Death Receptor Recruitment of Endogenous Caspase-10 and Apoptosis Initiation in the Absence of Caspase-8
2001; Elsevier BV; Volume: 276; Issue: 49 Linguagem: Inglês
10.1074/jbc.m105102200
ISSN1083-351X
AutoresFrank Kischkel, David A. Lawrence, Antoine Tinel, Heidi Leblanc, Arvind K. Virmani, Peter Schow, Adi F. Gazdar, John Blenis, David Arnott, Avi Ashkenazi,
Tópico(s)PARP inhibition in cancer therapy
ResumoCaspase-8 is believed to play an obligatory role in apoptosis initiation by death receptors, but the role of its structural relative, caspase-10, remains controversial. Although earlier evidence implicated caspase-10 in apoptosis signaling by CD95L and Apo2L/TRAIL, recent studies indicated that these death receptor ligands recruit caspase-8 but not caspase-10 to their death-inducing signaling complex (DISC) even in presence of abundant caspase-10. We characterized a series of caspase-10-specific antibodies and found that certain commercially available antibodies cross-react with HSP60, shedding new light on previous results. The majority of 55 lung and breast carcinoma cell lines expressed mRNA for both caspase-8 and -10; however, immunoblot analysis revealed that caspase-10 protein expression was more frequently absent than that of caspase-8, suggesting a possible selective pressure against caspase-10 production in cancer cells. In nontransfected cells expressing both caspases, CD95L and Apo2L/TRAIL recruited endogenous caspase-10 as well as caspase-8 to their DISC, where both enzymes were proteolytically processed with similar kinetics. Caspase-10 recruitment required the adaptor FADD/Mort1, and caspase-10 cleavage in vitrorequired DISC assembly, consistent with the processing of an apoptosis initiator. Cells expressing only one of the caspases underwent ligand-induced apoptosis, indicating that each caspase can initiate apoptosis independently of the other. Thus, apoptosis signaling by death receptors involves not only caspase-8 but also caspase-10, and both caspases may have equally important roles in apoptosis initiation. Caspase-8 is believed to play an obligatory role in apoptosis initiation by death receptors, but the role of its structural relative, caspase-10, remains controversial. Although earlier evidence implicated caspase-10 in apoptosis signaling by CD95L and Apo2L/TRAIL, recent studies indicated that these death receptor ligands recruit caspase-8 but not caspase-10 to their death-inducing signaling complex (DISC) even in presence of abundant caspase-10. We characterized a series of caspase-10-specific antibodies and found that certain commercially available antibodies cross-react with HSP60, shedding new light on previous results. The majority of 55 lung and breast carcinoma cell lines expressed mRNA for both caspase-8 and -10; however, immunoblot analysis revealed that caspase-10 protein expression was more frequently absent than that of caspase-8, suggesting a possible selective pressure against caspase-10 production in cancer cells. In nontransfected cells expressing both caspases, CD95L and Apo2L/TRAIL recruited endogenous caspase-10 as well as caspase-8 to their DISC, where both enzymes were proteolytically processed with similar kinetics. Caspase-10 recruitment required the adaptor FADD/Mort1, and caspase-10 cleavage in vitrorequired DISC assembly, consistent with the processing of an apoptosis initiator. Cells expressing only one of the caspases underwent ligand-induced apoptosis, indicating that each caspase can initiate apoptosis independently of the other. Thus, apoptosis signaling by death receptors involves not only caspase-8 but also caspase-10, and both caspases may have equally important roles in apoptosis initiation. tumor necrosis factor death-inducing signaling complex heat shock protein Fas-associated death domain deficient horseradish