Supplemental Data



Supplemental Data

Tension sensitive Plk1 phosphorylation on BubR1 regulates the stability of kinetochore-microtubule interactions

Sabine Elowe, Stefan Hümmer, Andreas Uldschmid, Xiuling Li, and Erich A. Nigg

Supplemental Materials and Methods:

Plasmids and recombinant protein production

Human BubR1 was amplified from cDNA clone IMAGp958G021430Q2, and inserted in-frame into a pCDNA3.1 vector (Invitrogen) with either an N-terminal Flag or mCherry tag (Shaner et al. 2004). For expression of recombinant BubR1 in E. coli, the cDNA was cloned into the pMAL vector (New England Biolabs), with an additional C-terminal Hexa histidine tag (His-tag). Mutagenesis was performed using Quickchange site-directed mutagenesis (Stratagene) according to the manufacturer’s instructions. All constructs were verified by sequencing. Recombinant Plk1 expression has been previously described (Neef et al. 2003). Aurora B and Aurora A purified from SF9 insect cells were a kind gift of U. Klein and E. Chan, respectively. For recombinant Bub1, BubR1, and Mps1, the respective kinase domains were cloned into the pVL1393 baculovirus transfer vector (Pharmingen) with an N-terminal GST tag, and expressed in SF9 cells.

Antibodies and antibody Production

Monoclonal anti-Plk1 (Baumann et al. 2007), and anti-Mps1 (Stucke et al. 2002) antibodies have been previously described. Polyclonal anti-Plk1 (Abcam), anti-Aurora B (AIM-1), anti-Flag (Sigma), anti-p55Cdc20 (Santa Cruz), anti-Bub3 (BD Transduction labs), anti-Cyclin B1 (Biomol), anti-α-tubulin (DM1A, sigma), anti-Hec1 (GeneTex), anti-APC7 (BioLegend), and anti-RanGAP1 (Zymed), as well as CREST anti-human auto-immune serum (Immunovision), were commercially obtained. Rabbit anti-GST was a kind gift of Dr. U. Gruneberg. A BubR1 monoclonal antibody (IgG2b κ) was generated against a GST-tagged fragment of BubR1 encompassing amino acids 1-247 and a CenpE polyclonal antibody was raised against a recombinant His-tagged fragment (amino acids 1-483) expressed in E. coli. Anti-pS676 polyclonal antibody was generated by immunizing rabbits with KLH-conjugated phosphopeptide (H-CLSPIIEDpSREATH-OH), and then isolated from a protein-A purified IgG fraction using the same peptide. For immunofluorescence experiments, all primary anibodies were detected with Cy2/Cy3-conjugated donkey antibodies (Dianova) and Alexa Fluor 647-conjugated goat antibodies (Ivitrogen). For all Western blots, signals were detected using HRP-conjugated anti-mouse or anti-rabbit antibodies (Pierce).

Peptide array synthesis and spots blotting

Peptide arrays were constructed according to the Spots-synthesis method as per the manufacturer’s directions (Intavis). Briefly, acid-hardened cellulose membranes pre-derivatized with polyethylene glycol (AbiMed) were spotted with a grid of Fmoc [pic]-alanine (Bachem) prior to peptide synthesis. Peptides were synthesized by standard Fmoc chemistry using an AbiMed ASP422 robot. Following peptide synthesis and side-chain deprotection, membranes were blocked overnight in 5% skim milk. Purified GST-PBD WT or GST-PBD AA fusion proteins were added at 1 µg/ml in TBS and incubated for 2 h at 4 °C. Membranes were washed three times in TBS and bound GST fusion proteins were visualized by blotting with anti-GST antibodies. The following peptides were synthesised, in both non-phosphorylated and phosphorylated forms: BubR1-T620: ARFVSTPFHEIM, ARFVSpTPFHEIM; BubR1-S435: AELLTSAEKRAE, AELLTpSAEKRAE; and Poloboxtide: GPMQSTPLNGAA, GPMQSpTPLNGAA.

SILAC labeling with 13C6 15N4-L-arginine and 13C6 15N2-L-lysine

HeLa S3 cells were cultured in DMEM formulated with either unlabeled L-lysine or L-arginine or labeled 13C6 15N4 –L-arginine and 13C6 15N2-L-lysine (Cambridge Isotope Laboratories) at the concentration of 44 and 86 µg/ml respectively, and supplemented with 10% dialyzed fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. Unlabeled HeLa cells were synchronized in mitosis using a sequential thymidine block/release, nocodazole block protocol. Labeled HeLa cells were harvested directly after five cell divisions. Extracts prepared from 4 x 107 unlabelled nocodazole arrested cells and of 4 x 107 isotopically labelled thymidine blocked cells were mixed at a ratio of 1:1. This mixture was divided into equal parts, and immunoprecipitation was performed with anti-BubR1 monoclonal antibody or 9E10 anti-myc monoclonal antibody as a negative control. Samples were separated by SDS-PAGE and excised gel fragments were processed for mass spectrometry

