Journal: Heliyon

Article Title: Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis

doi: 10.1016/j.heliyon.2016.e00131

Figure Lengend Snippet: LATS1 and LATS2 directly phosphorylate Ser894 in the TSS motif of INCENP in vitro . ( A ) Schematics of human INCENP and alignment of primary sequences around the TSS motif of INCENP from various organisms. The conserved TSS motifs of human and mouse INCENP contain a putative LATS phosphorylation site (S894 in human numbering). Red and green letters indicate putative phosphorylated amino acids of LATS and Aurora-B, respectively. Hs, human; Mm, mouse; Gg, chicken; Xl, frog; Ce, nematode; Dm, fruit fly; Sp, fission yeast; Sc, budding yeast. ( B ) In vitro kinase assays were performed with immunocomplexes of either 6Myc-LATS1 or LATS2 (kinases), 3FLAG-MOB1A (kinase activator), and purified GST-fused INCENP-C (amino acids 884–899)-WT or -S894A protein (substrate) in the presence of [γ- 32 P] ATP. Phosphorylation of LATS1, LATS2 (p-LATS1/2), and the C-terminus of INCENP (p-INCENP) was detected by autoradiography ( 32 P). Simply Blue staining was used as a loading control. WT and KD indicate wild-type and kinase dead, respectively. ( C ) Dot-blot analysis with two kinds of anti-pS894 polyclonal antibodies (#V5543 and #V5544). Phosphorylated and non-phosphorylated S894 peptides were blotted on PVDF membranes at the indicated concentrations, followed by western blotting. ( D ) In vitro LATS1/2-kinase assays were performed as in (B), except for the presence of [γ- 32 P] ATP. Phosphorylation of INCENP was assessed by western blotting with anti-pS894, anti-GST, and anti-Myc antibodies. Vec, vector; L1, LATS1; L2, LATS2. See Fig. S1 for uncropped gel images of SDS-PAGE (autoradiography and Simply Blue staining) in B and western blot in D. Black boxes indicate the cropped regions. Molecular sizes (kDa) are based on prestained protein markers.

Article Snippet: For A, a series of GST-INCENP-C was incubated with 1 μg of active Aurora-B kinase and/or 160 ng of active GST-LATS2 kinase (Carna Bioscience, Kobe, Japan).

Techniques: In Vitro, Purification, Autoradiography, Staining, Dot Blot, Western Blot, Plasmid Preparation, SDS Page

Journal: Heliyon

Article Title: Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis

doi: 10.1016/j.heliyon.2016.e00131

Figure Lengend Snippet: Influence of Thr892 and Ser893 phosphorylation by Aurora-B on phosphorylation of INCENP-Ser894. ( A ) In vitro kinase assays were performed using recombinant LATS2 and/or Aurora-B and purified GST-fused INCENP-C-WT, -T892A, -S893A, -T892A/S893A (AAS), or -T892A/S893A/S894A (AAA) protein as a substrate, followed by western blotting with the indicated antibodies. Bar graphs show the relative levels of pS894 normalized against the corresponding band intensity of GST-INCENP. ( B ) In vitro kinase assays were performed using recombinant Aurora-B-WT (wild-type) or KD (kinase dead) as the enzyme and purified GST-fused INCENP-C-WT, -T892A, -S893A, -T892A/S893A (AAS), or -T892A/S893A/S894A (AAA) protein as the substrate, followed by western blotting with the indicated antibodies. ( C ) Proposed model for the influence of phosphorylation of neighboring sites (T892 and S893) on S894 phosphorylation in the presence of Aurora-B alone (i), LATS alone (ii), LATS alone + INCENP with T892A/S893A mutations (iii), and both Aurora-B and LATS (iv). See Fig. S2 for uncropped western blot images in A, B. Black boxes indicate the cropped regions. Molecular sizes (kDa) are based on prestained protein markers.

Article Snippet: For A, a series of GST-INCENP-C was incubated with 1 μg of active Aurora-B kinase and/or 160 ng of active GST-LATS2 kinase (Carna Bioscience, Kobe, Japan).

Techniques: In Vitro, Recombinant, Purification, Western Blot

Journal: Heliyon

Article Title: Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis

doi: 10.1016/j.heliyon.2016.e00131

Figure Lengend Snippet: Validation of S894 phosphorylation on endogenous INCENP in vivo . ( A ) HEK293T cells were co-transfected with EGFP-tagged LATS2 and either 6Myc-tagged INCENP-WT, -S894A, or vector alone (vec), and then treated with nocodazole (Noc) and taxol (Tax). Western blotting of these cell lysates was performed with anti-pS894, anti-Myc, and anti-EGFP antibodies. ( B ) HeLa-S3 cell lysates were treated with λPPase (200 U) with or without protein phosphatase inhibitors. (-) indicates lysates incubated with neither λPPase nor protein phosphatase inhibitors, followed by western blotting with the indicated antibodies. Cyclin B1 is a mitotic marker. α-tubulin is a loading control. Arrowheads indicate bands corresponding to pS894-INCENP. ( C ) Proteins extracted from HeLa-S3 cells in TNE250 lysis buffer with or without protein phosphatase inhibitors were treated with or without λPPase (200 U), followed by western blotting with the indicated antibodies. Arrowheads indicate the bands corresponding to pS894-INCENP. See Fig. S3 for uncropped western blot images in A–C. Black boxes indicate the cropped regions. Molecular sizes (kDa) are based on prestained protein markers.