peroxidase immunoprecipitation polyacrylamide gel electrophoresis Western blot poly(A)DP-ribose polymerase CD95 ligand (CD95L, also called Fas or APO-1 ligand) is an important inducer of physiological apoptosis, acting primarily within the immune system (1Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4568) Google Scholar). A more recently discovered relative of CD95L, Apo2L/TRAIL (Apo2 ligand or tumor necrosis-related apoptosis-inducing ligand) (2Pitti R.M. Marsters S.A. Ruppert S. Donahue C.J. Moore A. Ashkenazi A. J. Biol. Chem. 1996; 271: 12687-12690Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar, 3Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Davis-Smith T. Rauch C. Smith C.A. Goodwin R.G. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2671) Google Scholar), is implicated as well in apoptosis control, although its precise biological role is less well defined (4Ashkenazi A. Dixit V.M. Curr. Opin. Cell Biol. 1999; 11: 255-260Crossref PubMed Scopus (1160) Google Scholar). Like most other tumor necrosis factor (TNF)1gene superfamily members, CD95L and Apo2L/TRAIL are homotrimeric, type II transmembrane proteins that interact with corresponding members of the TNF receptor superfamily. CD95L signals apoptosis through the “death” receptor CD95, whereas Apo2L/TRAIL signals apoptosis through the death receptors DR4 and DR5 (5Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5183) Google Scholar). Death receptors form a subgroup within the TNF receptor superfamily of type I transmembrane proteins that share sequence homology within a cytoplasmic region defined as the “death domain” (6Tartaglia L.A. Ayers T.M. Wong G.H.W. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1170) Google Scholar, 7Itoh N. Nagata S. J. Biol. Chem. 1993; 268: 10932-10937Abstract Full Text PDF PubMed Google Scholar). Signaling through death receptors can be modulated by “decoy” receptors that lack functional death domains; DcR3 binds CD95L (8Pitti R. Marsters S.A. Lawrence D.A. Roy M. Kischkel F.C. Dowd P. Huang A. Donahue C.J. Sherwood S.W. Baldwin D.T. Godowski P.J. Wood W.I. Gurney A.L. Hillan K.J. Cohen R.L. Goddard A.D. Botstein D. Ashkenazi A. Nature. 1998; 396: 699-703Crossref PubMed Scopus (686) Google Scholar, 9Yu K. Kwon B. Ni J. Zhai Y. Ebner R. Kwon B. J. Biol. Chem. 1999; 274: 13733-13736Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar), whereas DcR1 (10Pan G. Ni J. Wei Y.F., Yu, G.-L. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1385) Google Scholar,11Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1535) Google Scholar) and DcR2 (12Degli-Esposti M.A. Dougall W.C. Smolak P.J. Waugh J.Y. Smith C.A. Goodwin R.G. Immunity. 1997; 7: 813-820Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar, 13Marsters S.A. Sheridan J.P. Pitti R.M. Huang A. Skubatch M. Baldwin D. Yuan J. Gurney A. Goddard A.D. Godowski P. Ashkenazi A. Curr. Biol. 1997; 7: 1003-1006Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar) interact with Apo2L/TRAIL. CD95L (14Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1799) Google Scholar) and Apo2L/TRAIL (15Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, 16Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (586) Google Scholar, 17Sprick M.R. Weigand M.A. Rieser E. Rausch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar) use similar signaling mechanisms to transduce a pro-apoptotic signal into target cells (18Peter M.E. Cell Death Differ. 