Aurora Kinase-A Inactivates DNA Damage-Induced

Transcription

1 Cancer Cell, Volume 21 Supplemental Information Aurora Kinase-A Inactivates DNA Damage-Induced Apoptosis and Spindle Assembly Checkpoint Response Functions of p73 Hiroshi Katayama, Jin Wang, Warapen Treekitkarnmongkol, Hidehiko Kawai, Kaori Sasai, Hui Zhang, Hua Wang, Henry P. Adams, Shoulei Jiang, Sandip N. Chakraborty, Fumio Suzuki, Ralph B. Arlinghaus, Jinsong Liu, James A. Mobley, William E. Grizzle, Huamin Wang, and Subrata Sen Inventory of Supplemental Information Figure S1, related to Figure 1 Figure S2, related to Figure 3 Figure S3, related to Figure 4 Figure S4, related to Figure 5 Figure S5, related to Figure 7 Supplemental Experimental Procedures 1

2 Supplemental Data A 32 p- Np73 IB: Myc 32 p-aur-a Np73 TAp73 WT SA WT SA 32 p-tap73 IB: HA 32 p-aur-a B Myc- Np73: WT SA SD HA-p73: emp WT SA SD emp WT SA SD emp WT SA SD IB: Myc IP: HA IB: HA IB: Myc C Flag-AurA: HA-p73: IB: HA Direct IB: HA IP: Flag WT SA SD WT SA SD D MCF-10A IP: p73 NIG input IP: Aur-A NIG input E H1299 IP: p73 NIG input IP: Aur-A NIG input 1

3 Figure S1. Interaction between Aurora-A and p73, Related to Figure 1 A. Myc-ΔNp73 WT or Myc-ΔNp73 SA or HA-p73 WT or HA-p73 SA were transfected into Cos-1 cells. 24 hr after transfection, cells were subjected to immunoprecipitation with anti-myc or HA antibodies and immunoprecipitates were incubated with His-Aurora-A in the presence of [ 32P]- ATP. The reaction mixtures were subjected to immunoblotting with antibodies against Myc for Myc-ΔNp73, HA for HA-p73 and Aurora-A for His-Aurora-A (second and fourth) followed by [ 32 P]ATP incorporation visualized by autoradiography (top and third). B. Myc-ΔNp73 WT or mutants were co-transfected with either HA-p73 WT or mutants into Cos-1 cells. 24hr after transfection, cells were subjected to immunoprecipitation with anti-ha antibody followed by immunoblotting with the indicated antibodies (top and second). Expression level of Myc-ΔNp73 WT or mutants were analyzed by direct immunoblotting with anti-myc antibody (bottom). C. Empty vector or Flag-Aurora-A WT was co-transfected with HA-p73 WT, SA or SD into 293T cells. 24hr after transfection, cells were subjected to immunoprecipitation with anti-flag antibody followed by immunoblotting with the indicated antibodies. D. MCF-10A cells were immunoprecipitated with anti-p73 (left) or anti-aurora-a (right) antibodies or normal IgG (NIG). Immunoprecipitates were subjected to immunoblotting with anti-p73 and anti-aurora-a antibodies, respectively. Input indicates whole cell extracts. E. H1299 cells were analyzed as in (D). 2

4 A p53-wt p53-sd LMB: IB: GFP IB: -tub IB: PARP B interphase mitotic C interphase Noc treated IB: -tub IB: cycb IB: PARP IB: -actin Figure S2. Subcellular localization of p53 and p73 in the cells, Related to Figure 3 A. GFP-p53 WT or S215D mutant was transfected in H1299 cells. 24hr after transfection, cells were cultured in the presence (+) or absence (-) of leptomycin B for 6hr and then subjected to fractionation followed by immunoblotting with the indicated antibodies. B. MCF-7 cells were treated with nocodazole for 24 hr and then interphase cells and mitotic cells were separated by mechanical shake off. Cells were subjected to immunoblotting with the indicated antibodies. C. Cells prepared as in (B) were subjected to fractionations followed by immunoblotting with indicated antibodies. lear fraction in nocodazole treated cells (show in the panel with dotted line) expected to represent G2 phase cells prior to nuclear envelope breakdown. 3

