Marc Kvansakul, Mark F. van Delft, Erinna F. Lee, Jacqueline M. Gulbis, W. Douglas Fairlie, David C.S. Huang, and Peter M. Colman

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1 Molecular Cell, Volume 25 Supplemental Data A Structural Viral Mimic of Prosurvival Bcl-2: A Pivotal Role for Sequestering Proapoptotic Bax and Bak Marc Kvansakul, Mark F. van Delft, Erinna F. Lee, Jacqueline M. Gulbis, W. Douglas Fairlie, David C.S. Huang, and Peter M. Colman Supplemental Experimental Procedures Expression and retroviral constructs All mammalian expression vectors for HA-tagged BH3-only proteins, Bax and Bak subcloned into pef PGKhygro have been described previously (Chen et al., 2005; Huang et al., 1997; O'Connor et al., 1998; Willis et al., 2005), the FLAG-tagged vector for Bcl-x L was described previously (Huang et al., 1997). Similar constructs, made by subcloning into pef PGKpuro, were made for wild-type or mutant forms of M11L. Retroviral expression constructs (Noxa, Noxa3E, Bim S, Bim S 4E) were made by subcloning into the pmig vector (MSCV-IRES-GFP, GFP sequence is that of EGFP) and have been previously described (Chen et al., 2005; Willis et al., 2005). The GFP selection cassette was replaced with that of hygromycin (van Delft et al., 2006) for the constructs expressing FLAG-tagged Bcl-2, and wild-type or mutant M11L. All the cdnas used were of human origin except for Bad (mouse), Bid (mouse), Hrk (rat) and M11L (myxoma virus). Details of all oligonucleotides and constructs are available from the authors. Tissue culture, cell death induction, retroviral infections, and apoptosis assays All cell lines (HEK293T: immortalized human embryonal kidney cell line, Phoenix Ecotropic packaging cells (Kinsella and Nolan, 1996), FDC-P1: mouse myelomonocytic, and mouse embryonic fibroblasts: MEFs ) were cultured in Dulbecco s Modified Eagles (DME) medium supplemented with 10% fetal calf serum (FCS), and in some cases with 250 µm L-asparagine and 50 µm 2-mercaptoethanol. All MEFs (Chen et al., 2005; Willis et al., 2005; Willis et al., In Press) were generated from E embryos and immortalized (at passage 2-4) with SV40 large T antigen. All mice used were of C57BL/6 origin or have been

2 backcrossed (>10 generations) to this genetic background and their genotype determined as previously described (details are available from the authors). Cell death was induced with ABT-737 (4 µm, Abbott Laboratories; (Oltersdorf et al., 2005), UV-irradiation (200 J/m 2 ), Etoposide (100 µm), IL-3 deprivation (of FDC-P1 cells) or by retroviral infection. pmig retroviral constructs encoding BH3-only proteins were transiently transfected into Phoenix Ecotropic packaging cells and viral supernatants used to infect cells as described (Chen et al., 2005). Cell viability in both short-term assays and longterm assays of colony formation was determined as described (Chen et al., 2005). Long-term survival is expressed as a percentage of the number of colonies obtained relative to retroviral infection with empty parental retrovirus. Immunoprecipitation and immunoblotting The transfection and metabolic labeling of the human embryonic kidney (HEK) 293T cells with 35 S-methionine/cysteine (NEN) have been described (Huang et al., 1997; Moriishi et al., 1999; O'Connor et al., 1998). Immunoprecipitation was performed using mouse monoclonal anti-flag (M2: Sigma) or anti-ha (3F10; Roche) antibodies. Control immunoprecipitations were performed using an anti-mouse Glu-Glu (MMS-115R: CRP) antibody. Proteins were resolved by SDS:PAGE (Novex gels, Invitrogen), transferred onto nitrocellulose membranes and proteins detected by immunoblotting using mouse monoclonal anti-bax (2D2; Sigma), FLAG (9H1; (Wilson-Annan et al., 2003); rabbit polyclonal anti-bak (B5929: Sigma); rat monoclonal anti-bim (3C5; Alexis). Secondary antibodies included HRP-conjugated anti-rat or anti-mouse IgG (Chemicon). The proteins were detected using Enhanced ChemiLuminescence (ECL, GE Healthcare). Supplemental References Chen, L., Willis, S. N., Wei, A., Smith, B. J., Fletcher, J. I., Hinds, M. G., Colman, P. M., Day, C. L., Adams, J. M., and Huang, D. C. S. (2005). Differential targeting of pro-survival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 17, Huang, D. C. S., O'Reilly, L. A., Strasser, A., and Cory, S. (1997). The anti-apoptosis function of Bcl-2 can be genetically separated from its inhibitory effect on cell cycle entry. EMBO J 16, Kinsella, T. M., and Nolan, G. P. (1996). Episomal vectors rapidly and stably produce hightiter recombinant retrovirus. Hum Gene Ther 7,

