Supplemental data
Plk1 regulates mitotic Aurora A function through TrCP-dependent degradation of hBora
Eunice H.Y. Chan, Anna Santamaria, Herman H.W. Silljé and Erich A. Nigg
Supplemental Experimental Procedures
Plasmid constructions and site directed mutagenesis
cDNA clones for human Bora and -TrCP1 (ID: IRAKp961O2411Q2 and HU3_p983C04289D) were obtained from the “Deutsches Ressourzentrum für Genomforschung” (RZPD) and sub-cloned in-frame into a pcDNA3.1 vector (Invitrogen) encoding either an N-terminal 3xMyc tag or a FLAG tag. Site-directed mutagenesis was performed by PCR using specific oligonucleotides and pfu Turbo (Stratagene, La Jolla, CA). Mutations in phosphodegron and PBD docking site were created by changing conserved serine or theonine residues to alanine. All constructs were verified by sequencing.
Antibody production and protein purification from E.coli
Antibodies against human Bora were generated by immunization of rabbits with 4 injections of 250g of N-terminally His6-tagged full-length protein (Charles River Laboratories, Wilmington, Mass.). Antibodies were affinity-purified by applying serum to a Ni2+ column containing the antigen used for immunization. After washing and elution of the column, antibodies were dialysed against PBS, essentially as described before (Chan, et al. 2005). MBP-tagged proteins were purified from E. coli as described (Baumann, et al. 2007).
Cell culture and synchronization
HeLa S3 and HEK293T cells were grown at 37C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (Invitrogen), supplemented with 10% heat-inactivated FCS and penicillin-streptomycin (100IU/ml and 100mg/ml, respectively). To arrest cells at prometaphase, they were treated with 50ng/ml or 500ng/ml nocodazole (Sigma, St. Louis, MO) for 16h and mitotic cells were collected by mechanical shake-off, washed twice with PBS, and released into normal growth medium. Samples were taken either from arrested cells or after release for various time intervals. Alternatively, cells were treated with 150µg/ml Monastrol (Sigma) for 16h.
Cell Extracts, Immunoprecipitation and Western Blot Analysis
Lysates were prepared using HEPES lysis buffer (50mM Hepes pH7.4, 150mM NaCl, 0.5% Triton X-100) containing 30g/ml RNase A, 30g/ml DNase, 1M Okadaic acid and protease and phosphatase inhibitors (Sillje, et al. 1999). Protein concentrations were determined using the Dc protein assay (Bio-Rad Laboratories, Hercules, CA). Lysates were incubated for 2-4 h at 4°C with 9E10 anti-Myc, anti-FLAG, anti-GFP or anti-Aurora A antibodies. In each case 1g of antibody was couple to 1l Sepharose-G or A beads (20 l beads in total) (Pierce, Rockford, IL, USA). After protein capture, beads were washed 4x with HEPES buffer and resuspended in gel sample buffer and proteins were analysed by SDS-PAGE and immunoblotting. Membranes were probed with the following antibodies: affinity-purified rabbit anti-hBora (1g/ml), rabbit anti-FLAG (Sigma), monoclonal 9E10 anti-Myc (undiluted culture supernatant), monoclonal anti-GFP (undiluted culture supernatant), anti-cyclin B1 or anti -tubulin (Sigma). Primary antibodies were detected with HRP-conjugated anti-mouse or anti-rabbit anitibodies (Pierce). Signal was detected by ECL SuperSignal (Pierce).
In vitro kinase assays
In vitro phosphorylation of hBora by Plk1 and Cdk1 was carried out in a total volume of 20l BRB80 kinase buffer for 30min at 30C using 500ng hBora, 100ng Cdk1/cyclinB (Upstate Biotechnology, Lake Placid, NY) or 100ng Plk1 (Kelm, et al. 2002), supplemented with 10M ATP and 2Ci [-32P] ATP (Amersham Corp., Arlington Heights, IL) . Kinase reactions to be used subsequently in Far Western analyses were performed under identical conditions except that [-32P] ATP was omitted. After stopping reactions by the addition of SDS sample buffer and heating to 95°C, samples were resolved by SDS-PAGE followed by autoradiography or transferred to nitrocellulose membranes (Amersham) for Far Western blotting.
