Kinase Activity of Fission Yeast Mph1 Is Required for Mad2 and Mad3 to Stably Bind the Anaphase Promoting Complex

Summary Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6–8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.


Inventory of Supplementary Data:
A) mph1 kinase dead alleles are benomyl (anti-microtubule drug) sensitive. The indicated strains were diluted, plated on rich media (YES) with or without benomyl and then grown for 3 days at 30 o C. B) mph1 kinase dead alleles mis-segregate chromosome 2. The indicated strains containing cen2-GFP were grown in liquid cultures and then bi-nucleate cells carefully analysed and scored for segregation of chromosome 2. N was >900 cells for all strains, with the experiment repeated at least 3 times for each strain. C) The indicated strains, containing the Ch16 mini-chromosome, were plated on low adenine containing media and colonies analysed for sectoring. Loss of Ch16 in the first division results in a half-sectored colony (half pink, half white). Total colony number was >3000 for all strains, and is the combination of at least 3 experiments for each strain. D) mph1 kinase dead alleles display lagging chromosomes. Strains were grown, fixed and analysed by tubulin immunofluorescence and DAPI staining. The percentage of anaphase cells containing lagging chromosomes were scored (these are frequently merotelically attached single sisters in fission yeast [1]). N of cells was >1000 for each strain, and was repeated at least 3 times for each strain. These data are also summarised in Fig1B. Figure S1 and Fig 1B demonstrate significant chromosome loss rates for the mph1-kd alleles, for example they are far higher than mad2Δ or mad3Δ strains. The loss rates are even higher for the complete deletion, which argues that the N-terminal domains of this protein are likely to have additional, kinase-independent functions. These data also demonstrate that the double mutants, bub1Δ mph1Δ, and bub1-∆K mph1∆K, have higher rates of chromosome loss than single mutants. Such synthetic phenotypes can be interpreted in various ways: (i) the two protein kinases may share substrates (in this scenario, absence of post-translational modification on both Mph1 and Bub1 sites in "substrate X" leads to a more severe loss of function than loss of solely Mph1-dependent or Bub1-dependent modification); (ii) the kinases may share some but not all substrates, or (iii) the kinases have distinct targets thereby affecting multiple pathways. Unfortunately the only way to distinguish between these possibilities is to identify their respective kinetochore targets, and this has yet to be done for Mph1 kinase. We conclude that Mph1 and Bub1 both have roles in chromosome segregation that are distinct from spindle checkpoint arrest and to an extent distinct from one another. This is supported by known genetic interactions: whilst many genetic interactions are shared there are some differences, for example bub1Δ is synthetic lethal with klp5Δ but this is not true for mph1Δ [2]. We have confirmed this observation and were unable to construct an nda3, mph1Δ, bub1Δ triple mutant strain (LM, data not shown) again suggesting non-redundant segregation functions for these two protein kinases.

Figure S2 (related to main text Fig 3). Mad2 and Mph1 phosphorylation site schematics and sequence alignments.
A) Mph1 schematic indicating the auto-phosphorylation sites identified by mass spectrometry from the in vitro Mph1 kinase assay. The kinase-dead point mutation is also indicated. B) Mad2 schematic indicating the five in vitro phosphorylation sites (the two confirmed in vivo are labelled in red). The dimerisation mutant is also shown. C) Mad2 sequence alignments with conserved phospho-sites indicated in red. Fig 3). mad2-S187D displays reduced Mad1 binding.

Figure S3 (related to main text
A) Structural models explaining the importance of S187 modification. See supplementary discussion for further details and explanation of these models. B) Mad1 pull downs from mad2-S187D and mad2-S187N alleles.
Whilst an aspartic acid substitution at position 187 disrupts the Mad2-Mad1 interaction, this is not the case for an asparagine substitution, indicating that it is the charge, rather than the size, that is disruptive. C) The phospho-specific anti-Mad2 antibodies confirm that serines 92, 187 and 189 can all be modified by Mph1 kinase in vitro. Unfortunately they are not sensitive enough to detect modifications on Mad2p purified from fission yeast cells.