Mass spectrometry and peptide identification

Peptide separation in nanoLC-MS/MS experiments was performed on a CAPLC system (Waters). Samples were loaded onto an AQUASIL C18 phase (Dr. Maisch GmbH) column and separated on a ReproSil-Pur C18-AQ reverse-phase material (Newobjective). Peptides were separated by a stepwise 60-min gradient of 0-100 % between buffer A (2 % acetonitrile, 0.5 % formic acid) and buffer B (80 % acetonitrile, 0.5 % formic acid) at a flow rate of 170 nl/min. The mass spectrometer was operated in data dependent MS/MS mode to automatically switch between 1.5 sec MS survey and 5.4 sec MS/MS fragmentations of the four most abundant precursor ions. Peak lists were generated using Mascot Distiller (MatrixScience) and searched against the human International Protein Index (IPI) database () with carbamidomethyl cysteine as a fixed modification and oxidized methionine, phosphorylation, 13C6 15N4-L-arginine, and 13C6 15N2-L-lysine as variable modifications. Searches were performed with precursor and fragment ion tolerances of 0.15 Da. Phosphopeptides identified with a score 15 or higher were considered significant. For all sequenced phosphopeptides, Mascot search hits and exact phosphorylation sites were confirmed by manually interpreting MS/MS ion spectra. For phosphopeptide quantification, the ratios between the monoisotopic peaks of labeled and unlabeled forms of phosphopeptides were calculated either by MSQuant () or visual examination.

Image processing, quantification, and statistical analysis

Images were taken at identical exposure times within each experiment, acquired as 8-bit RGB images, and processed in Adobe Photoshop. Images shown in the same panel have been identically scaled. Measurement of microtubule and kinetochore intensities was performed in ImageJ () on non-deconvolved images. Quantification of kinetochore intensities was performed as previously described (Lenart et al. 2007). Essentially, a circular region with fixed diameter was centered on each kinetochore, and unless indicated otherwise, CREST intensity was measured in the same region and used for normalization after subtraction of background intensity measured outside the cell. For Fig. 2A and Fig. 7C, the average of Z-stack projections was used for quantification. Similarly, α-tubulin intensity in Fig. 3C was normalized against the area of the cell, after subtraction of background intensity. Statistical significance of quantification experiments was verified by Student’s t-test using GraphPad software.

Elowe et al. Supplemental Figure legends.

Figure S1. BubR1 is displaced from the kinetochore after CenpE siRNA

(A) HeLa S3 cells were transfected with an siRNA duplex against CenpE or GL2 for 36 h and stained for BubR1 (green) and DAPI (blue). Scale bar = 10 µm. Histogram shows quantification of BubR1 intensity at kinetochores. Normalized BubR1 intensity from at least 60 kinetochores from 5 different cells is shown. Error bars indicate standard error (SE).

(B) and (C): HeLa S3 cells were transfected with siRNA duplexes for 36 h and stained for Aurora B (B) or pCenpA (C), together with CREST and DAPI staining. Scale bar = 10 µm.

Figure S2. Plk1 localization in BubR1 rescue experiments

HeLa cells were depleted of endogenous BubR1 (using a 3’-UTR-directed siRNA) and simultaneously transfected with Flag-tagged WT and T620A BubR1 constructs. After 36 h, the cells were fixed and stained with anti-Flag (green) and anti-Plk1 (red) antibodies. DNA was stained with DAPI. Scale bar = 10 µm.

Figure S3. BubR1 cold treatment and rescue experiments

(A) and (B) HeLa cells were transfected with GL2 or siRNA duplexes targeting BubR1 (siBubR1, siBubR1-3’). BubR1 depletion in HeLa cells is demonstrated by immunofluorescence (A) and Western blotting (B).

(C) HeLa S3 cells were treated as described in the legend to Figure 3. Cells were fixed directly (T=0) or after a 20 min (T=20) or 30 min (T=30) incubation at +4°C and then stained with anti-Flag (M2) monoclonal antibody.

(D) HeLa cells were incubated at +4°C for the indicated times, before they were fixed and stained for Plk1 (red), α-tubulin (green), and CREST (blue). Representative metaphase plates from each time point are shown. Scale bars = 10 µm.

Figure S4. Characterization of anti-pS676 polyclonal antibody

(A) Preimmune serum and serum from day 95 bleed of immunized rabbit #720 were tested for reactivity on asynchronously growing HeLa cells, using immunofluorescence microscopy. Antibodies from this bleed were then affinity purified on the phosphopeptide antigen and used for all subsequent studies.

(B) Phosphospecificity of the anti-pS676 antibody. Lysates from aphidicolin and nocodazole arrested cell were incubated in the presence of calf intestinal phosphatase (+CIP), or buffer alone (-CIP), resolved by SDS-PAGE, and probed with the anti-pS676 antibody (upper panel). The membrane was then stripped and reprobed with antibodies against BubR1 (middle panel) and α-tubulin (bottom panel).

(C) Endogenous BubR1 was depleted by siRNA and simultaneous rescue experiments were performed with empty Flag vector, as well as Flag-tagged BubR1 WT, KD, and T620A constructs. Cells were fixed after 36 h and stained with anti-pS676 (red), anti-BubR1 (green), and DAPI (blue). Scale bar = 10 µm.

(D) Quantification of the phenotype observed in (C). Error bars indicate SE.

(E) Asynchronously growing cells and cells treated for 16 with either nocodazole or taxol were fixed and stained with anti-pS676 and anti-BubR1 antibodies.

Supplemental Movies S1-S4

Selected movies showing segregation of GFP-Histone H2B labeled chromosomes in BubR1 depleted cells expressing mCherry empty vector (movie S1), mCherry-BubR1WT (movie S2), mCherry-BubR1KD (movie S3), or mCherry-BubR1T620A (movie S4).

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