5 A (kda) well C IB: GFP Direct IB: GFP D 24hr 48hr emp WT KDemp WT KD IB: mortalin IB: -actin emp Full del-bd emp Full del-bd WT SD WT SD B IP: Flag Flag-Mot: Full del-bd GFP-p73-mortalin complex GFP-p53: WT SD WT SD GFP-p73 tetramer GFP-p73 monomer IB: GFP IB: mortalin IP: p73 Input Figure S3. Interaction between p73 and mortalin, Related to Figure 4 A. GFP-p73a WT or SD were transfected in Hela cells and cell extracts were subjected to native gel electrophoresis followed by immunoblotting with anti-gfp antibody (left) and anti-mortalin antibody (right). B. Full length Flag-mortalin (Full) or p53 binding domain deletion mutant of Flag-mortalin (del-bd) were co-transfected with GFP-p53 WT or with SD into cells. 24 hr after transfection, cells were subjected to immunoprecipitation with anti-flag antibody followed by immunoblotting with anti- GFP (top) and anti-flag (bottom) antibodies, respectively. Whole cell extracts were directly immunoblotted with anti-gfp antibody (middle). C. Empty vector (emp) or Flag mortalin full (Full) or deletion mutant (del-bd) were transfected into cells for 24 hr. Cell extracts were then subjected to immunoprecipitation with anti-p73 antibody followed by immunoblotting with the indicated antibodies. Whole cell extracts (Input) were also directly immunoblotted. D. Empty vector or Flag-Aurora-A WT or Flag-Aurora-A KD were transfected into cells for 24hr. Cell extracts were then subjected to immunoblotting with the indicated antibodies. 4

6 empty Flag-mot delta-bd IB: -tub IB: PARP Figure S4. Subcellular localization of Aurora-A in mortalin deletion mutant cells, Related to Figure 5 Panc-1 cells were transfected with empty vector or Flag-mortalin deletion mutant (Flag-mot del-bd). 24 hr after transfection, cells were subjected to protein fractionations followed by immunoblotting with the indicated antibodies. Arrow indicates Aurora-A. 5

7 A sirna-p73 B prophase metaphase ana/telophase IB: Cyc B IB: ß-actin IP: p73 IB: ppka paur-a Aur-A Direct IB: CycB Hsp90 Figure S5. The role of p73 in mitosis, Related to Figure 7 A. Hela/GFP-H2B cells were transfected with control or with p73 sirna for 48 hr and then grown in the presence of nocodazole for 18 hr. Then cells were subjected to immunoblotting with the indicated antibodies. B. MCF-10A cells were synchronized by double thymidine block and release method. The cells synchronized at G1/S boundary after second thymidine block were released in fresh culture media. 5 hr after release, monastrol (200mM) was added. At 9 hr after release, monastrol arrested prophase cells were collected by shake-off. For metaphase cells, shaken off were washed with warm PBS 3 times and cultured in media containing MG132 (20mM). 2 hr later, MG132 arrested metaphase cells were collected. Anaphase/telophase cells were collected 1.5 hr after MG132 arrested cells were washed and cultured in fresh media. The cells were subjected to immunoprecipitation with anti-p73 antibody followed by immunoblotting with the indicated antibodies (top and second). Whole cell extracts were also directly immunoblotted. 6

8 Supplemental Experimental Procedures Plasmids pegfp-tap73, pgl3-luc, pgl3-p21-luc, and pgl3-mdm2-luc vectors were kindly provided by Dr. Zhimin Yuan. HA-TAp73 and Myc- Np73 were kindly provided by Dr. Elsa Flores. pegfp- and pcdna3-flag-mortalin and mortalin deletion mutant of p53 binding domain (Mot delta-p53bd) were kindly provided by Dr. Kenji Fukasawa (Ma et al., 2006). S235A and S235D mutations were generated by using Quik mutagenesis kit (Stratagene) and mutations were confirmed by sequencing. p73 cdnas were subcloned into pgex4t vector to produce GST fusion protein and into pmcherry vector (kindly provided by Dr. Richard Behringer) for time-lapse microscopic analysis. Antibodies and chemicals Antibodies used in this study were rabbit polyclonal anti-aurora-a (Zhou et al., 1998, Genetax), mouse monoclonal anti-aurora-a (BD Biosciences), anti-p73 (IMG-246, IMGENEX), anti-p73 (Ab-4, NeoMarker), anti-p73 (S-20), anti- -actin, anti-cdc20, anti-cyclin B1, anti-gfp anti-ha, anti-hsp90, anti-mad2, anti-mortalin, control IgG (Santa Cruz Biotechnology), anti-p21, anti-parp, anti-phospho- PKA substrate, anti-phosphothreonine-288 Aurora-A (Cell Signaling Biotechnology), anti-bubr1 (MBL International), anti-mad2 (Covance Research Products), and anti-α-tubulin (EMD Biosciences). The specificities of anti-p73 antibodies against TA-p73 have been described (Sayan et al., 2005, Rosenbluth et al., 2009). Chemicals used in this study were Aurora-A inhibitor MLN8054 (Millennium Pharmaceuticals), cisplatin (Teva Parenteral Medicines), MG132 (A.G. Scientific), monastrol, and nocodazol (Sigma). Cell culture Cos-1, Hela, GFP-H2B stably expressing HeLa, H1299, MCF-7, and 293T cells were maintained in DMEM with 10% FBS and antibiotics. McCoy5A or DMEM/F-12 with the same supplements was used for Saos-2 cells or Panc-1 cells, respectively. GST- fusion protein GST fusion protein production and purification were performed as described earlier (Katayama et al., 2001). Native gel electrophoresis WT or mutant p73 transfected cells were lysed in native page sample buffer (Invitrogen) containing 1% n- dodecyl-β-d-maltoside and 1% Triton X-100. Cell extracts were subjected to native gel electrophoresis, followed by immunoblotting according to the manufacturer s protocol (Invitrogen). Immunohistochemical staining To retrieve antigenicity, we treated tissue sections at 100ºC in a steamer containing 10 mmol citrate buffer (ph 6.0) for 60 min. The sections were immersed in methanol containing 0.3% hydrogen peroxidase for 20 min to block endogenous peroxidase activity and incubated in 2.5% blocking serum to reduce nonspecific binding. The sections were incubated with antibodies against Aurora A (Genetax, 1:200 dilution), p53 (DAKO DO-7, 1:100 dilution), and p73 (IMG-246, 1:100 dilution) at 4 C overnight, washed, and incubated with secondary antibody at R.T. for 60 min. Standard avidin-biotin immunohistochemical analysis of the sections was performed according to the manufacturer s recommendations (Vector Laboratories). The staining results were evaluated by two pathologists (H.W. for MD Anderson samples and W.E.G. for UABCC samples) on the basis of the percentage of positive tumor cells (0, <5%; 1, 5%-20%; 2, 20%-50%; and 3, >50%) and staining intensity (0-negative, 1-weak, 2-moderate, and 3-strong). For the statistical analysis, Aurora-A and p73 expression levels were categorized as low (combined score 3) or high (combined score 4). 7

9 Supplemental References Katayama, H., Zhou, H., Li, Q., Tatsuka, M., and Sen, S. (2001). Interaction and feedback regulation between STK15/BTAK/Aurora-A kinase and protein phosphatase 1 through mitotic cell division cycle. J Biol. Chem. 276, Rosenbluth, J.M., Johnson, K., Tang, L., Triplett, T., and Pietenpol, J.A. (2009). Evaluation of p63 and p73 antibodies for cross-reactivity. Cell Cycle. 8, Sayan, A.E., Paradisi, A., Vojtesek, B., Knight, R.A., Melino, G., Candi, E. (2005). New antibodies recognizing p73: comparison with commercial antibodies. Biochem. Biophys. Res. Commun. 330,