Article Snippet: For A, a series of GST-INCENP-C was incubated with 1 μg of active Aurora-B kinase and/or 160 ng of active GST-LATS2 kinase (Carna Bioscience, Kobe, Japan).

Techniques: In Vivo, Transfection, Plasmid Preparation, Western Blot, Incubation, Marker, Lysis

Journal: Heliyon

Article Title: Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis

doi: 10.1016/j.heliyon.2016.e00131

Figure Lengend Snippet: LATS and Aurora-B kinases phosphorylate Ser894 of INCENP in vivo . LATS2 -knockout HeLa-S3 cells were transfected with siRNAs targeting LATS1 (siLATS1) and INCENP (siINCENP), and then treated with nocodazole (lanes 1–5 and 11–15) and taxol (lanes 6–10 and 16–20) in the presence or absence of Hesperadin (Hesp). siControl is a universal scrambled negative control siRNA duplex. The intensity of the pS894-specific band was reduced by knockout of LATS2 alone, and further reduced by knockdown of LATS1 or INCENP in LATS2 -knockout cells (arrowhead in upper panels). The level of pS894 was normalized against the level of α-tubulin or INCENP. A double asterisk shows the degradation products of pS894-INCENP. These intensities were substantially reduced by additional treatment with Hesperadin (lanes 11–20). The slower-migrating bands of INCENP were converted to faster bands upon depletion of LATS1 and LATS2 (second panels from top). See Fig. S3 for uncropped western blot images in . Black boxes indicate the cropped regions. Molecular sizes (kDa) are based on prestained protein markers.

Article Snippet: For A, a series of GST-INCENP-C was incubated with 1 μg of active Aurora-B kinase and/or 160 ng of active GST-LATS2 kinase (Carna Bioscience, Kobe, Japan).

Techniques: In Vivo, Knock-Out, Transfection, Negative Control, Western Blot

Journal: Heliyon

Article Title: Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis

doi: 10.1016/j.heliyon.2016.e00131

Figure Lengend Snippet: LATS2 -knockout HeLa-S3 cells exhibit multinucleated phenotype, telophase delay, and chromosome missegregation with lagging chromosomes. ( A ) Exponentially growing LATS2 -knockout (LATS2 KO) HeLa-S3 cells and parental HeLa-S3 cells were fixed, and immunostained with anti–α-tubulin antibody (red) and Hoechst 33258 (gray or blue). Scale bar, 10 μm. ( B ) Percentage of cells with the indicated nuclear number in LATS2 KO (black) and parental (gray) HeLa-S3 cells. Data represent the average of ∼200 cells from three independent experiments. ( C ) Percentage of mitotic cells (including cells in prometaphase, metaphase, anaphase, and telophase) in asynchronous LATS2 KO and parental HeLa-S3 cells (blue bars). Data represent the average of ∼240 cells from three independent experiments. ( D ) LATS2 KO and parental HeLa-S3 cells were fixed 1 h after release from nocodazole arrest, immunostained with anti–α-tubulin antibody and Hoechst 33258, and counted by microscopy. Graphs show the percentage of mitotic cells at the indicated stage. Data represent the average of ∼80 mitotic cells from three independent experiments. ( E ) Percentage of cells with a chromosome bridge or lagging chromosomes during anaphase. LATS2 KO and parental HeLa-S3 cells were synchronized, stained, and counted as in D. Data represent the average of ∼40 anaphase cells from three independent experiments. ( F ) Percentage of cells undergoing multipolar division during telophase. LATS2 KO and parental HeLa-S3 cells were synchronized, stained, and counted as in D. Data represent the average of ∼30 telophase cells from three independent experiments. ( G ) Percentage of cells with multipolar spindles during metaphase. LATS2 KO and parental HeLa-S3 cells were synchronized, stained, and counted as in D. Data represent the average of ∼40 metaphase cells from three independent experiments.

Article Snippet: For A, a series of GST-INCENP-C was incubated with 1 μg of active Aurora-B kinase and/or 160 ng of active GST-LATS2 kinase (Carna Bioscience, Kobe, Japan).

Techniques: Knock-Out, Microscopy, Staining

Journal: Heliyon

Article Title: Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis

doi: 10.1016/j.heliyon.2016.e00131

Figure Lengend Snippet: A proposed model for the phospho-regulation of INCENP by LATS1 and LATS2 during cytokinesis.

Article Snippet: For A, a series of GST-INCENP-C was incubated with 1 μg of active Aurora-B kinase and/or 160 ng of active GST-LATS2 kinase (Carna Bioscience, Kobe, Japan).

Techniques: