Protein interaction domain mapping of human kinetochore protein Blinkin reveals a consensus motif for binding of spindle assembly checkpoint proteins Bub1 and BubR1 - PubMed (original) (raw)

Protein interaction domain mapping of human kinetochore protein Blinkin reveals a consensus motif for binding of spindle assembly checkpoint proteins Bub1 and BubR1

Tomomi Kiyomitsu et al. Mol Cell Biol. 2011 Mar.

Abstract

The kinetochore is a supramolecular structure essential for microtubule attachment and the mitotic checkpoint. Human blinkin/human Spc105 (hSpc105)/hKNL1 was identified originally as a mixed-lineage leukemia (MLL) fusion partner and later as a kinetochore component. Blinkin directly binds to several structural and regulatory proteins, but the precise binding sites have not been defined. Here, we report distinct and essential binding domains for Bub1 and BubR1 (here designated Bubs) at the N terminus of blinkin and for Zwint-1 and hMis14/hNsl1 at the C terminus. The minimal binding sites for Bub1 and BubR1 are separate but contain a consensus KI motif, KI(D/N)XXXF(L/I)XXLK. RNA interference (RNAi)-mediated replacement with mutant blinkin reveals that the Bubs-binding domain is functionally important for chromosome alignment and segregation. We also provide evidence that hMis14 mediates hNdc80 binding to blinkin at the kinetochore. The C-terminal fragment of blinkin locates at kinetochores in a dominant-negative fashion by displacing endogenous blinkin from kinetochores. This negative dominance is relieved by mutations of the hMis14 binding PPSS motif on the C terminus of blinkin or by fusion of the N sequence that binds to Bub1 and BubR1. Taken together, these results indicate that blinkin functions to connect Bub1 and BubR1 with the hMis12, Ndc80, and Zwint-1 complexes, and disruption of this connection may lead to tumorigenesis.

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Figures

FIG. 1.

FIG. 1.

Mitotic associations between the hMis12 complex and Ndc80 were abolished by the mutation of the HP1-binding site on hMis14. (A) Summary cartoon of hMis12 complex-interacting proteins in interphase and mitosis (17). (B and C) FLAG-hMis12 was immunoprecipitated from asynchronous (B) and TN16-treated (C) mitotic extracts and was subsequently dissociated by a 3× FLAG peptide. The solubilized protein complexes, which coprecipitated with FLAG-hMis12, were fractionated by a Superose 6 HR 10/30 size-exclusion chromatography column and immunoblotted by the antibodies indicated. The migrations of the molecular mass marker are indicated above the blots (in kilodaltons). (D) GFP-hMis12-expressing cells (green) were fixed and stained with anti-Ndc80 antibody (red) and Hoechst 33342 (blue). Interphase (Int), prophase (Pro), and prometaphase (PM) cells are shown. (E) Immunoprecipitation of GFP-tagged hMis14 wild-type (WT) and hMis14 m2E mutant proteins. Cultures of HeLa cells that stably expressed GFP-tagged hMis14 WT and m2E were used. The expression of GFP-hMis14 m2E was repressed in a subpopulation of the cells. Equal amounts of GFP-hMis14 WT and m2E were applied in the immunoprecipitation lanes. Cells were treated with nocodazole for 18 h. Input and immunoprecipitates (IP) were immunoblotted using the antibodies indicated.

FIG. 2.

FIG. 2.

Identification of the Zwint-1- and hMis14-binding regions in blinkin. (A) The BLKC mutant constructs are shown. The Zwint-1- and hMis14-binding regions are shown in dark purple and orange, respectively. (B) Y2H interaction between BLKC mutants and Zwint-1, hMis14, or hMis13. Plasmids pGBD and pGAD carried the DNA-binding and -activating domains of yeast GAL4, respectively. (C) Amino acid sequences of BLKC-4 (aa 1981 to 2316). Highly conserved amino acids and similar amino acids are boxed in dark and light purple, respectively. Amino acids indicated by red triangles are replaced by alanine in BLKC-4 mutants. (D) Y2H interaction between BLKC-4 mutants and Zwint-1 or hMis14.