Figure S4 (related to main text Fig 3). MS/MS spectra from mass spectrometric analyses of Mad2p.
Examples of MS/MS spectra used to identify and assign phosphorylation of each phosphopeptide identified. Fragment ions containing the peptide's N-(b-ions) or C-(y-ions) termini are labelled. A number of the identified peptides show neutral loss of phosphoric acid from the full length peptide, supporting the conclusion of peptide phosphorylation. Significant fragmentation has also occurred allowing peptide sequencing and assignment of the phosphorylated residue. A) Examples of spectra for the Mad2 phosphorylation sites identified from Mad2-HTB purified from nda3 arrested and/or cycling fission yeast cells (in vivo). B) One example (S92) spectrum for Mad2 phosphorylation sites identified from in vitro Mph1 kinase assays. Spectra for other sites (S69, S187, T188, S189) are available upon request.

Supplemental Discussion
A possible structural explanation for the effects of S187D mutation The 2:2 tetrameric Mad1-Mad2 complex is significantly asymmetric, with different orientations of Mad1 with respect to Mad2 (see Figure S3). The structure of two Mad1-Mad2 dimers that make up the human complex are shown in the same orientation with respect to Mad2 (PDB id 1GO4, [3]). Several residues that are identical in human and S.pombe are shown as sticks. The left dimer shows a compact association with the N-terminal helix of Mad1. This interaction is mediated by the burial of Leu531Mad1 in a hydrophobic pocket on the last two β-strands of Mad2. The interaction is further stabilised by the packing of the N-terminal helix of Mad1 against residues Arg99Mad2 and Gln101Mad2. The right dimer shows looser packing, where the end of the N-terminal helix is partially melted and the close contact with Leu531Mad1 is lost. In this conformation the integrity of the 8th β-strand is also disrupted. This asymmetry has been hypothesized to be functionally significant as it may provide a "fracture line" or weak point where Mad1-Mad2 complexes could be disassembled [3].
Thr187 and Thr189, the human equivalents of S187 and S189 in S.pombe, lie at the end of the

Generation of Mad2 phosphorylation mutants
Phosphorylation mutants were created from pJK-Mad2, which is based on the vector pJK148 [4].
It contains 250 bp of 5'UTR, followed by the mad2 ORF and 150 bp of 3'UTR. All mad2 strains were generated by integration of pJK-Mad2 vectors into a mad2Δ strain at the endogenous mad2 locus. Point mutations were introduced to replace S69, S92, S187, T188, S189 with alanine or aspartic acid using the Quikchange Kit for Site Directed Mutagenesis (Stratagene).

Generation of mph1-kd and mph1-kd-SZZ
The mph1-kd strain was generated by integration of pJK mph1-kd into a wild-type mph1 strain at the endogenous mph1 locus. pJK mph1-kd is based on the vector pJK148 [4]. It contains the mph1 sequence from base 408 to the end of mph1 followed by ~200bp of 3'UTR. The kinasedead mutation was generated by introducing a single point-mutation at base A1376C. The mph1kd-SZZ strain was generated similar to mph1-kd with the addition of the insertion of a C-terminal SZZ prior to the 3'UTR.

Mitotic arrests
Yeasts strains are listed in Table 1. nda3-KM311(cs) cells were grown overnight in yeast extract plus supplements (YES) medium at 30°C to mid-log phase and then shifted to 18°C for 6 h. For microscopy cells were briefly fixed at -20°C in 100 % methanol and mounted with 1 mg/ml DAPI (Sigma). For viability assays cells were arrested at 18°C for 6 h and single cells plated on YES plates and incubated at 32°C. For nuf2-3 arrests cells were grown overnight in YES medium at 25°C to mid-log phase and then shifted to 32°C for 4 h followed by fixation in -20°C 100 % methanol.

G2 arrests (cdc25-22)
For cdc25-22 arrests cells were grown overnight in YES medium at 25°C to mid-log phase and then shifted to 36°C for 3.5 h. To release cells from the G2 arrest cultures were cooled down rapidly to 25°C and then incubated at 25°C.

Chromosome mis-segregation assays
Cen2-gfp: Overnight cultures were grown to mid-log phase in YES and the rate of cen2-gfp missegregation scored in binucleate live cells.

CH16 minichromosome loss:
The CH16 strain used in this assay contains a ura4 marked bub1∆ [5]. Overnight cultures were grown to mid-log phase in PMG lacking uracil and adenine.
250 cells were plated on YES plates containing low adenine (6.6 μg/ml). Plates were incubated for 4 days at 30°C and the percentage of CH16 mini-chromosome loss scored as a percentage of half-sectored colonies to white colonies. For more details on chromosome segregation assays see [5].
Immunoprecipitations were carried out as described above with the following modifications. No phosphatase inhibitors were added to the lysis buffer. Immunoprecipitated complexes were analyzed by immunoblotting with a sheep anti-Mad2 antibody and Peroxidase-Anti Peroxidase (Sigma) antibody to detect Mad1-TAP.

Mph1-SZZ kinase purification
Cycling Reactions were typically carried out with 1.5 µg substrate/recombinant protein. The reaction was incubated at 30°C for 30 mins. Cold kinase assays were carried out with 100 mM ATP and further analysed by mass spectrometry or by immunoblotting with phosphoantibodies generated against phosphorylated residues S92 (Covance), S187, S189 (Eurogentec) of Mad2.

Protein digestion and Phosphopeptide enrichment
Proteins were electrophoresed into NovexNuPAGE 4-12% Bis-Tris gels (Invitrogen). Proteins were stained with a colloidal blue kit (Invitrogen). Proteins were excised and standard trypsin digestion procedure carried out as described previously [6]. The supernatant of samples for MS analysis without phosphopeptide enrichment were loaded onto StageTips [7]. Supernatants of samples for phosphopeptide enrichment were removed and peptides further extracted from the gel using 3% TFA/30% ACN solution followed by 100% ACN. Before enrichment approximately 3 mg TiO 2 beads were pre-incubated in 20 µl of 85 mg/ml lactic acid in 80% ACN/0.1%TFA. Preincubated bead mixture was added to the peptide mixture and incubated for 1 h at room temperature. After washing the beads once with 10% ACN/0.1% TFA, and twice with 80% ACN/0.1% TFA, peptides were eluted with 2% ammonium hydroxide in 40% ACN (pH 10.5).
Eluate was concentrated to 100 µl and 100 µl of 2% TFA was added. All samples were loaded onto StageTips and stored at -20ºC for MS analysis.

Nano-LC-MS/MS and Data analysis
An LTQ-Orbitrap (Thermo Scientific), 1200 series nanoflow HPLC pump (Agilent Technologies) and HTC PAL auto sampler (CTC Analytics) were used for LC-MS/MS analysis. Mobile phases were (A) 5% acetonitrile, 0.5% acetic acid and (B) 99.5% acetonitrile, 0.5% acetic acid. A flow rate of 300 nl/min was used with a spray voltage of 1.8 kV. Analysis was done using either a twostep linear gradient of 0%-20% B in 35 min, 20%-80% B in 4 min and 80% B for 2 min or a twostep linear gradient of 0%-20% B in 75 min, 20%-80% B in 13 min and 80% for 10 min. A column needle was prepared by packing 3 µm Reprosil C18 materials (Dr. Maisch, Germany) into Pico Tip Emitter silica tips (8±1µm, New Objectives) under the pressure of nitrogen [8]. Each cycle consisted of one full MS scan acquired in the Orbitrap analyser, followed by MS/MS of the six most abundant peptides in the ion trap.

Database searching
DTAsupercharge (V1.18) was used to create peak lists from raw data. Peak lists were then used within Mascot daemon (V2.2.0) to search against the UniProt/SwissProt Schizosaccharomyces pombe database. Search parameters were set to: precursor mass tolerance of 10 ppm, fragment ion mass tolerance to 0.8 Da, enzyme as trypsin, allowing 3 missed cleavages.
Carboamidomethylation of cysteine was set as a fixed modification, with oxidation of methionines, phosphorylation of serine, threonine, and tyrosine as variable modifications.