2000; 7: 759-760Crossref PubMed Scopus (46) Google Scholar). Both ligands trigger a series of protein-protein interactions that assemble a death-inducing signaling complex (DISC) at the cytoplasmic death domain of the receptor. Upon ligand binding, CD95, DR4, and DR5 recruit the adaptor molecule FADD/Mort1 (19Boldin M.P. Varfolomeev E.E. Pancer Z. Mett I.L. Cmonis J.H. Wallach D. J. Biol. Chem. 1995; 270: 387-391Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 20Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2173) Google Scholar) through homophilic death domain interactions. In turn, FADD recruits the zymogen form of the apoptosis-initiating protease caspase-8, through homophilic interaction of “death effector domains” (21Boldin M. Goncharov T. Goltsev Y. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2116) Google Scholar, 22Muzio M. Chinnaiyan A. Kischkel F. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J. Zhang M. Gentz R. Mann M. Krammer P. Peter M. Dixit V. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2748) Google Scholar). The proximity of caspase-8 zymogens in the DISC facilitates activation through self-processing, leading to cleavage of downstream effector caspases that execute the apoptotic death program. Caspase-10 is closely related by sequence to caspase-8 (23Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (697) Google Scholar, 24Vincenz C. Dixit V.M. J. Biol. Chem. 1997; 272: 6578-6583Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). The two enzymes are encoded on the same region of human chromosome 2q33–34 (23Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (697) Google Scholar, 25Kischkel F.C. Kioschis P. Weitz S. Poustka A. Lichter P. Krammer P.H. Cytogenet. Cell Genet. 1998; 82: 95-96Crossref PubMed Google Scholar, 26Grenet J. Teitz T. Wei T. Valentine V. Kidd V.J. Gene. 1999; 226: 225-232Crossref PubMed Scopus (83) Google Scholar), suggesting that they arose by duplication of one ancestral gene. In vitro experiments demonstrate the ability of caspase-10 to process caspase-3 and caspase-7 (23Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (697) Google Scholar), and overexpression of caspase-10 induces apoptosis (24Vincenz C. Dixit V.M. J. Biol. Chem. 1997; 272: 6578-6583Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). By analogy to caspase-8, it has been proposed that caspase-10 may be involved in apoptosis signaling by death receptors; however, studies investigating this hypothesis have yielded conflicting results. Upon overexpression in transfected cells, TNF receptor 1 and CD95 interacted with caspase-10 (24Vincenz C. Dixit V.M. J. Biol. Chem. 1997; 272: 6578-6583Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), and DR4 and DR5 favored recruitment of caspase-10versus caspase-8 through an adaptor that appeared to be distinct from FADD (10Pan G. Ni J. Wei Y.F., Yu, G.-L. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1385) Google Scholar, 27McFarlane M. Ahmad M. Srinivasula S.M. Fernandes-Alnemri T. Cohen G.M. Alnemri E.S. J. Biol. Chem. 1997; 272: 25417-25420Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). In addition, investigation of patients with the type II autoimmune lymphoproliferative syndrome, who carry at least one inactivating mutation in the caspase-10 gene (28Wang J. Zheng L. Lobito A. Chan F. Dale J. Sneller M. Yao X. Puck J. Straus S. Lenardo M. Cell. 1999; 98: 47-58Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 29Gronbaek K. Dalby T. Zeuthen J. Ralfkiaer E. Guidberg P. Blood. 2000; 95: 2184-2185Crossref PubMed Google Scholar), provided evidence for an essential role of caspase-10 in apoptosis induction by Apo2L/TRAIL in lymphocytes and dendritic cells and by CD95L in lymphocytes (28Wang J. Zheng L. Lobito A. Chan F. Dale J. Sneller M. Yao X. Puck J. Straus S. Lenardo M. Cell. 1999; 98: 47-58Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). On the other hand, the results from biochemical analyses of the Apo2L/TRAIL DISC in nontransfected BJAB B lymphoma and Jurkat T leukemia cells, which express both caspase-8 and -10, indicated that DR4 and/or DR5 recruit and activate caspase-8 but not caspase-10 (16Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (586) Google Scholar, 17Sprick M.R. Weigand M.A. Rieser E. Rausch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar). Furthermore, studies with caspase-8-deficient brain cancer cell lines (30Grotzer M.A. Eggert A. Zuzak T.J. Janss A.J. Marwaha S. Wiewrodt B.R. Ikegaki N. Brodeur G.M. Phillips P.C. Oncogene. 2000; 19: 4604-4610Crossref PubMed Scopus (161) Google Scholar, 31Teitz T. Wei T. Valentine M.B. Vanin E.F. Grenet J. Valentine V.A. Behm F.G. Look A.T. Lahti J.M. Kidd V.J. Nat. Med. 2000; 6: 529-535Crossref PubMed Scopus (705) Google Scholar), mutant Jurkat T cell lines (16Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (586) Google Scholar, 17Sprick M.R. Weigand M.A. Rieser E. Rausch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar,32Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 33Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (275) Google Scholar), or mouse embryonic fibroblasts (34Varfolomeev E.E. Schuchmann M. Luria V. Chiannlkulchai N. Beckmann J.S. Mett I.L. Rebrikov D. Brodianski V.M. Kemper O.C. Kollet O. Lapidot T. Soffer D. Sobe T. Avraham K.B. Goncharov T. Holtmann H. Lonai P. Wallach D. Immunity. 1998; 9: 267-276Abstract Full Text Full Text PDF PubMed Scopus (1042) Google Scholar) suggested resistance toward apoptosis induction by Apo2L/TRAIL or CD95L. Transfection of caspase-8-deficient Jurkat cells with a caspase-8 expression plasmid restored sensitivity to both ligands (16Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (586) Google Scholar, 17Sprick M.R. Weigand M.A. Rieser E. Rausch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 32Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 33Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (275) Google Scholar). These studies implied that caspase-8 has an obligatory role in death receptor signaling, whereas caspase-10 is neither involved nor important for this function. To reassess the involvement of caspase-10 in death receptor action, we characterized a panel of antibodies that recognize this protein in its three functional isoforms and used these antibodies to examine death receptor-mediated recruitment and processing of caspase-10 in nontransfected cells. Our results demonstrate that both Apo2L/TRAIL and CD95L recruit caspase-10 to their respective DISCs, where it is processed with kinetics similar to those of caspase-8. We found that the commercially available caspase-10/b antibodies that were used in previous studies to demonstrate that caspase-10 is present in certain cell lines but is not recruited by stimulated death receptors (16Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (586) Google Scholar, 17Sprick M.R. Weigand M.A. Rieser E. Rausch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar,32Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar) can cross-react to heat shock protein 60 (Hsp60), which is similar in mass to caspase-10/b, thus providing a new perspective on earlier observations. By investigating cell lines that express either caspase-8 alone or caspase-10 alone, we demonstrate that each caspase can be recruited to the DISC and processed to initiate apoptosis independently of the other. These results suggest that caspase-8 and caspase-10 both may have important physiologic roles in apoptosis initiation downstream of death receptors. Human B cell lymphoma BJAB were a gift from E. Humke (Genentech); human T cell leukemia Jurkat (parental), Jurkat (FADD def.) (clone E1), and Jurkat (Casp8 def.) (clone I9.2) were obtained by J. Blenis (Harvard Medical School). The following human nonsmall cell lung carcinoma cell lines: H1437, H2O09, H727, H1171, H2347, HCC1171, H2126, H226, H2O87, HCC515, HCC95, H290, H2122, H522, HCC44, HCC78, HCC193, H2887, H1417, HCC1395, and H1395; small cell lung carcinoma cell lines: H524, H345, H889, H2171, H69, H1618, H592, H2141, H1045, H378, H33, H2195, H1607, and H125; and breast carcinoma cell lines: HCC2688, HCC2157, HCC1143, HCC712, HCC1500, HCC1419, HCC2911, HCC1187, HCC38, HCC1739, HCC1395, HCC1569, HCC1428, HCC70, HCC1937, HCC1954, HCC1599, HCC2713, and HCC1806 were obtained by A. Virmani and A. Gazdar (University of Texas Southwestern Medical School) (35Gazdar A.F. Kurvari V. Virmani A. Gollahon L. Sakaguchi M. Westerfield M. Kodagoda D. Stasny V. Cunningham H.T. Wistuba I.I. Tomlinson G. Tonk V. Ashfaq R. Leitch A.M. Minna J.D. Shay J.W. Int. J. Cancer. 1998; 78: 766-774Crossref PubMed Scopus (241) Google Scholar, 36Phelps R.M. Johnson B.E. Ihde D.C. Gazdar A.F. Linnoila R.I. Matthews M.J. Bunn P.A. Carney D. Minna J.D. Mulshine J.L. J. Cell. Biochem. 1996; 24 (suppl.): 32-91Crossref Scopus (259) Google Scholar, 37Virmani A.K. Fong K.M. Kodagoda D. McIntire D. Hung J. Tonk V. Minna J.D. Gazdar A.F. Genes Chromosomes Cancer. 1998; 21: 308-319Crossref PubMed Scopus (180) Google Scholar). Human MCF-7 breast carcinoma cells were a gift from E. Sauseville (NCI, National Institutes of Health). All cell lines were cultured in RPMI 1640 + 10% heat-inactivated fetal bovine serum + 1000 units/ml penicillin-streptomycin (Life Technologies, Inc.). The primary antibodies anti-FADD (catalog number F36620) and anti-PARP (catalog number P76420) were purchased from Transduction Laboratories (Lexington, KY); anti-caspase-3 (catalog number 556425) was from PharMingen (San Diego, CA); anti-caspase-8 (catalog number 05-477) was from Upstate Biotechnology (Lake Placid, NY); anti-Hsp60 (catalog number 804-069-C100) was from Alexis Corporation (San Diego, CA); and anti-FLAG (M2) was from Sigma. Anti-caspase-10 had the following sources: C10.1 (mouse monoclonal, 4B2 (number 2411, a gift from J. Kim, Genentech), generated against the large subunit (p22)); C10.2 (rabbit polyclonal, generated against the N-terminal sequence: PPVDKEAESYQGEEELVSQ, Genentech); C10.3 (mouse monoclonal, catalog number M059–3, MBL International, Watertown, MA); C10.4 (mouse monoclonal, catalog number MAB834, R&D Systems, Minneapolis, MN); C10.5 (rabbit polyclonal, generated against a common sequence of the small subunit (p12): ALNPEQAPTSLQDSIPAEA, Genentech); and C10.6 (rabbit polyclonal, catalog number 06-836, Upstate Biotechnology). Anti-caspase-10/b antibodies besides C10.6 that showed cross-reactivity to Hsp60 were from Alexis, Upstate Technologies, PharMingen, Oncogene Sciences, and Stressgen; these antibodies cross-reacted also to the bacterial homolog of Hsp60, GroEL (data not shown). Anti-DR4 (3G1) and anti-DR5 (3H1) monoclonal antibodies were generated by using receptor-Fc fusion proteins as antigens. As secondary reagents we used: horseradish peroxidase (HRP)-conjugated goat α-mouse IgG2b (catalog number 1090-05) from Southern Biotechnology Associates, Inc. (Birmingham, AL), HRP-conjugated goat anti-mouse IgG1 (catalog number 02287E) from PharMingen, HRP-conjugated sheep anti-mouse IgG (catalog number NA931), and HRP-conjugated streptavidin (catalog number RPN1231) (Amersham Pharmacia Biotech), and HRP-conjugated goat anti-rabbit IgG (catalog number 111-035-144) from Jackson ImmunoResearch (West Grove, PA). Purified Hsp60 protein (catalog number SPP-742) was purchased from StressGen Biotechnologies, Inc. (Collegeville, PA). The ligands, each containing an N-terminal FLAG epitope tag, were purified over an M2-agarose column. Apo2L/TRAIL-FLAG (amino acids 114–281 in pFLAG-MAC; Sigma) was expressed in Escherichia coli. CD95L-FLAG (amino acids 131–281 in pCMV-1; Sigma) was expressed in Chinese hamster ovary cells). As substrates for immunodetection, either ECLTM(Amersham Pharmacia Biotech) or SuperSignal® West Dura Extended Duration Substrate (Pierce) were used. All other chemicals used were of