FIG. 3.

FIG. 3.

The overexpression of the BLKC fragment causes chromosome misalignment and mitotic arrest. (A) TRex-HeLa chromosomally integrated GFP-BLKC was treated with (+) or without (−) tetracycline (tet), and extracts were immunoblotted using the antibodies indicated. The loading control was tubulin (TUB). (B) Immunoblot of GFP, histone H3S10ph, and tubulin after treatment with tet (left). Frequency of nonflat cells determined by phase-contrast microscopy was measured (n > 1,000 cells, right). (C) TRex-HeLa chromosomally integrated GFP-BLKC was treated with tet (+ tet) or without (− tet) for 24 h and fixed with cold ethanol. After RNase treatment, cells were stained with PI (50 μg/ml) and analyzed by flow cytometry. The G2/M population was measured by counting 20,000 cells. (D) Immunoblot of BLKC and TUB with serial dilutions of BLKC-GFP extract. Antibody against the C-terminal region of blinkin was used to detect both endogenous blinkin and BLKC-GFP. One and two asterisks indicate endogenous blinkin and BLKC-GFP, respectively. (E) Cells were incubated for 24 h with (+) or without (−) tetracycline (tet) and stained with the antibodies indicated. Cells without tet treatment were stained as the control.

FIG. 4.

FIG. 4.

Dominant-negative effect of BLKC. (A and B) TRex-HeLa chromosomally integrated GFP-BLKC was incubated for 24 h with (+) or without (−) tetracycline (tet) and stained with the antibodies indicated. Antiblinkin antibody recognizes the N terminus of blinkin; thus, it stains endogenous blinkin but not BLKC. Bars, 10 μm. (C) Model of the dominant-negative effect by BLKC. See details in the text.

FIG. 5.

FIG. 5.

Blinkin RNAi abolishes the mitotic arrest by BLKC expression. (A) TRex-HeLa chromosomally integrated GFP-BLKC was treated with tet and blinkin siRNA as indicated. Extracts were obtained 48 h after treatment and immunoblotted using the antibodies indicated. The loading control was tubulin. (B and C) Cells were fixed 48 h after the treatment of tet and blinkin siRNA and stained with antibodies as indicated. (D) Time-lapse micrographs of GFP-BLKC-expressing TRex-HeLa cells after control and blinkin RNAi treatment. TRex-HeLa without integration was treated with the control and BubR1 RNAi without the addition of tet. DNA was stained with Hoechst 33342. Numbers indicates the time (in minutes) after nuclear envelope breakdown (NEBD). Bars, 10 μm. (E) Frequency of round cells determined by phase-contrast microscopy was measured 18 h after nocodazole treatment. Each cell line was treated by siRNA and tet as indicated. Bars indicate standard deviation. Control, 60.6 ± 8.8 (n = 2,060 cells); BubR1, 16.8 ± 2.0 (n = 1,484); Blinkin+tet, 27.0 ± 1.3 (n = 1,801).

FIG. 6.

FIG. 6.

The BLKC mutant deficient in hMis14 binding suppresses the dominant-negative effect by BLKC. (A) The BLKC mutant constructs chromosomally integrated in Flip-In TRex 293 cells are shown. A gray X indicates m7 and m15 mutation sites. (B) Each cell line was treated with tet to express GFP-tagged BLKC constructs. Extracts were obtained 24 h after tet treatment and immunoblotted by antibodies as indicated. Nonintegrated cells were used as controls. (C) Each cell line was fixed and stained by Hoechst 33342 and antiblinkin and anti-CENP-C antibodies 24 h after tet treatment. Bar, 10 μm. (D) Each cell line was cultured for 12 h with tet and for an additional 18 h after the addition of nocodazole. Extracts were immunoprecipitated with anti-GFP antibodies. Input and immunoprecipitates were immunoblotted using the antibodies indicated.

FIG. 7.

FIG. 7.

