Mo M , Arnaoutov A , Dasso M .

ZIAHD008816 PHS HHS

	Figure 1. p31comet depletion causes an SAC-dependent mitotic exit delay. (A) p31comet-depleted XEE reactions containing DSN, with or without 15 nM His6-p31comet were incubated at 23°C for 15 min., followed by CaCl2 addition. At intervals after CaCl2 addition, samples were collected for Western blotting with the indicated antibodies. Mock lanes show samples from a mock-depleted reaction, without added His6-p31comet. (B) XEEs depleted of p31comet, Mad2 or both proteins were subjected to analysis as in (A). (C) Cyclin B levels were quantified for reactions as described in Panel A and B, and normalized relative to the initial level at time = 0. Values represent the mean ± SD derived from 3 independent assays.
	Figure 2. IKK-β phosphorylates Xenopus p31comet. (A) Samples from cycling XEEs with 1,000 DSN/µl were collected at intervals after shift to 23°C. GST-p31comet beads and [γ−32P]ATP were added to an aliquot of the sample for analysis of kinase activity as described in Methods (top panel, bottom histogram). In parallel, another aliquot was directly analyzed by Western blotting for Cyclin B (second panel). The arrow indicates32P-labeled GST-p31comet. The stage of the cycling XEE is shown above (I: interphase, M: mitosis). (B) GST-p31comet or GST were incubated with ATP-γ-S and Aurora B, Mps1 or IKK-β. Samples from each reaction were subject to Western blotting using an anti-thiophosphate ester specific antibody (upper panel) and to CBB staining (lower panel). Black arrows within the upper panel indicate bands resultant from kinase autophosphorylation. Arrow to the right of the upper panel indicates phosphorylated GST-p31comet. (C) CSF-XEE containing [γ−32P]ATP and either DMSO or TCPA (final concentration 300 µM) were incubated with GST-p31comet or Histone H1. Each sample was subjected to SDS-PAGE and autoradiography (upper panels. Arrows indicate phosphorylated p31comet or Histone H1) and to Western blotting to assure equal loading (lower panels). (D) Samples as in panel A were subjected to Western blotting using phospho-specific antibody (anti-p31comet-S4p,T6p). Positive control (+ve) lane contains in vitro phosphorylated GST-p31comet as in Figure S3A, except ATP was used instead of [γ−32P]ATP. Negative control (-ve) lane includes untreated purified GST-p31comet.
	Figure 3. Phosphomimetic p31comet accelerates M-phase exit. (A) 15 nM GST-tagged p31comet-wt, p31comet-AAA or p31comet-EEE were added to p31comet-depleted CSF-XEE containing DSN. The reactions were incubated for 20 min. at 23°C, followed by CaCl2 addition. Samples were taken at the indicated times after CaCl2 addition (in minutes), and subjected to Western blotting with anti-Cyclin B (top panel), anti-GST (second panel), anti-p31comet (third panel) or anti-CENP-A (bottom panel, loading control). Graph shows Cyclin B levels, which were quantified at each point, and normalized relative to initial levels at time = 0. Values represent the mean ± SD derived from 3 independent assays. (B) 75 nM His-tagged p31comet-wt, p31comet-AAA or p31comet-EEE were added to p31comet-depleted CSF-XEE containing DSN, and, where indicated, 60 µM nocodazole (Noc.). The reactions were incubated for 30 min. at 23°C, followed by CaCl2 addition. Cyclin B destruction was monitored as in Panel A.
	Figure 4. p31comet phosphorylation by IKK-β enhances Mad2 association. (A) 50 nM recombinant His-tagged p31comet-wt, p31comet-AAA or p31comet-EEE were incubated with 100 nM recombinant Xenopus Mad2L12A. Mad2L12A was immunoprecipitated using anti-Mad2 antibodies coupled to protein A Agarose beads. Mad2L12A and co-precipitating p31comet were detected by CBB staining. The level of each p31comet variant was quantified and normalized to the amount of Mad2L12A. The relative yield of p31comet-AAA or p31comet-EEE was compared to the yield of p31comet-wt; the ratio of each mutant to the wild type protein is shown (lower graph). Values represent the mean ± SD from three independent assays. (B) His-tagged p31comet-wt, p31comet-AAA or p31comet-EEE were added at increasing concentrations (3× = 45 nM, 6× = 90 nM, 12× = 180 nM) to XEEs containing DSN and nocodazole. After 30 min. at 23°C, chromatin was removed by pelleting, and MCC was isolated from the soluble fraction by immunoprecipitation using anti-Cdc20 antibodies. Co-precipitating Mad2 was assayed by Western blotting. After quantification, Mad2 signals at each concentration for every p31comet variant were normalized to the samples without p31comet addition, as shown in the graph below. Values represent the mean ± SD from three independent assays. (C) Reactions were assembled as in (B), with increasing concentrations of His-tagged p31comet-wt, p31comet-AAA or p31comet-EEE (1× = 15 nM, 5× = 75 nM, 10× = 150 nM). After 30 m in. at 23°C, chromosomes were removed by pelleting and washed. The chromatin fraction was subjected to SDS-PAGE and Western blotting with the indicated antibodies. After quantification, Mad2 signals at each concentration for every p31comet variant were normalized to the samples with 1× p31comet (lower graph). Values represent the mean ± SD from three independent assays.
	Figure 5. IKK-β loss compromises SAC silencing. (A) DMSO or TPCA (final concentration 300 µM) were added to CSF-XEE with DSN. The reactions were incubated for 10 min. at 23°C before CaCl2 addition. Samples were taken at indicated times after CaCl2 addition, and subjected to SDS-PAGE and Western blotting with anti-Cyclin B and -CENP-A (loading control) antibodies. The graph shows Cyclin B levels, which were quantified in each reaction and normalized relative to the initial level at time = 0. Values represent the mean ± SD derived from 3 independent assays. (B) XEEs were depleted of IKK-β, or both IKK-β and Mad2. Reactions were carried out as in Panel A, and samples were subjected to SDS-PAGE and Western blotting with the indicated antibodies. The graph shows Cyclin B levels, which were quantified in each reaction and normalized relative to the initial level at time = 0. Values represent the mean ± SD derived from 3 independent assays. ΔIgG lanes show samples from a mock-depleted reaction.

