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Figure 2. Sequential phosphorylation of human Bub1 by Cdk1 and Mps1 enhances its binding to Mad1.(A) Domains and motifs of Mad1. Schematic domain structures and tested fragments of Mad1 protein. CTD, C-terminal domain; MIM, Mad2-interacting motif; RLK, the arginine-leucine-lysine motif. The E fragment of Mad1 containing both MIM and CTD was used to make the Mad1–Mad2 complex in this study. (B) In vitro pull-down of Mad1CTD using empty beads or beads conjugated to the indicated Bub1 peptides. Proteins bound on beads were separated on SDS-PAGE and visualized by Coomassie blue staining. Relative band intensities were quantified and indicated below the gel. (C) Isothermal titration calorimetry (ITC) assays of binding between the C-terminal domain (CTD) of human Mad1 and the human Bub1 peptides containing either phospho-T461 alone or both phospho-S459 and phospho-T461. Kd, dissociation constant. (D) In vitro kinase assays of recombinant Bub1ΔK–Bub3 WT or S459A/T461A (SATA) treated with Cdk1 or Mps1 or both. The kinase reactions were resolved on SDS-PAGE and blotted with indicated antibodies. pSpT, a phospho-specific antibody recognizing both phospho-S459 and phospho-T461. (E) In vitro kinase assays similar to (D), except that the kinases were added in the indicated orders. In lanes 1 and 2, Cdk1 or Mps1 was first incubated with the substrate for 30 min before being inhibited by RO3306 or reversine, respectively. (F) Schematic drawing of the sequential phosphorylation of Bub1 at S459 and T461 by Cdk1 and Mps1. (G) Bub1ΔK–Bub3 WT and SATA were first phosphorylated by both Cdk1 and Mps1, and then assayed for binding to GST-Mad1E–Mad2 beads. The bound proteins were blotted with the indicated antibodies.DOI:
http://dx.doi.org/10.7554/eLife.22513.004Figure 2—figure supplement 1. Binding of scBub1 phosphorylated at T455 by Mps1 to scMad1 CTD.(A) In vitro pull-down of the scMad1E–scMad2 complex with beads bound to GST or the indicated GST-scBub1 fragments, which were expressed alone or co-expressed with the kinase domain of scMps1. Proteins bound to the beads were analyzed with SDS-PAGE and stained with Coomassie blue. (B) Isothermal titration calorimetry (ITC) assay of binding between Mps1-phosphorylated scBub1449–530 and the scMad1E–scMad2 complex or the C-terminal domain (CTD) of scMad1. Kd, dissociation constant. (C) In vitro pull-down of scMad1CTD WT and RLK/AAA with beads conjugated to the indicated scBub1 peptides. The phosphorylated residues of Bub1 peptides were denoted. hs, Homo sapiens. (D) ITC assays of binding between the C-terminal domain (CTD) of scMad1 and the scBub1 peptides containing either phospho-T455 alone or both phospho-T453 and phospho-T455. Kd, dissociation constant.DOI:
http://dx.doi.org/10.7554/eLife.22513.005 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 |