Identification of minimal Bub1- and BubR1-binding sites in blinkin. (A) Blinkin contains the conserved motifs [S/G]ILK (light blue), RRVSF (blue), MELT repeat (red), and coiled coil (black). Three regions of blinkin, BLKN (aa 1 to 728), BLKM (aa 729 to 1833), and BLKC (aa 1834 to 2316), and 150-aa fragments are shown in the schematic. (B) Repeat sequences of human blinkin. Highly conserved amino acids and similar amino acids are boxed in black and gray, respectively. Number indicates the amino acid position of blinkin. (C) Yeast two-hybrid interaction between 150-aa fragments of blinkin and Bubs. For the control, p53 and simian virus 40 (SV40) T antigen were used. (D) Y2H interaction between blinkin151-300 mutants and Bub1 or BubR1. A MDLT motif on blinkin151-300 was replaced by alanine (4A) or deleted (ΔMDLT). (E) Y2H analysis between 50-aa fragments of blinkin151-300 and Bubs. Bub1- and BubR1-binding fragments are shown in purple and green, respectively. (F) Y2H assay between BLKN deletion mutants and Bub1 or BubR1.

FIG. 8.

FIG. 8.

Identification of the Bubs-binding consensus motif in blinkin. (A) Amino acid sequences of Bub1- and BubR1-binding regions in putative blinkin family members in Homo sapiens (GenBank accession no. NP_653091), Rattus norvegicus (XP_230465.2), Mus musculus (NM_029617), Gallus gallus (XM_420938), and Danio rerio (XM_001921844). Conserved amino acids and similar amino acids are highlighted in dark and light purple, respectively. Essential amino acids for the interaction with Bubs identified by a Y2H assay are indicated by red circles. The Bubs recognition KI motif, KI(D/N)XXXF(L/I)XXLK, is enclosed in a red rectangle. (B) Y2H analysis between blinkin151-250 mutants and Bub1 or BubR1. Amino acids replaced by alanine are indicated. The 50-aa fragments of blinkin151-200 and blinkin201-250 are shown in purple and green, respectively.

FIG. 9.

FIG. 9.

BLKN fused to BLKC suppresses the dominant-negative effect by BLKC. (A) The blinkin mutant constructs chromosomally integrated in Flip-In TRex 293 cells are shown. Minimal Bub1- and BubR1-binding regions identified by a Y2H assay are indicated in purple and green, respectively. (B) Each cell line was treated by tet to express GFP-tagged blinkin constructs. Extracts were obtained 24 h after the addition of tet and immunoblotted by antibodies as indicated. Nonintegrated cells was used as controls. (C) Each cell line was fixed and stained by Hoechst 33342 and anti-TUB and anti-CENP-C antibodies 24 h after the tet treatment. (D) Each cell line was fixed and stained by Hoechst 33342, anti-blinkin(M), which specifically recognizes the middle region of blinkin, and anti-CENP-A antibodies 24 h after the tet treatment. (E) Time-lapse micrographs of each cell from 24 h after the addition of tet are shown. Hoechst 33342 was used for DNA staining. The number indicates the time (in minutes). Bars, 10 μm. (F) Summary of the phenotypes in four different cell lines. (G) Each cell line was cultured for 12 h after tet treatment and for an additional 18 h after the addition of nocodazole. Extracts were immunoprecipitated with anti-GFP antibodies. Input and immunoprecipitates (IP) were immunoblotted using the indicated antibodies.

FIG. 10.

FIG. 10.

Deletion of the minimal Bubs-binding domain in BLKN+BLKC fails to suppress chromosome misalignment after blinkin RNAi treatment. (A) Schematic of experimental procedures. Each cell line was synchronized using a double-thymidine block protocol. Tetracycline (tet) and siRNA were added at the indicated time points. (B) Each cell line was treated with tet to express GFP-tagged blinkin constructs. Extracts were obtained 23 h after the addition of tet and immunoblotted by antibodies as indicated. Nonintegrated cells were used as controls. (C) Summary of the phenotypes of five different RNAi cells. Chr., chromosomes. (D) Time-lapse micrographs of each cell in control RNAi or blinkin RNAi are shown. Hoechst 33342 was used for DNA staining. Misaligned and lagging chromosomes are indicated by the red and yellow arrows, respectively. The number indicates the time (in minutes). Bar, 10 μm.

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