TUCAN, an Antiapoptotic Caspase-associated Recruitment Domain Family Protein Overexpressed in Cancer

Abstract

Caspase-associated recruitment domains (CARDs) are protein interaction domains that participate in activation or suppression of CARD-carrying members of the caspase family of apoptosis-inducing proteases. A novel CARD-containing protein was identified that is overexpressed in some types of cancer and that binds and suppresses activation of procaspase-9, which we term TUCAN (tumor-up-regulatedCARD-containing antagonist of caspasenine). The CARD domain of TUCAN selectively binds itself and procaspase-9. TUCAN interferes with binding of Apaf1 to procaspase-9 and suppresses caspase activation induced by the Apaf1 activator, cytochrome c. Overexpression of TUCAN in cells by stable or transient transfection inhibits apoptosis and caspase activation induced by Apaf1/caspase-9-dependent stimuli, including Bax, VP16, and staurosporine, but not by Apaf1/caspase-9-independent stimuli, Fas and granzyme B. High levels of endogenous TUCAN protein were detected in several tumor cell lines and in colon cancer specimens, correlating with shorter patient survival. Thus, TUCAN represents a new member of the CARD family that selectively suppresses apoptosis induced via the mitochondrial pathway for caspase activation. Caspase-associated recruitment domains (CARDs) are protein interaction domains that participate in activation or suppression of CARD-carrying members of the caspase family of apoptosis-inducing proteases. A novel CARD-containing protein was identified that is overexpressed in some types of cancer and that binds and suppresses activation of procaspase-9, which we term TUCAN (tumor-up-regulatedCARD-containing antagonist of caspasenine). The CARD domain of TUCAN selectively binds itself and procaspase-9. TUCAN interferes with binding of Apaf1 to procaspase-9 and suppresses caspase activation induced by the Apaf1 activator, cytochrome c. Overexpression of TUCAN in cells by stable or transient transfection inhibits apoptosis and caspase activation induced by Apaf1/caspase-9-dependent stimuli, including Bax, VP16, and staurosporine, but not by Apaf1/caspase-9-independent stimuli, Fas and granzyme B. High levels of endogenous TUCAN protein were detected in several tumor cell lines and in colon cancer specimens, correlating with shorter patient survival. Thus, TUCAN represents a new member of the CARD family that selectively suppresses apoptosis induced via the mitochondrial pathway for caspase activation. caspase-associated recruitment domain tumor-upregulated CARD-containing antagonist of caspase nine expressed sequence tag 4′,6-diamidino-2-phenylindole aminofluorocoumarin hemagglutinin dithiothreitol glutathioneS-transferase in vitro translated granzyme B staurosporine polyacrylamide gel electrophoresis Apoptosis is a cell suicide process that plays important roles in multiple facets of normal development and physiology as well as many diseases (reviewed in Refs. 1Vaux D. Korsmeyer S. Cell. 1999; 96: 245-254Abstract Full Text Full Text PDF PubMed Scopus (1367) Google Scholar and 2Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6191) Google Scholar). Apoptosis is caused by caspases, a family of cysteine proteases that cleave target proteins at aspartyl residues (3Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1936) Google Scholar, 4Thornberry N. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6159) Google Scholar). Caspases are initially produced as inactive zymogens, which become activated typically by proteolytic cleavage at aspartic acid residues, generating the catalytic subunits of the active enzymes. Caspases can be broadly divided into two classes: upstream initiators and downstream effectors (3Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1936) Google Scholar, 4Thornberry N. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6159) Google Scholar). The downstream caspases are thought to be activated primarily by cleavage by upstream caspases. The proforms of initiator and effector caspases differ principally in the length of their N-terminal prodomains, the region preceding the catalytic portion of these polypeptides. Upstream initiator caspases contain large prodomains, and these domains serve as protein interaction motifs that participate in zymogen activation. A common method of activating upstream caspases involves the induced proximity mechanism, where the N-terminal prodomains are used for achieving clustering of caspase zymogens. Because uncleaved procaspases commonly display at least weak protease activity, bringing them into close apposition results in transproteolytic cleavage and activation (reviewed in Ref. 5Salvesen G.S. Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (774) Google Scholar). The caspase-associated recruitment domain (CARD)1 is a protein interaction motif found in the N-terminal prodomains of several caspases and in apoptosis-regulatory proteins that either activate or suppress activation of CARD-containing procaspases (6Hofmann K. Bucher P. Tschopp J. Trends Biochem. Sci. 1997; 22: 155-156Abstract Full Text PDF PubMed Scopus (448) Google Scholar). In mammals, eight CARD-carrying caspases have been identified, including procaspase-1, -2, -4, -5, -9, -11, -12, and -13. To date, multiple noncaspase CARD-containing proteins have been discovered and functionally characterized, including Apaf1, Nod1 (CARD4), NAC (DEPCAP), Raidd (CRADD), Cardiak (Rip2, RICK), Bcl10 (CIPER), ARC (Nop30), Asc, CARD9, CARD10, CARD11, CARD14, cIAP1, cIAP2, and CLAN. The CARD domains of many of these proteins are capable of binding the CARD-containing prodomains of specific CARD-carrying caspases, either facilitating or inhibiting protease activation. Here we describe a new antiapoptotic member of the human CARD family that is overexpressed in some types of cancer and selectively binds and inhibits activation of procaspase-9. Starting from the sequence of the CARD domain of Apaf1 (7Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar), a cascade of PSI-BLAST (8Altschul S.F. Madden T.L. Schaeffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59933) Google Scholar) searches was performed, using new “hits” as queries for subsequent searches, until no new hits were found, as described elsewhere (9Li, W., Pio, F., Pawlowski, K., and Godzik, A. (2001)Bioinformatics, in press.Google Scholar). Lower significance hits from this procedure (“saturated BLAST”) were confirmed (or rejected) using the profile-to-profile alignment algorithm FFAS (10Rychlewski L. Jaroszewski L. Li W. Godzik A. Protein Sci. 2000; 9: 232-241Crossref PubMed Scopus (452) Google Scholar) against a library of apoptosis-related domains. The detailed analysis of three-dimensional models of the putative CARD domains was a final criterion in accepting/rejecting distant members of the family. After the complete list of proteins in the CARD family was assembled, representative members of the family were used to scan genomic and EST data bases with the TBLASTN program. Genomic regions where similarity to the CARD domain was found were again verified against the FFAS server and later analyzed with gene finding programs (9Li, W., Pio, F., Pawlowski, K., and Godzik, A. (2001)Bioinformatics, in press.Google Scholar) for the presence of other domains until a complete protein sequence was assembled. Three-dimensional models of CARD domains were generated using the MODELLER program (11Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10565) Google Scholar), based on FFAS alignments. The TUCAN cDNA sequence was found using PSI-BLAST and the CARD sequence of Nod1 as a query. This search revealed homology with a predicted protein of EST clone KIAA0955 in the KDRI brain genomic data base (available on the Internet at www.kazusa.or.jp/en/database.html). Human cDNA clones encoding the entire open reading frame of TUCAN were amplified using Pfu polymerase (Stratagene) using two-step polymerase chain reaction with two sets of oligonucleotide primers: set 1 (5′-GACCCTTTGTGACATCTCACA-3′ (forward) and 5′-CCAATGACAACGCTGGATTC-3′ (reverse)) and set 2 (5′- GGAATTCAAGATGATGAGACAGAGGCAG-3′ (forward) and 5′-CCCTCGAGCCAATGAGAACGCTGGATTC-3′ (reverse)). The resultant cDNA fragments were digested with EcoRI/XhoI and ligated into mammalian expression vectors pcDNA3-Myc, pcDNA3-HA, and pcDNA-3-FLAG at EcoRI andXhoI cloning sites. The nucleotide sequence of the assembled full-length TUCAN was confirmed by DNA sequencing analysis (www.sciencereagents.com). The region encoding the CARD domain was polymerase chain reaction-amplified from the cDNA using the primers 5′-GGAATTCTCAGGTGCAGCCTTTGT-3′ (forward) and 5′- CCCTCGAGCCAATGAGAACGCTGGATTC-3′ (reverse). Resulting fragments were digested with EcoRI/XhoI and ligated into pcDNA3-Myc, pcDNA3-HA, pJG4–5, pGilda, and pGEX-4T1 using EcoRI and XhoI sites. The ΔCARD construct was made using a similar strategy and the primers 5′- GGAATTCAAGATGATGAGACAGAGGCAG-3′ (forward) and 5′- AGGCTCGAGTCATGCAGCTACAAGCTGGAG-3′ (reverse). Cells were cultured at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum, 1 mml-glutamine, and antibiotics. For transient transfection assays, 293T cells in log phase were transfected with plasmids encoding full-length or fragments of TUCAN, procaspase-9, Apaf1, or other proteins using SuperFect transfection reagent (Qiagen). Cells were harvested 48 h later and used for immunoprecipitation and immunoblotting studies. Jurkat cells were maintained in culture in RPMI 1640, 10% fetal bovine serum, 1 mml-glutamine medium. For stable transfections, Jurkat cells were subjected to electroporation (2.5 kV/cm; 950 microfarads; Bio-Rad Gene Pulsar) using 10 µg ofPvuI-linearized pcDNA3 or 10 µg of linearized pcDNA3-Myc-TUCAN. After 2 days, cells were seeded at 106/ml in medium containing 1 mg/ml G418 (Omega Scientific, Inc.). Medium was replaced twice weekly, until colonies of stably transfected clones arose. Multiple clones were pooled and expanded in culture. Cells were transfected with pEGFP (CLONTECH) and effector plasmids using SuperFect transfection reagents (Qiagen) as indicated. After 24 h, both floating and adherent cells were pooled, fixed with 3.7% formaldehyde/phosphate-buffered saline, and stained with 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI), and the percentage of green fluorescent protein-positive cells with apoptotic morphology (nuclear fragmentation, chromatin condensation) was determined by fluorescence microscopy. Caspase activity was measured in cell lysates from HEK293T and Jurkat cells (normalized for total protein content), monitoring release of AFC from fluorogenic substrate acetyl-aspartyl-glutamyl-valinyl-aspartyl-aminofluorocoumarin (Ac-DEVD-AFC) using a spectrofluorimeter in continuous mode, as described (12Deveraux Q.L. Takahashi G.S. 1997; PubMed Scopus Google Scholar). For some cytochrome or granzyme B was as described (12Deveraux Q.L. Takahashi G.S. 1997; PubMed Scopus Google Scholar). from cells were in of caspase and with of or and of protein at °C for 1 were by and of the were with 1 of in vitro translated Wang S. A. Res. 1997; Google Scholar), either 10 cytochrome and 1 or 10 of granzyme B by for caspase activity as were generated in using and with to the residues of TUCAN or CARD domain of TUCAN as for or tag were from and been described S. K. Z. Q.L. G.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). was from For immunoprecipitation and immunoblotting cells were in with 1 1 1 and protease lysates were by and subjected to immunoprecipitation using specific and protein or A were in to and with by with lysates were analyzed by immunoblotting for total protein A protein containing the CARD of procaspase-9 was expressed from in Cells were in of saline, 1 1 1 and µg/ml and and were by The resultant were with of for 1 was and the from the The was twice with saline, 1 1 and were with 1 1 10 The final protein was against 1 1 to to or of TUCAN in to protein In vitro translated proteins were using the A total of µg of or DNA was with of in the presence of at °C for proteins of similar length and that Apaf1 was in a to the CARD of procaspase-9. proteins were based on and were in and the were to for of of full-length TUCAN or or proteins on of were with of proteins in of binding 1 and at °C for 2 The were with 1 of binding and proteins were in and subjected to by analysis of the using either a or by to and binding to was from specific and the of binding to was for in the of TUCAN and which have and human for analysis were from and specimens, fixed in and in were from the of of from and with as by the on and were by of the of was initially in was for some data a of To construct colon cancer of were from representative of containing tumor and into a new with a were to as described B. H. J. J. PubMed Scopus Google Scholar). were using a method as described in the using S. K. Z. Q.L. G.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). was at with with µg/ml of either or protein was used to of the The of tumor was based on the percentage of cells by or of were analyzed using the method and were used for of TUCAN data with the patient survival. the CARD domain of Nod1 as a searches of the data bases were performed, EST containing open reading frame for a acid The this open reading frame in a for Biol. PubMed Scopus Google Scholar) and was by The predicted which we have as TUCAN of for a CARD by a region similarity to other domains A The CARD of this protein is similar to the CARD of Apaf1 acid by procaspase-9 and 1 The N-terminal region of this CARD family protein a acid sequence with a region in the protein NAC of the human data base (available on the Internet at revealed that the gene encoding TUCAN on and is predicted to contain 10 chain reaction analysis is expressed in human (available on the Internet at www.kazusa.or.jp/en/database.html). are for their to we TUCAN for with the CARD-containing proteins procaspase-1, procaspase-9, Apaf1, Nod1 (CARD4), NAC Cardiak Raidd (CRADD), Bcl10 cIAP1, cIAP2, CARD9, and TUCAN with procaspase-9 and representative results from that were using TUCAN containing either or expressed by transient transfection in HEK293T cells with procaspase-9 or other inactive of procaspase-9 in which the catalytic cysteine was with was for these to of apoptosis N. H. S. J. Science. 