Immunofluorescence Microscopy
HeLa S3 cells were grown on coverslips and HEK293T cells on fibronectin coated coverslips. Cells were fixed and permeabilized by PTEMF solution containing 4% formaldehyde; 0.2% Triton-X-100; 10mM EGTA and 1mM MgCl2 in 20mM PIPES, pH6.8 for 10min at room temperature To visualize centrosomal proteins, cells were fixed by cold methanol at -20°C for 5min. Coverslips were incubated for 30min at room temperature in PBS, 1% BSA. All antibody incubations were carried out for 1h at room temperature followed by three washes in PBS. Primary antibodies used were: anti-Aurora A (BD Transduction Laboratories, Lexington, KY), anti-pT288 Aurora A (Cell Signaling), anti-Aurora B (AIM1) (BD Transduction Laboratories), anti-TPX2 (Abcam, Cambridge, UK), anti-Wee1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti--tubulin (Sigma), anti-pericentrin (Abcam), FITC-labeled anti--tubulin (Sigma) and TRITC or FITC-labeled anti-Myc 9E10 (Santa Cruz Biotechnology). Primary antibodies were detected with Alexa488 and Alexa568-conjugated goat antibodies (Sigma). DNA was visualized using with 2µg/ml diamidino-2-phenylindole DAPI (Sigma). Immunofluorescence microscopy was performed with a Zeiss Axioplan II microscope (Carl Zeiss, Jena, Germany) with a 63x oil immersion objective and images were acquired using a Micromax charge couple device (CDD) camera (Princeton Instruments, Trenton, NJ). Alternatively, a Deltavision microscope on a Nikon Eclipse TE200 base (Applied Precision, Issaquah, WA) equipped with an Apo 60_/1.4 oil immersion objective and a CoolSnap HQ camera (Photometrics) was used for collecting 0.15-m distanced optical sections in the z-axis. Images at single focal planes were processed with a deconvolution algorithm depending on the objective used. Settings were conservative, with noise filtering set to low and 2 deconvolution cycles. The number of z-stacks collected was variable (between 5 and 20), depending on the height of the individual cell. Images were projected into one picture using the Softworx software (Applied Precision). Exposure times and settings for image processing (deconvolution) were constant for all samples to be compared within any given experiment. Images were processed with Adobe Photoshop CS (AdobeSystems).
Supplemental Figures
Fig. S1
A)Western blotting of mitotic HeLa S3 cells treated for 48 h with GL2 (control) or βTrCP1/2 siRNA oligonucleotides, and arrested in G1/S (thymidine block) or mitosis (nocodazole shake-off). Membranes were probed for hBora, Wee1, Cyclin B and for -tubulin as loading control. Note the accumulation of hBora in βTrCP1-depleted cells treated with nocodazole.
B)In vitro kinase assay were performed with Cdk1, Aurora A, Aurora B or Plk1 and MBP-hBora as a substrate in the presence of [-32P] ATP and the samples were subjected to SDS-PAGE and autoradiography is shown. Coomassie blue (CBB) staining shows protein loading.
C)Experiment performed as in B, but samples were subjected to 7.5% SDS-PAGE to better reveal hBora upshifts upon phosphorylation.
Fig. S2
A) Histogram summarizing the mitotic indices of HeLa S3 cells treated with GL2 (control) or hBora1 or 2 siRNA for 24h, 48h and 72h in 3 independent experiments (200 per experiment); bars indicate SD.
B) Histogram illustrating percentages of mitotic cells with spindle abnormalities. HeLa cells treated with GL2 or hBora siRNA duplexes for 72h and released from double thymidine block were fixed after 10h and stained with -tubulin and DAPI to determine spindle morphology. Results are from 3 individual experiments (300-350 per experiment), and bars indicate SD.
C) Histogram summarizing percentages of mitotic cells with cold-stable K-fibers. Results are from three independent experiments (100-150 per experiment); bars indicate SD.
Fig. S3
A)HEK293T cells were transfected with constructs expressing Myc-tagged hBora, hBoraN and hBoraC and arrested by nocodozole. Aurora A-immunoprecipitates were immunoblotted with antibodies against Aurora A and Myc. Single asterisk (*) denotes unphosphorylated hBora and doube asterisk (**) indicates phosphorylated hBora. Note preferential binding of the phosphorylated, upshifted hBora N-terminus.
B)HEK293T cells were co-transfected with plasmids expressing Myc-hBora, FLAG-Aurora Afor 48h and arrested by nocodozole. FLAG-immunoprecipitates were treated with or without alkaline phosphatase (AP) immunoblotted with antibodies against FLAG and Myc. Single asterisk (*) denotes unphosphorylated hBora and doube asterisk (**) indicates phosphorylated hBora.
Fig. S4
A)Evolutionary conservation of the putative phospho-degron motif in hBora (underlined). The phosphorylated serine and threonine is marked in red. Numbers refer to their position.
B)HEK293T cells were co-transfected with various constructs of Myc-hBora (wild-type, phosphodegron mutant; S497A/T501A, and PBD docking mutant; S252A) and Myc-Plk1 WT or KD for 48h. Cell extracts were probed by Western blotting with antibodies against 9E10 anti-Myc and -tubulin antibodies. The expression between different hBora constructs is not comparable, because of variations in transfection efficiencies.
C)Various constructs of Myc-hBora (as in A) and FLAG--TrCP1 were co-expressed in HEK293T cells for 48h and treated with nocodozole for the last 16h. FLAG-immunoprecipitates were analysed by immunoblotting with Myc and FLAG antibodies. Note that although little phosphorylated hBora is present in the lysate containing FLAG--TrCP1, this population selectively binds to immunoprecipitated FLAG--TrCP1.
Supplemental References
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