Mitotic Hyperphosphorylation of the Fission Yeast SIN Scaffold Protein cdc11p Is Regulated by the Protein Kinase cdc7p  Andrea Krapp, Elena Cano, Viesturs Simanis  Current Biology  Volume 13, Issue 2, Pages 168-172 (January 2003) DOI: 10.1016/S0960-9822(02)01417-3

Figure 1 Activation of the SIN Correlates with Hyperphosphorylation of cdc11p, which Requires cdc7p Function cdc11p phosphorylation was assessed by probing Western blots of protein extracts from various cdc11-HA-tagged strains with 12CA5. Since many of the mutants used are mainly heat sensitive, synchronous cultures were generated by hydroxyurea (HU) arrest-release. Wild-type cells were synchronized in early S phase by the addition of hydroxyurea to 12 mM (HU) at 25°C. In these cells (I), cdc11p was seen in the hypophosphorylated and phosphorylated interphase forms (marked 1 and 2, respectively). After 5 hr, the HU was washed out, and cells were incubated at 36°C. Samples (M) were taken after approximately 100 min, when cells had entered anaphase, unless otherwise stated. In extracts from these cells (M), the hyperphosphorylated forms of cdc11p were also seen. The kinetics of anaphase entry after release from HU arrest are given in the Supplementary Material. Since the various phosphorylated forms of cdc11p do not always resolve identically, all gels have a sample marked wild-type (I) from interphase-arrested cdc11-HA cells (forms 1 and 2) and a sample (M) from anaphase cdc11-HA cells (forms 1, 2, and 3). For each mutant, I and M are the interphase and anaphase samples, respectively. (A) cdc11-HA cells were arrested by incubation in yeast extract (YE) containing 12 mM HU at 25°C for 5 hr. Cells were resuspended in YE without HU and were incubated at 36°C. Samples were taken before release (0 min) and during anaphase (approximately 100 min) after release. cdc11-HA or cdc16-116 cdc11-HA cells were grown at 25°C to exponential phase and were shifted to 36°C for 4 hr. cdc11-HA byr4::ura4+ mob1-R4 cells were grown at 25°C. (B) cdc11-HA cdc2-17 leu1::pint5(ura4+)-spg1 cells were incubated in medium without thiamine for 12 hr at 25°C, then shifted to 36°C for 5 hr to impose the cell cycle arrest as spg1p expression was induced (spg1OE). M is a mitotic wild-type sample, spg1OE corresponds to cells expressing increased levels of spg1p. cdc2-17 cdc11-HA cells grown in the same medium served as control (C). (C) The mutant plo1-ts4 cdc11-HA was arrested in interphase by incubation in HU at 25°C and was then released from the block at 36°C. Samples were taken at the time of release (I) and at the time of anaphase (M). Anaphase cells were fixed, and indirect immunofluorescence was performed with antibody to byr4p. (D and E) Cells of the indicated mutant strains, all of which also have the cdc11-HA allele, were arrested in interphase by incubation in HU and were released from the block at 36°C. Samples were taken at the time of release (I) and at the time of anaphase (M). (F) cdc25-22 cells transformed with pREP3-cdc7 were induced to express cdc7 for 12 hr at 25°C and were then shifted to 36°C for 4 hr to impose the G2 arrest as cdc7 expression peaked. (G) cdc11-HA cdc16::ura4+ cdc7-A20 cells were grown at 32°C and were arrested by incubation in HU. Cells were released from the block at the indicated temperatures, and samples were taken as cells passed through anaphase at the indicated temperatures. Current Biology 2003 13, 168-172DOI: (10.1016/S0960-9822(02)01417-3)

Figure 2 Spindle Assembly Checkpoint Activation Blocks cdc11p Hyperphosphorylation, and byr4p Is in a Complex with the Interphase Forms of cdc11p Western blots of the protein samples were probed with 12CA5 to detect cdc11-HAp or antibody to byr4p. In (A) and (E), wild-type I and M are protein samples prepared from cdc11-HA cells in interphase and anaphase, respectively. (A) nda3-KM311 cdc11-HA cells were arrested in mitosis by incubation at 19°C for 5 hr. The exponentially growing control at 32°C served as control. (B) nda3-KM311 cells were fixed by the addition of paraformaldehyde to 3% (v/v), and indirect immunofluorescence was performed by using antibody to byr4p, followed by Fluorescein-conjugated secondary antibody. Under the conditions used, cells have not yet condensed their chromosomes, and 80% of cells showed staining for both byr4p and cdc7p. (C) Protein extracts were prepared from either wild-type or cdc11-HA cells, and immunoprecipitation was performed with 12CA5 antibody. Western blots of the extracts and precipitated proteins (IPP) were probed with antiserum to byr4p. (D) Protein extracts were prepared from either byr4::ura4+ mob1-R4 cdc11-HA or cdc11-HA cells, and immunoprecipitation was performed by using antibody to byr4p. Western blots of the extracts and precipitated proteins (IPP) were probed with antiserum to 12CA5. (E) cdc11-HA cdc25-22 cells were synchronized by arrest-release, and a protein extract was prepared from cells 80 min after release. Immunoprecipitation was performed by using antibody to byr4p. Western blots were probed with 12CA5. E is the extract from the cdc25-22 arrest-release used for the immunoprecipitation shown in the adjacent lane. I is the interphase extract, and M is a mitotic extract. Current Biology 2003 13, 168-172DOI: (10.1016/S0960-9822(02)01417-3)

Figure 3 PP2A-par1p Contributes to Dephosphorylation of cdc11p at the End of Mitosis, and SPB Association of cdc11p Is Required for Its Phosphorylation In Vivo In all the panels, Western blots were probed with 12CA5 to detect cdc11-HAp. (A) cdc25-22 cdc11-HA par1::ura4+ or cdc25-22 cdc11-HA cells were arrested for 4 hr at 36°C and were released to 25°C. Samples were taken at the indicated time points (in minutes) after release. (B) sid4-SA1 cdc11-HA cells were grown at 19°C to exponential phase and were then shifted to 36°C for 5 hr. (C) cdc11-HA cells were transformed with pREP3 containing a fragment of cdc11 expressing amino acids 442–1045 of cdc11p. Cells were grown to exponential phase, without induction of the nmt1 promoter (+T) or with induction for 5 generations at 25°C (−T). Current Biology 2003 13, 168-172DOI: (10.1016/S0960-9822(02)01417-3)