3 Moriishi, K., Huang, D. C. S., Cory, S., and Adams, J. M. (1999). Bcl-2 family members do not inhibit apoptosis by binding the caspase-activator Apaf-1. Proc Natl Acad Sci U S A 96, O'Connor, L., Strasser, A., O'Reilly, L. A., Hausmann, G., Adams, J. M., Cory, S., and Huang, D. C. S. (1998). Bim: a novel member of the Bcl-2 family that promotes apoptosis. EMBO J 17, Oltersdorf, T., Elmore, S. W., Shoemaker, A. R., Armstrong, R. C., Augeri, D. J., Belli, B. A., Bruncko, M., Deckwerth, T. L., Dinges, J., Hajduk, P. J., et al. (2005). An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, van Delft, M. F., Wei, A. H., Mason, K. D., Vandenberg, C. J., Chen, L., Czabotar, P. E., Willis, S. N., Scott, C. L., Day, C. L., Cory, S., et al. (2006). The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell 10, Willis, S. N., Chen, L., Dewson, G., Wei, A., Naik, E., Fletcher, J. I., Adams, J. M., and Huang, D. C. (2005). Pro-apoptotic Bak is sequestered by Mc1-1 and Bcl-x L, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev 19, Willis, S. N., Fletcher, J. I., Kaufmann, T., van Delft, M. F., Chen, L., Czabotar, P. E., Ierino, H., Lee, E. F., Fairlie, W. D., Bouillet, P., et al. (In Press). Apoptosis is induced when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science. Wilson-Annan, J., O'Reilly, L. A., Crawford, S. A., Hausmann, G., Beaumont, J. G., Parma, L. P., Chen, L., Lackmann, M., Lithgow, T., Hinds, M. G., et al. (2003). Proapoptotic BH3- only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity. J Cell Biol 162,

4 Figure S1. Lack of mammalian sequence homologs of M11L. The top ten hits from a NCBI BlastP search using the M11L amino acid sequence. Other than related viral proteins, the highest ranked mammalian protein (shown in bold) did not show any significant similarity with M11L. +: significant hits analyzed in greater detail in Fig. S3. Figure S2. Intramolecular disulfide bond in M11L. (A) Experimental electron density for the M11L:Bak BH3 complex contoured at 1.5σ around the M11L C33-C127 disulfide bond. Unexpectedly, a buried disulfide bond is present in M11L, despite the presence of reducing agents throughout the protein purification and crystallization. (B) Viability of FDC-P1 cells stably expressing FLAG-tagged wild-type M11L or a double cysteine mutant (C33S/C127S) 0 or 24 h following IL-3 withdrawal. Data represent means ± SD from 3 independent experiments.

5 Figure S3. Viral homologs of Myxoma virus M11L. Sequence alignment (using ClustalW) of the most relevant BlastP hits (indicated by + in Fig. S1). Fully conserved (*), highly conserved (:) and well conserved (.) residues are marked. & - residues forming the M11L binding groove (Fig. 3A). Residues shaded in yellow form part of the M11L binding groove and are fully conserved. # highlights valine 79 in M11L; this position is usually an arginine (R) in Bcl-2 family proteins (see Fig. 3A). Note the high conservation of key residues that form the hydrophobic groove of M11L.