1998; PubMed Scopus Google Scholar). lysates were and were using or by TUCAN with procaspase-9 but not the CARD-containing protein Apaf1 TUCAN not with the CARD-containing proteins procaspase-1, CARD9, cIAP1, and TUCAN not with caspases, to with the caspases, and 2 To the of the CARD domain TUCAN for with procaspase-9, fragments of TUCAN were expressed of the CARD or the CARD with full-length TUCAN and the but not the ΔCARD of TUCAN 2 Thus, the CARD domain of TUCAN is and for with procaspase-9. of TUCAN was confirmed by using and proteins and the and ΔCARD TUCAN with full-length TUCAN and the but not the ΔCARD 2 and data not Thus, the CARD domain of TUCAN is and for to Apaf1, TUCAN binds selectively to procaspase-9, as the of the CARD of Apaf1 with a three-dimensional of the CARD of TUCAN The of the CARD of Apaf1 in with the CARD of procaspase-9 revealed that and in Apaf1 important with the CARD of procaspase-9, a of P. J. J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). of these residues are or in the CARD of TUCAN, to and the CARD of Apaf1 the to with procaspase-9 and two P. J. J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). In TUCAN, for in the which or two and with the CARD of procaspase-9. of the that at this not be to the other and of the nine the be for with of the of other this and other in the CARD of procaspase-9 be Thus, these that of the of Apaf1 in with procaspase-9 are in TUCAN, the data specific of TUCAN with procaspase-9. To the of TUCAN and Apaf1 for binding to the CARD of procaspase-9, we in vitro protein binding For binding protein or was with of TUCAN or Apaf1 which been by in vitro and and TUCAN and specific binding to some binding to these TUCAN was to the CARD of procaspase-9 with of to Apaf1, based on of the of proteins by analysis for binding to and for in the of the TUCAN and Thus, TUCAN to the CARD of procaspase-9 with with The of TUCAN to with procaspase-9 the of this protein for binding with Apaf1 in To this HEK293T cells were transfected with of DNA encoding TUCAN and fixed of plasmids encoding procaspase-9 and Apaf1 Apaf1 that is active in the of cytochrome Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). were using lysates from these transfected in which were by and procaspase-9 by In the of TUCAN, of procaspase-9 with was In TUCAN in HEK293T cells in in the of procaspase-9 that with The total of procaspase-9 and in lysates were not by TUCAN a specific in caused by the TUCAN Apaf1 as a cytochrome of P. D. S. Wang X. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). TUCAN interferes with binding of Apaf1 to procaspase-9, we of this protein inhibits activation of caspases by cytochrome in cell HEK293T cells were transfected with either or and were for total protein and with 10 cytochrome and 1 to activation of Apaf1 (12Deveraux Q.L. Takahashi G.S. 1997; PubMed Scopus Google Scholar, P. D. S. Wang X. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). In cytochrome induced activation of caspases, as measured by cleavage of the fluorogenic caspase substrate caspase activity was induced by cytochrome in from analysis of of the using confirmed of the protein in cells transfected with but not In to caspase activity induced by the of granzyme B was in from and cells and many caspase family N. D. P. S. J. K. D. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), this that cells not have in caspase activation the of these of TUCAN caspase activation induced by cytochrome we these by the of the endogenous TUCAN protein from cells contain endogenous levels of TUCAN protein were with or serum, and were by to protein analysis confirmed in the levels of endogenous TUCAN protein from lysates of procaspase-9 were with the that TUCAN with this In Apaf1 levels were by the of that been with with to cytochrome activation of caspases that of TUCAN was with a in cytochrome of Thus, of some of the procaspase-9 from of TUCAN in a in the of cytochrome to activate a for TUCAN as endogenous of cytochrome procaspase-9 activation. Apoptosis can be induced by procaspase-9 and Apaf1 in HEK293T cells Y. D. N. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), to TUCAN can HEK293T cells were transfected with plasmids encoding procaspase-9, Apaf1, or TUCAN, and in and apoptosis was measured 24 h later based on of fixed In the of TUCAN, the of procaspase-9 and Apaf1 induced apoptosis of the transfected cells In cells with a apoptosis to the CARD domain of TUCAN was for suppression of apoptosis induced by the of procaspase-9 and Apaf1 The protein release of endogenous from apoptosis and (reviewed in Ref. Science. 1998; 281: PubMed Google Scholar). the of TUCAN on apoptosis induced by transient transfection of HEK293T cells with a in in HEK293T cells induced apoptosis of of the transfected In TUCAN with the percentage of cells analysis confirmed that TUCAN not with expression of the protein The CARD domain of TUCAN was for apoptosis in which DNA was transfected in from to to DNA of apoptosis In to Bax, the Fas apoptosis by and H. A. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Y. H. P. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Apoptosis induced by transient of Fas in HEK293T cells was not by TUCAN that TUCAN selectively apoptotic To the of TUCAN as apoptosis Jurkat cells were stably transfected with either or analysis of the cell lines using of protein in Jurkat cells the but not the the and the staurosporine have been to apoptosis and H. A. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Y. H. P. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. Z. S. H. J. 2000; PubMed Scopus Google Scholar), these were in the and Jurkat in cell was induced by and in with Jurkat In to and cell induced by was not by of TUCAN, with data that Fas can apoptosis of Apaf1 and H. A. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Y. H. P. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). in cell were with in caspase activity, measured in lysates from Jurkat with VP16, or or was used as the caspase activity was in Jurkat cells with cells B In was the of caspase activity were measured in and Jurkat cells the of to from stable Jurkat not a apoptotic in of caspase activity the of TUCAN and that for caspase activation antiapoptotic proteins are produced at levels in apoptosis (reviewed in Ref. J. J. 1999; PubMed Google Scholar). To into the expression of TUCAN in the of of human tumor cell lines J. P. S. K. S. L. N. J. W. A. D. D. B. K. Science. 1997; Scopus Google Scholar) was analyzed by immunoblotting using specific for TUCAN were for total protein to levels of TUCAN protein the tumor lines with some cell lines containing levels of this protein as and cancer In some of these tumor TUCAN in as a or as the of that to be the levels of endogenous TUCAN protein in some of these cancer cell lines were with the transfected HEK293T and Jurkat The levels of TUCAN produced in transfected HEK293T cells were with the endogenous levels of TUCAN found in cancer cells of TUCAN produced in the stably transfected Jurkat cells were in with endogenous TUCAN measured in and cancer cell from these that the levels of TUCAN produced by transfection are similar to that in some tumor cell the expression of this antiapoptotic protein was analyzed by in a of colon cancer from with and A was that tumor be analyzed on a in to these tumor normal in the of the of TUCAN in tumor normal TUCAN was in the cancer cells with normal cells in of of these specimens, that of colon their levels of TUCAN TUCAN was the of these cells with either or with that been with TUCAN confirmed the of these results results were with to on a of percentage and is the of and and these data were with patient TUCAN was of their cancer with or with of TUCAN with tumor was found that TUCAN expression is in a of colon correlating with shorter patient of TUCAN results for colon with from tumor were for were using but similar results were using the results significance was determined by analysis of the of with in a new tumor were for were using but similar results were using the results significance was determined by analysis of the of with have identified a new member of the human CARD which we have TUCAN antagonist of The TUCAN protein binds to procaspase-9 and interferes with interaction with Apaf1, apoptosis in the TUCAN apoptosis induced by that are to activate the mitochondrial pathway for caspase including Bax, and In apoptosis induced via including and Fas family is not by The sequence of TUCAN was as EST clone KIAA0955 (available on the Internet at from sequencing of brain N. A. H. N. DNA Res. 1998; PubMed Scopus Google Scholar). other have the predicted protein sequence into including and in to This represents the of this including the that this protein is produced in and predicted CARD binding to the of other proteins The CARD domain of TUCAN is for binding procaspase-9 and TUCAN N-terminal domain that similarity to other domains. This region of the TUCAN protein acid similarity with a of the NAC a CARD-carrying of the Apaf1 Z. K. S. Godzik A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The of this domain is but serve to TUCAN in a of as with other protein or protein the N-terminal region of TUCAN several including at and at and and protein at and with of multiple of TUCAN based on in we that of this protein a in TUCAN TUCAN a caspase cleavage at residues levels of TUCAN are found in several human cancer cell with normal TUCAN was in of colon of of this antiapoptotic protein in with data from of cells from Apaf1 and procaspase-9 have that these proteins as tumor in a pathway H. A. S. Science. 1999; PubMed Scopus Google Scholar). Overexpression of TUCAN be functionally to of Apaf1 or procaspase-9, that levels of TUCAN tumor or with this colon cancer levels of TUCAN were to from their based on analysis using analysis of the expression and of TUCAN in is for