Arnaoutov, The Ran GTPase regulates kinetochore function. 2003, Pubmed, Xenbase

Arnaoutov, The Ran GTPase regulates kinetochore function. 2003, Pubmed , Xenbase
Blazkova, The IKK inhibitor BMS-345541 affects multiple mitotic cell cycle transitions. 2007, Pubmed
Boyarchuk, Bub1 is essential for assembly of the functional inner centromere. 2007, Pubmed , Xenbase
Choi, Phosphorylation propels p31(comet) for mitotic exit. 2015, Pubmed
Date, Phosphorylation regulates the p31Comet-mitotic arrest-deficient 2 (Mad2) interaction to promote spindle assembly checkpoint (SAC) activity. 2014, Pubmed
Dobles, Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. 2000, Pubmed
Fava, Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint. 2011, Pubmed
Foley, Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore. 2013, Pubmed
Habu, p31(comet) inactivates the chemically induced Mad2-dependent spindle assembly checkpoint and leads to resistance to anti-mitotic drugs. 2013, Pubmed
Hagan, p31(comet) acts to ensure timely spindle checkpoint silencing subsequent to kinetochore attachment. 2011, Pubmed
Hegemann, Systematic phosphorylation analysis of human mitotic protein complexes. 2011, Pubmed
Irelan, A role for IkappaB kinase 2 in bipolar spindle assembly. 2007, Pubmed
Jia, Tracking spindle checkpoint signals from kinetochores to APC/C. 2013, Pubmed
Jia, Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31(comet). 2011, Pubmed
Kitagawa, Components of the spindle-assembly checkpoint are essential in Caenorhabditis elegans. 1999, Pubmed
Landais, Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors. 2009, Pubmed , Xenbase
Lara-Gonzalez, The spindle assembly checkpoint. 2012, Pubmed
Ledoux, NF-κB and the cell cycle. 2014, Pubmed
Ma, Depletion of p31comet protein promotes sensitivity to antimitotic drugs. 2012, Pubmed
Matsuoka, ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. 2007, Pubmed
Mistry, NF-kappaB promotes survival during mitotic cell cycle arrest. 2004, Pubmed
Murray, Cell cycle extracts. 1991, Pubmed
Rosette, Cytoskeletal control of gene expression: depolymerization of microtubules activates NF-kappa B. 1995, Pubmed
Scott, Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae. 2005, Pubmed
Teichner, p31comet Promotes disassembly of the mitotic checkpoint complex in an ATP-dependent process. 2011, Pubmed
Westhorpe, p31comet-mediated extraction of Mad2 from the MCC promotes efficient mitotic exit. 2011, Pubmed
Xia, Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint. 2004, Pubmed , Xenbase
Yang, p31comet blocks Mad2 activation through structural mimicry. 2007, Pubmed