1. Volume 17, Issue 1, Pages 49-59 (January 2005)
Inactivation of the Cdc25 Phosphatase by the Stress-Activated Srk1 Kinase in Fission Yeast 
Sandra López-Avilés, Maribel Grande, Marta González, Ase-Lill Helgesen, Vicenç Alemany, Maribel Sanchez-Piris, Oriol Bachs, Jonathan B.A. Millar, Rosa Aligue 
Molecular Cell 
Volume 17, Issue 1, Pages (January 2005)
DOI: /j.molcel

2. Figure 1 Overexpression of srk1+ Causes Cells to Arrest in G2
(A and B) Wild-type cells expressing pREP1-srk1+ or pREP1-srk1KA were grown either in the presence (+B1) or absence (−B1) of thiamine on (A) plates or (B) in liquid media. Bar, 14 μm.
(C) Western blot of extracts from cells in (B) were probed either with anti-HA antibodies (top) or anti-cdc2 antibodies (bottom).
(D) Wild-type cells expressing pREP1-srk1+ grown either in the presence (+B1) or absence (−B1) of thiamine were fixed and stained with DAPI. Bar, 14 μm.
Molecular Cell  , 49-59DOI: ( /j.molcel )

3. Figure 2 Srk1 Inhibits Tyrosine Dephosphorylation of Cdc2
(A) Wild-type (wt), wee1Δ, cdc2-3w, and cdc2-3w cdc25Δ cells transformed with pREP41-srk1+ were grown on selective plates either in the absence of thiamine (−B1) or presence of thiamine (+B1) for 3 days at 30°C.
(B) wee1 Δ, mik1Δ, cdc2-3w, and cdc2-3w cdc25Δ cells transformed with pREP41-srk1+ were grown in liquid culture cells either in the absence of thiamine (−B1) or presence of thiamine (+B1) for 24 hr. Cell morphology was analyzed microscopically. Bar, 10 μm.
(C) Cell extracts from the cells in (B) were analyzed by Western Blot with either anti-HA antibodies (top) or anti-cdc2 antibodies (bottom).
(D) Wild-type cells transformed with pREP1-srk1+ (top), pREP2-pyp3+ (middle), or both pREP1-srk1+ and pREP2-pyp3+ plasmids (bottom) were grown in the absence of thiamine for 24 hr and visualized microscopically. Bar, 14 μm.
(E) Level of Srk1 protein from cells in (D) was detected by Western blot with anti-HA antibodies.
(F) Wild-type, srk1Δ, wee1-50, and srk1Δ wee1-50 cells were grown at 35°C in YES liquid medium until mid-log phase and stained with DAPI to visualize nuclear DNA. Bar, 10 μm.
Molecular Cell  , 49-59DOI: ( /j.molcel )

4. Figure 3 Srk1 Interacts with and Phosphorylates Cdc25
(A) cdc25-12myc cells were transformed with pREP1-srk1KA and grown in the presence (+ B1) or absence (− B1) of thiamine. Srk1KA-HA was immunoprecipitated from cell extracts and analyzed by Western blot for the presence of Srk1 and Cdc25 with anti-HA antibodies or anti-myc antibodies, respectively.
(B) GST-Srk1 and GST-Srk1KA fusion proteins were assayed for kinase activity with myelin basic protein (MBP) as a substrate (left). Right panel shows the Coomassie blue staining of purified proteins.
(C) Graphical representation of Cdc25 fragments expressed as GST fusion proteins.
(D) Equal concentrations of the purified GST-Cdc25 fusion proteins were used as substrates for GST-Srk1 kinase or inactive GST-Srk1KA kinase. The arrow indicates full-length GST-Cdc25.
(E) Srk1 (lanes 1, 2, and 3) and Srk1KA (lanes 4, 5, and 6) were immunoprecipitated from fission yeast cell extracts, and kinase activity was measured (left) with GST-Cdc25 (lanes 1 and 4), GST-Cdc2556–145 (lanes 2 and 5), or GST (lanes 3 and 6) fusion proteins as substrates. Coomassie blue gel staining of the samples is shown in the right panel.
(F) The amount of Srk1 (lane 1) and Srk1KA (lane 2) proteins in the immunoprecipitates from (E) was determined by Western blot with anti-HA antibodies.
Molecular Cell  , 49-59DOI: ( /j.molcel )

5. Figure 4
Srk1 Phosphorylates and Inhibits Cdc25 to Induce Cell Cycle Arrest in G2
(A) GST-Srk1 was incubated in the presence of either full length GST-Cdc25 or GST-Cdc25-9A proteins and analyzed for incorporation of γ-32P (top) or amount of fusion protein in the assay (bottom).
(B) cdc25-9A cells transformed with pREP1-srk1+ were grown in liquid culture for 24 hr either in the presence (+B1) or absence (−B1) of thiamine and visualized microscopically. Bar, 15 μm.
(C) The amount of Srk1 protein in cell extracts of cells in (B) was determined by Western blot with anti-HA antibodies (top) or anti-cdc2 antibodies (bottom).
(D) GST-Srk1 was incubated with synthetic human Cdc25C peptide containing amino acids 211 to 221 (Ser214/216) (lane 2) or the same peptide in which Ser216 is mutated to alanine (Ser216A) (lane 1) or phosphorylated Ser216 (Ser216-P) (lanes 5 and 7) or the same peptide in which Ser214 is mutated either to alanine (Ser214A) (lane 3) or aspartic acid (Ser214D) (lane 4) or phosphorylated Ser214 (Ser214-P) (lanes 5 and 6). Control reactions contain only Srk1 (lane 8) or only buffer (lane 9). The phosphorylated peptide is marked with an arrow. Incorporation of γ-32P was normalized to the quantity of peptide loaded and expressed as a percentage of that incorporated into the S214A peptide.
Molecular Cell  , 49-59DOI: ( /j.molcel )