analytical grade and were purchased from Sigma. These experiments were done essentially as described (15Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, 16Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (586) Google Scholar, 17Sprick M.R. Weigand M.A. Rieser E. Rausch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar). If not otherwise stated, the cells (107) were either stimulated for 10 min or the indicated time with ligand (1 μg/ml Apo2L/TRAIL-FLAG or CD95L-FLAG + 2 μg/ml M2) or left untreated (unstimulated). After one wash with phosphate-buffered saline the cells were lysed for 30 min on ice with lysis buffer (1% Triton X-100, 150 mm NaCl, 10% glycerol, 20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 0.57 mm phenylmethylsulfonyl fluoride, protease inhibitor mixture (CompleteTM, Roche Molecular Biochemicals) and centrifuged at 15,000 × g for 15 min at 4 °C. The postnuclear supernatants were collected and rotated at 4 °C for 4–16 h in the presence of 20 μl of protein A/G beads (Pierce) alone or with an indicated antibody. After seven washes with lysis buffer, the immunoprecipitations (IPs) were analyzed by SDS-PAGE followed by electroblotting and detection through Western blot (WB). Apo2L/TRAIL or CD95L DISCs were immunoprecipitated from 5 × 106 BJAB cells. The IPs were washed four times with lysis buffer and three times with reaction buffer (1× phosphate-buffered saline, 5 mm dithiothreitol). Then 40 μl of reaction buffer and 0.5 μl of [35S]methionine-labeled, in vitro translated caspase-8/a, -10/a, -10/b, or -10/d were added to the beads and incubated at room temperature. The in vitro translations were done following the manufacturer's protocol (TNT®quick coupled transcription/translation system) (Promega, Madison, WI) using an 35S-labeled mix of cysteine and methionine (Tran35S-LabelTM, ICN). Alternatively, the DISC IPs were substituted by adding 0.1 μg of purified caspase-8/a or caspase-10/a to the reaction mixture (from PharMingen and BioVision, Palo Alto, CA, respectively). Various time points were taken and analyzed by SDS-PAGE using a precast 10% Bis Tris NuPAGETMgel system, and the gels were then electrotransferred onto nitrocellulose membranes following the manufacturer's instructions (Novex, San Diego, CA). The blots were air dried for 2 h at 37 °C and exposed to BAS-III imaging plates (Fuji Photo Film Inc., Greenwood, SC). The plates were read using the BAS-2000 IP Scanner with the Image Reader V1.2 software and analyzed with the MacBAS V.2.4 software (Fuji Photo Film Inc.). After SDS-PAGE and electroblotting the membranes were blocked with either 5% milk or 2% fish gelatin (for rabbit antibodies) in phosphate-buffered saline containing 0.5% Tween 20. The dilutions for the primary and secondary antibodies and the developing reagent are indicated in parentheses below. For C10.1, we used either a secondary antibody (1 μg/ml, 1:5000 anti-mouse IgG-HRP, ECL) or coupled the antibody directly to HRP (1:200, ECL). We used the following conditions for the other caspase-10 antibodies: C10.2, C10.5, and C10.6 (1 μg/ml, 1:40000 anti-rabbit IgG-HRP, ECL); C10.3 (1 μg/ml, 1:10000 anti-IgG1-HRP, SuperSignal or 1 μg/ml, 1:5000 anti-mouse IgG-HRP, ECL); or C10.4 (1 μg/ml, 1:5000 anti-mouse IgG-HRP, ECL). For caspase-8 and FADD analysis the membranes were cut at about 32 kDa, and the upper part was probed with anti-caspase-8 (1 μg/ml, 1:20000 anti-IgG2b-HRP, ECL), whereas the lower part was probed with anti-FADD (1 μg/ml, 1:10000 anti-IgG1-HRP, SuperSignal). Other blots were treated with anti-Hsp60 (5 μg/ml, 1:5000 anti-mouse IgG-HRP, ECL), anti-caspase-3 (1:1000, 1:40000 anti-rabbit IgG-HRP, ECL), anti-PARP (1:500, 1:10000 anti-IgG1-HRP, SuperSignal), biotinylated anti-DR4 (3G1), and anti-DR5 (3H1) (1 μg/ml, 1:5000, ECL and SuperSignal). Jurkat (Casp8 