A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling
- Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
Phosphorylation of Bub1 S459 is critical for spindle checkpoint activation.
(A) Domains and motifs of Bub1 and sequence alignment of its conserved motif (CM). TPR, tetratricopeptide repeat; GLEBS, Gle2-binding sequence; Phe, phenylalanine-containing motif, also known as …
Figure 1—figure supplement 1
Identification of mitotic phosphorylation sites in human Bub1 and characterization of the Bub1 S459A mutant.
(A) Domain structure of human Bub1 and a summary of all 30 phosphorylation sites identified by mass spectrometry. Phosphorylated residues are denoted in red. (B) Lysates of cells in Figure 1D were …
Figure 2 with 1 supplement
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 …
Figure 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. …
Figure 3
The Bub1–Mad1 interaction is crucial for checkpoint activation in human cells.
(A) HeLa cells expressing Myc-Bub1 transgenes were treated with nocodazole and MG132 in the presence or absence of reversine. Myc-Bub1 proteins were immunoprecipitated and blotted with the indicated …
Figure 4 with 1 supplement
The Bub1–Mad1 complex promotes APC/CCdc20 inhibition by MCC components.
(A) Flow charts of the in vitro reconstitution of Mps1-stimulated APC/C inhibition by MCC components. The incubation times of each reaction step are indicated. All processes were performed at room …
Figure 4—figure supplement 1
Characterization of monomeric and dimeric Mad2 in the APC/CCdc20 inhibition assay.
(A) UV trace of recombinant purified Mad2 fractionated on a Resource Q column. The first peak belongs to the Mad2 monomer (Mad2Mono), whereas the second peak contains the Mad2 dimer (Mad2Di). (B) …
Figure 5 with 1 supplement
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 …
Figure 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 …
Figure 6
Phosphorylation of Mad1 T716 promotes its binding to Cdc20.
(A) Domains and motifs of Cdc20. C box, a conserved APC/C-binding motif; MIM, Mad2-interacting motif; BM1, basic motif 1 (27RWQRK31); BM2, basic motif 2 (54RTPGRTPGK62). (B) In vitro pull-down of …
Figure 7
A sequential multi-target phosphorylation cascade by Mps1 promotes the assembly and activation of the Bub1–Mad1 scaffold.
(A) Mps1 recognizes unattached kinetochores (KT) through its direct binding to the Ndc80 complex (Ndc80C). At kinetochores, Mps1 first phosphorylates Knl1 at multiple MELT motifs to recruit the …