6. Figure 5 Srk1 Causes Accumulation of Cdc25 in the Cytoplasm
(A) pREP1-srk1+ cells were grown in the absence (−B1) or presence (+B1) of thiamine for 24 hr. Cells extracts were prepared and analyzed by Western blot with anti-Cdc25 antibodies (top), anti-HA antibodies (middle), or anti-tubulin antibodies as a loading control (bottom).
(B) Cell extracts were prepared from cells expressing empty vector pREP, pREP1-srk1+, or pREP1-srk1KA and analyzed by Western blot to monitor the level of Srk1 expression (top), anti-Cdc25 antibodies (middle), or anti-PSTAIR antibodies as a loading control (bottom).
(C) cdc25-12myc cells expressing either pREP1-wee1+ or pREP1-srk1+ were grown in the presence or absence of thiamine to either induce (−B1) or repress (+B1) wee1 or srk1 expression, respectively. Cell extracts were analyzed by Western blot with anti-myc antibodies to visualize Cdc25 (top) or anti-Cdc2 antibodies as a loading control (bottom).
(D) Cells from (C) were visualized microscopically. Bar, 14 μm.
(E) cdc25-12myc cells expressing empty pREP1 vector (top panels) or pREP1-Srk1-HA (middle panels) or pREP1-Wee1-HA (bottom panels) were subjected to fluorescence microscopy to analyze the cellular localization of Cdc25 protein with anti-myc antibodies (red) and localization of Srk1-HA and Wee1-HA proteins with anti-HA antibodies (green). Cells were stained with DAPI to visualize DNA (blue). Bar, 10 μm.
Molecular Cell  , 49-59DOI: ( /j.molcel )

7. Figure 6
Srk1-Induced Cell Cycle Arrest and Stabilization of Cdc25 Is Dependent on Rad24
(A) The phenotype of wild-type (middle) or rad24Δ (right) cells overexpressing Srk1 was compared. rad24Δ cells are shown as a control (left). Bar, 10 μm.
(B) Extracts from the cells in (A) were analyzed by Western blot with anti-Cdc25 antibodies (top) and anti-HA antibodies to visualize Srk1 (middle) or anti-PSTAIR antibodies to detect Cdc2 (bottom).
(C) GST-Cdc25 was incubated for 30 min with Srk1 or Srk1KA in the presence of ATP and then for an additional 15 min with purified Rad24-6His protein. Rad24 was immunoprecipitated, and the level of Cdc25 and Rad24 in the immunoprecipitates was assessed by Western blot with either anti-GST or anti-Rad24 antibodies, respectively.
Molecular Cell  , 49-59DOI: ( /j.molcel )

8. Figure 7
Srk1 Controls Cdc25 Stability and Interaction with Rad24 in Response to Osmotic Stress
(A) srk1-HA cells were synchronised in S phase and released to the following cell cycle. Cell cycle position was monitored by the percentage of septated cells (top graph). At the times indicated, cell extracts were analyzed by Western blot with anti-HA (Srk1) or anti-PSTAIR (Cdc2) antibodies. Srk1 was immunoprecipitated from the remainder of the extract and assayed for in vitro kinase activity with GST-Cdc2556–145 as a substrate (bottom graph). Srk1 kinase activity was normalized to the level of Srk1 in the immunoprecipitates.
(B) Srk1 was immunoprecipitated from extracts of srk1-HA cells either before (−KCl) or after addition of 1 M KCl for 30 min (+KCl) and assayed for in vitro kinase activity with GST-Cdc2556–145 as a substrate. Srk1 kinase activity was normalized to the level of Srk1 in the immunoprecipitates.
(C) Log phase wild-type and srk1Δ cells were incubated in the absence (−) or presence of 1 M KCl (+) for 30 min. Rad24 was immunoprecipiated from cells extracts, and the level of Cdc25 and Rad24 in the immunoprecipitates was monitored by Western blot with anti-Cdc25 antibodies or anti-Rad24 antibodies, respectively.
(D) Serial dilutions of log phase cultures of wild-type, sty1Δ, and srk1Δ cells were plated on rich medium containing 0.8 M KCl. Colony formation was analyzed after 3 days growth at 30°C.
(E) Wild-type and srk1Δ cells were grown in 1 M KCl for the times indicated. The level of Cdc25 (top) or Cdc2 (bottom) was determined by Western blot with anti-Cdc25 or anti-PSTAIR antibodies, respectively.
Molecular Cell  , 49-59DOI: ( /j.molcel )