def.) were transfected using LipofectAMINE 2000 (Life Technologies, Inc.) essentially following the manufacturer's instructions with slight modifications. Briefly, 5 × 105 cells were transfected in 500 μl with 8 μl of LipofectAMINE 2000 and 5.5 μg of total DNA (4 μg of green fluorescent protein in pRK5 + 1.5 μg of caspase in pCI). After each transfection cells were split and either left untreated or stimulated with 1 μg/ml Apo2L/TRAIL-FLAG or CD95L-FLAG (both ligands cross-linked with 2 μg/ml M2. After 14 h of incubation, apoptosis was quantified by the percentage of propidium iodide (5 μg/ml)-stained cells in the green fluorescent protein-positive population on a FACScan using the CELLQuest software (Becton Dickinson, Franklin Lakes, NJ). The cells (2 × 106) were treated with 1 μg/ml Apo2L/TRAIL-FLAG plus 2 μg/ml M2 for the indicated time, split, and analyzed for phosphatidylserine exposure, mitochondrial membrane potential (ΔΨm), and DNA fragmentation by FACScan using the CELLQuest software. Following the manufacturer's instructions Annexin V-fluorescein isothiocyanate staining was assayed to detect phosphatidylserine exposure on the cell surface (Trevigen, Inc., Gaithersburg, MD). To measure ΔΨm, the cells were incubated with 3,3-dihexyloxacarbocyanine iodide (460 ng/ml; FL-1) (Molecular Probes, Inc., Eugene, OR) for 10 min at 37 °C in the dark and analyzed by FACScan. The percentage of DNA fragmentation was quantified through analysis of propidium iodide-stained nuclei as described (38Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4438) Google Scholar). Changes in morphology upon apoptosis induction were visualized by confocal microscopy and by Hoechst 33342 staining (Molecular Probes). The Hoechst dye was incubated with the cells for 20 min at room temperature and immediately evaluated by fluorescence microscopy. Caspase-8 has two main isoforms: 8/a and 8/b (39Scaffidi C. Medema J.P. Krammer P.H. Peter M.E. J. Biol. Chem. 1997; 272: 26953-26958Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar); both isoforms are recruited and activated by the CD95L and Apo2L/TRAIL DISCs (15Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, 16Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (586) Google Scholar, 17Sprick M.R. Weigand M.A. Rieser E. Rausch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 39Scaffidi C. Medema J.P. Krammer P.H. Peter M.E. J. Biol. Chem. 1997; 272: 26953-26958Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Caspase-10 has four described isoforms, of which three (10/a, 10/b, and 10/d) contain a functional protease domain (40Ng P.W. Porter A.G. Janicke R.U. J. Biol. Chem. 1999; 274: 10301-10308Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) (for schematic representation, see Fig. 2 A). To investigate directly whether the DISCs assembled by Apo2L/TRAIL or CD95L can process caspase-10, we isolated each ligand-induced DISC from BJAB lymphoma cells. BJAB cells express DR4, DR5, CD95, caspase-8 (15Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, 16Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (586) Google Scholar, 17Sprick M.R. Weigand M.A. Rieser E. Rausch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar), and caspase-10 (see below). We incubated each DISC with in vitro translated, [35S]methionine-labeled caspase-8/a, caspase-10/a, caspase-10/b, or caspase-10/d. SDS-PAGE and radiography revealed that each DISC processed caspase-8/a and the three caspase-10 isoforms with similar kinetics (Fig.1 A). We detected all of the previously proposed processed forms of caspase-10 (p25, p20 or p17, and p12) (40Ng P.W. Porter A.G. Janicke R.U. J. Biol. Chem. 1999; 274: 10301-10308Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) (Figs. 1 A and2 A). In contrast to the ligand-induced DISC, purified active caspase-
Referência(s)