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First published online 5 May 2004
doi: 10.1242/jcs.01107

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A role for the Cdc14-family phosphatase Flp1p at the end of the cell cycle in controlling the rapid degradation of the mitotic inducer Cdc25p in fission yeast

¹ Centro de Investigación del Cáncer, Departamento de Microbiología y Genética, Campus `Miguel de Unamuno', Universidad de Salamanca/CSIC, 37007 Salamanca, Spain
² Cell Cycle Control Laboratory, Swiss Institute for Experimental Cancer Research (ISREC), Chemin des Boveressess 155, 1066 Epalinges, Switzerland

View larger version (64K): [in a new window]   Fig. 1. Wee1p and Cdc25p proteins in flp1 mutant S. pombe cells. (A) Wee1p is partially dephosphorylated in a flp1 mutant strain. Cells from asynchronous cultures of wee13HA and wee13HA flp1 (incubated at 30°C) were processed for western analysis and probed with -Ha monoclonal or -tubulin antibodies. (B) Cdc25p is hyperphosphorylated in the flp1 deletion strain. Cells from asynchronous cultures of wt and flp1 (incubated at 30°C) were processed for western analysis and probed with -Cdc25 polyclonal antibodies. The asterisk marks a reference protein that cross-reacts with the antibody. (C) Cdc25p is hyperphosphorylated in flp1 deletion strains at different stages of the cell cycle. Western blot analysis of Cdc25p protein extracts from cells block in S phase (hydroxyurea, HU, 4 hours), in G2 (cdc2-33, 4 hours at 35.5°C), in mitosis (nda3-KM311, 6 hours at 19°C) or in G1 (cdc10-129, 4 hours at 35.5°C). Blots were probed with -Cdc25 and -Cdc2 polyclonal antibodies, the latter for a loading and proper migration control.

View larger version (65K): [in a new window]   Fig. 2. Cell division is delayed in flp1 mutant S. pombe cells. nda3-KM311 and nda3-KM311 flp1 (otherwise isogenic strains) were blocked in metaphase by incubation of asynchronously growing cultures (Asn) at 20°C for 6 hours. Cells were then released from the block at 32°C and samples were taken at the indicated intervals and processed for cytological and biochemical analysis. (A) Plots of binucleate (top plot, Mitotic Index), septated cells (middle plot, Septation Index) and relative kinase activity (lower plot) are shown. (B) Cdc13p-associated H1 kinase activity (top panels) and relative amounts of immunoprecipitated Cdc2 for the kinase assays (lower panels). (C) Cdc13p, Cdc25p and -tubulin western analysis (as indicated) from samples processed from nda3-KM311 and nda3-KM311 flp1 cells. (D) Elutriated G2 nda3-KM311 (control) or nda3-KM311 flp1 cells were blocked at metaphase by incubating them for 4 hours at 20°C and then released at 32°C. Samples were taken at the indicated intervals and processed for -tubulin and Cdc25p western analysis.

View larger version (38K): [in a new window]   Fig. 3. Cdc25p localisation and stability are altered in flp1 mutant fission yeast cells. (A) Asynchronous exponentially growing cultures of myc12-cdc25 (reference control) and flp1 myc12-cdc25, otherwise isogenic strains, were processed for indirect immunofluorescence microscopy using anti-Myc antibodies. Nuclei were stained with DAPI. Approximate phase of the cell division cycle is indicated. Cells at different stages of the cell cycle were scored to quantify the defect observed in proper Cdc25p localisation as compared with the reference control (myc12-cdc25 cells). (B) Equal numbers of cells from asynchronous cultures of two different strains (myc12-cdc25 GFP-atb2+ and flp1 myc12-cdc25) were mixed and processed for indirect immunofluorescence microscopy using anti-Myc antibodies, DAPI and GFP-Atb2 (the latter to distinguish flp1+ wild-type from flp1 mutant). flp1 mutant cells are indicated by white arrowheads in the photomicrographs. Scale bars: 10 µm. Note that GFP-Atb2p-positive cells contain less Myc-Cdc25p (as judge by the relative intensity of anti-Myc staining). (C) Ten-fold dilution of the indicated strains from log-phase cultures at 25°C were inoculated in YES Petri dishes and incubated for 48 hours at the indicated temperatures.

View larger version (44K): [in a new window]   Fig. 4. Flp1p interacts with and dephosphorylates Cdc25p. (A) GSTp, GST-Flp1p and GST-Flp1CSp proteins were expressed under the control of the nmt1 promoter in a cdc25 cdc2-3w strain and purified with glutathione-Sepharose (GSH) beads. An aliquot of each sample was processed for PAGE and stained with Coomassie Blue to visualise proteins. (B) GST-Flp1p and GST-Flp1CSp but not GSTp associate in vivo with Cdc25p. GSTp (negative control, lanes 1), GST-Flp1p (lanes 2) or GST-Flp1CSp (lanes 3) were purified and processed for the detection of Cdc25p from cdc25+ (wt) or cdc25 cdc2-3w (cdc25) cells expressing them (20 hours of induction of the nmt1 promoter). A total extract control from nda3-KM311 metaphase-arrested cells is shown for the detection of Cdc25p (lane C). Note that the Cdc25p protein that associates with the GST-Flp1CSp form is hyperphosphorylated as compared with the migration of Cdc25 from metaphase-arrested cells shown in lane C. (C,D) Purified GSTp, GST-Flp1p and GST-Flp1CSp proteins from A were assayed in vitro for their ability to dephosphorylate an artificial substrate, pNPP (C), or Cdc25p immunoprecipitated from nda3-KM311 metaphase-arrested cells (D). Note in D the mobility shift observed when immunoprecipitated Cdc25p is incubated with GST-Flp1p, in comparison with GSTp and GST-Flp1CSp negative controls.

View larger version (19K): [in a new window]   Fig. 5. GST-Flp1p dephosphorylates in vitro CDK-phosphorylated GST-Cdc25p. Purified GST-Flp1p and GST-Flp1CSp proteins from cdc25 cdc2-3w strains (as in Fig. 4A) were assayed in vitro, in the linear range of the assay, for their ability to dephosphorylate purified GST-Cdc25p from bacteria and previously phosphorylated in vitro by immunoprecipitated Cdc2/Cdc13 complexes. The relative amount of radioactive GST-Cdc25p was quantified in three different and independent experiments (in which every component was de novo purified or immunoprecipitated) and plotted. 110 µM CDK-phosphorylated GST-Cdc25p were incubated with 0 µM (sample 1 and 6), 55 µM (sample 2), 110 µM (sample 3) or 220 µM (sample 4) of GST-Flp1p or 330 µM GST-Flp1CSp (sample 5). Samples were run on PAGE gels and stained with Coomassie Blue. Relative phosphorylation levels in GST-Cdc25p were quantitated using a phosphorimager and plotted as a percentage of the phosphorylation levels measured/observed in reactions without GST-Flp1p. Note that the extent of GST-Cdc25p dephosphorylation does increase slightly with addition of more GST-Flp1p. Bars indicate standard error.

View larger version (57K): [in a new window]   Fig. 6. The phosphorylation state of Wee1p is not affected by loss of flp1p function. (A) A metaphase block- and-release experiment of nda3-KM311 wee13HA (control) and nda3-KM311 wee13HA flp1 (otherwise) isogenic strains. Cells were synchronously released from an nda3 block (6 hours at 20°C), samples were taken at the indicated (in minutes) intervals to analyse Wee1p protein phosphorylation by shift mobility after western analysis with -Ha monoclonal antibody. The plot of septated cells (Septation Index) is shown. (B) Cdc25p localises at the nucleolus when flp1+ is overexpressed. Photomicrographs of nmt1: flp1+ or nmt1: flp1+ myc12-cdc25 cells induced for the expression of Flp1 for 18 hours. (C) Overexpression of flp1+ induces Cdc25p dephosphorylation. Expression of Flp1 was induced in an nmt1: flp1+ strain by removal of thiamine from the medium, samples were taken at indicated intervals and processed for western analysis and immunofluorescence (see B).