylated peptides (Figure 4B). They also recognized wild-type Fkh2p but not the Fkh2p(T314A,S462A) mutant expressed in fission yeast cells (Figure 4C). Fkh2p exhibited multiple forms because of phosphorylation (Buck et al, 2004; Bulmer et al, 2004). Similarly, multiple bands appeared in Fkh2p(T314A,S462A) mutant (Figure 4C; Supplementary Figure 8), suggesting that multiple bands come from phosphorylation other than these sites. We, therefore, next examined whether Cdc2p is required for phosphorylation of Fkh2p on T314 or S462 in vivo. We first examined a temperature-sensitive cdc2 mutant. Inactivation of cdc2+ by a temperature shift resulted in a decrease in the level of Fkh2p phosphorylation on each of these two residues (Figure 4D). We then examined a strain in which the B-type cyclin gene cig2+ is deleted and found that the level of Fkh2p phosphorylation on T314 and S462 was also decreased (Figure 4D). In addition, shut off of expression of the B-type cyclin gene cdc13+ induced a slight decrease in the level of Fkh2p phosphorylation on each of these two residues (Figure 4E). To test whether Fkh2p is phosphorylated depending on the cell-cycle stage, cells were transiently arrested in G1 by the inactivation of cdc10+ to induce cyclin degradation and released to the cell cycle (Figure 4F and G). In G1, Fkh2p was found to be dephosphorylated on T314 and S462. On release from G1, S462 was phosphorylated earlier than T314, although both of these residues were eventually phosphorylated. This may be due to the facts that the major cyclin responsible for phosphorylating these residues may be different and that the expression of the cyclin may vary during the cell cycle. On the basis of these results, we concluded that Cdc2p and the B-type cyclins Cig2p and Cdc13p are required for phosphorylation of Fkh2p on T314 and S462 in vivo. To examine whether phosphorylation of Fkh2p on T314 affects its ability to bind to the upstream region of ste11+ containing the FLEX1 and FLEXL1 sites, we performed ChIP analysis with cells expressing phosphomimetic mutants of Fkh2p (Figure 5). The mating efficiency and the induction of ste11+ were low in GFP-tagged Fkh2p(S462E) cell-like control cells (Supplementary Figure 2; see Figure 3B and C). At 2 h after nitrogen withdrawal, the amount of Fkh2p(T314E) associated with this genomic region failed to increase compared with that of the wild-type protein. These results thus suggested that the poor mating efficiency of, as well as the impaired induction of ste11+ mRNA in, fkh2-T314E mutant cells is due to the reduced ability of the Fkh2p(T314E) mutant protein to bind to the upstream region of ste11+. Given
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