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Figure 5. Phosphorylation of Mad1 T716 by Mps1 promotes MCC assembly and checkpoint signaling.(A) Schematic drawing of the assay examining MCC assembly facilitated by Mps1-phosphorylated Mad1–C-Mad2. (B) MCC assembly and APC/C inhibition were performed as depicted in (A). The ubiquitination reaction mixtures were resolved on SDS-PAGE and blotted with the anti-Myc antibody that detected Myc-Securin. The slow-migrating species represented the poly-ubiquitinated forms of Securin. FL, full-length. Cdc20ΔN60 lacks residues 1–60. (C) Recombinant human Mad1E–Mad2 complex was incubated with Mps1 in the presence or absence of reversine. The reactions were resolved on SDS-PAGE and stained with Coomassie. Note that Mps1 underwent autophosphorylation in the absence of reversine. (D) Cartoon drawing of the crystal structure of human Mad1CTD (PDB ID: 4DZO) with the RLK motif, T644, and T716 shown in sticks. (E) Mitotic indices of HeLa cells expressing the indicated Mis12–Mad1 fusion proteins. Error bars, s.d. (n = 5 independent experiments). ****p<0.0001; Student’s t-test. (F) HeLa cells expressing Myc-Mad1 MIM/5A or MIM/5A;T716A were synchronized with thymidine and released into nocodazole-containing medium. MIM/5A, mutant with the Mad2 Interacting Motif replaced by alanine. Because overexpression of Mad1 inactivates the spindle checkpoint, we used Mad1 MIM/5A to prevent cells from escaping nocodazole-mediated mitotic arrest. Myc-Mad1 proteins were immunoprecipitated and blotted with indicated antibodies. (G) The MCC mixtures were prepared as depicted in (A) with the indicated Mad1E proteins, and then applied to the APC/C-dependent ubiquitination assay. The ubiquitination reaction mixtures were resolved on SDS-PAGE and blotted with the anti-Myc antibody that detected Myc-Securin. The slow-migrating species represented the poly-ubiquitinated forms of Securin. In reactions containing reversine, reversine was added prior to the addition of ATP.DOI:
http://dx.doi.org/10.7554/eLife.22513.009Figure 5—figure supplement 1. Characterization of Mad1 phosphorylation and its function in APC/C inhibition by MCC components.(A) Summary of all 15 Mps1-phosphorylation sites of Mad1E identified by mass spectrometry. Phosphorylated residues are depicted in red. (B) Lysates of cells in Figure 5E were blotted with anti-Mad1 and anti-Tubulin antibodies. (C) In vitro kinase assays of recombinant Mad1E–Mad2 WT or indicated mutants treated with Mps1 in the absence or presence of ATP. YF-13A-T716, Mad1 mutant with all phosphorylation sites except T716 mutated to phenylalanine or alanine. The kinase reactions were resolved on SDS-PAGE and blotted with indicated antibodies. pT716, a phospho-specific antibody recognizing phospho-T716 of Mad1. (D) The MCC mixtures were prepared as depicted in Figure 5A with the indicated Mad1E proteins, and then applied to the APC/C ubiquitination assay. The ubiquitination reaction mixtures were resolved on SDS-PAGE and blotted with the anti-Myc antibody that detected Myc-Securin. The slow-migrating species represented the poly-ubiquitinated Securin. YF-5A, Y535F/S538A/T540/S546A/T550A/S551A; YF-14A, mutant with all Mps1 phosphorylation sites of Mad1E mutated to phenylalanine or alanine.DOI:
http://dx.doi.org/10.7554/eLife.22513.010 Image published in: Ji Z et al. (2017) © 2017, Ji et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |