Cell Death and Differentiation (2002) 9, 439 ± 447 ã 2002 Nature Publishing Group All rights reserved 1350-9047/02 $25.00 www.nature.com/cdd Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC Y Zhan1,2,3, R Hegde1,2,3, SM Srinivasula1,2,3, Abbreviations: DED, death effector domain; EST, expressed T Fernandes-Alnemri1,2 and ES Alnemri*,1,2 sequence tags; CTD, C-terminal domain 1 Center for Apoptosis Research, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA Introduction 2 Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA Death effector domain (DED)-containing proteins are im- 3 These authors contributed equally to this work portant components of the cell death receptor-signaling * Corresponding author: ES Alnemri, Thomas Jefferson University, Kimmel pathway.1,2 Ligation of the death receptors, such as TNF, Cancer Institute, Bluemle Life Sciences Building, Room 904, 233 S 10th Street, Philadelphia, PA 19107, USA. Tel: 215-503-4632; Fax: 215-923-1098; CD95 and TRAIL receptors, results in the recruitment of the E-mail: [email protected] adaptor molecule FADD/Mort-1 through homotypic interac- tions of its C-terminal death domain with the cytoplasmic Received 8.2.02; accepted 8.2.02 death domain of the death receptors.1,3,4 FADD, which also Edited by G Melino contains an N-terminal DED, transduces the activation signal from the death receptors to the DED-containing precursors (procaspases) of the initiator caspases 8 and 10, by direct Abstract interaction of its DED with their corresponding DEDs.1,5 The Death effector domain-containing proteins are involved in association of FADD with procaspase-8 and procaspase-10 important cellular processes such as death-receptor induced results in their auto-processing/activation and the release of apoptosis, NF-kB activation and ERK activation. Here we the mature caspases 8 and 10 from the membrane associated death receptor signaling complex into the cytoplasm.5±7 The report the identification of a novel nuclear DED-containing mature caspase-8 and -10 could then cleave several protein, FLAME-3. FLAME-3 shares significant sequence important cellular substrates such as Bid and the effector (46.6% identical) and structural homology to another DED- caspases 3, 6 and 7 resulting in initiation of the caspase containing protein, DEDD. FLAME-3 interacts with DEDD and cascade and apoptosis.8,9 Thus, the DED regions of the c-FLIP (FLAME-1) but not with the other DED-containing adaptor molecule FADD and the initiator caspases 8 and 10 proteins FADD, caspase-8 or caspase-10. FLAME-3 translo- represent the link between the death receptors and the cates to, and sequesters c-FLIP in the nucleus upon downstream cell death machinery. overexpression in human cell lines. Using the yeast two- Although some members of the family of DED-containing hybrid system to identify DEDD-interacting proteins, the proteins, such as FADD, caspase-8 and caspase-10, are TFIIIC102 subunit of human transcription factor TFIIIC was agonists of the death receptor pathway, yet others, such as identified as a DEDD- and FLAME-3-specific interacting the viral and cellular FLIP proteins, are dominant negative inhibitors or antagonists to this pathway.10 The viral and protein. Co-expression of either DEDD or FLAME-3 with cellular FLIP proteins inhibit the death receptor pathway by hTFIIIC102 in MCF-7 cells induces the translocation from the binding to the DED regions of FADD and/or caspases 8 cytoplasm and sequestration of hTFIIIC102 in the nucleus, and 10 preventing the formation of a productive death indicating that DEDD and FLAME-3 form strong heterocom- receptor signaling complex.10 c-FLIP-deficient mice do not plexes with hTFIIIC102 and might be important regulators of survive,11 suggesting that the anti-apoptotic activity of c- the activity of the hTFIIIC transcriptional complex. Consistent FLIP plays an important role in embryonic development. with this, overexpression of DEDD or FLAME-3 in 293 cells Beside regulation of apoptosis, other important functions inhibited the expression of a luciferase-reporter gene under for DED-containing proteins have been reported. Several the control of the NF-kB promoter. Our data provide the first studies have shown that the DED protein PEA-15 is direct evidence for the involvement of DED-containing involved in the regulation of integrin activation, ERK proteins in the regulation of components of the general activation, phospholipase D expression and glucose transport.12±15 Another DED protein, DEDD, was reported transcription machinery in the nucleus. to bind to DNA, inhibit RNA polymerase I activity in vivo Cell Death and Differentiation (2002) 9, 439 ± 447. DOI: 10.1038/ and inhibit transcription in an in vitro transcription sj/cdd/4401038 assay.16,17 DED-containing proteins FADD, cFLIP, Cas- pase-8 and vFLIP can also activate NF-kB pathway through their DEDs.18±20 These findings indicate that Keywords: Apoptosis; Death effector domain; TFIIIC; DEDD; DED-proteins are involved in diverse cellular processes FLAME-1; FLIP ranging from cell survival to cell death. DEDD and FLAME-3 interact with the TFIIIC transcriptional complex YZhanet al 440 In order to identify novel cellular DED-containing and DED-interacting proteins, we searched the National Center for Biotechnology Information GenBank expressed-se- quence tags (EST) database for sequences encoding uncharacterized DED-containing proteins related to DEDD. We report here the cloning and functional characterization of a previously uncharacterized DED-containing protein designated FLAME-3, which has high homology to DEDD. Using the yeast two-hybrid system we identified hTFIIIC102 subunit of TFIIIC transcription factor as DEDD- and FLAME-3- interacting protein. Consistent with this result we found that DEDD and FLAME-3 can sequester TFIIIC102 in the nuclei of transfected cells. Our results suggest that the interaction of DEDD and FLAME-3 with this subunit may regulate transcription of class III genes. Results Cloning, sequence analysis and tissue distribution of FLAME-3 Death effector domain (DED)-containing proteins play important roles in apoptosis and cellular signaling.1,10,21 To identify new DED-containing proteins, we searched the entire public GenBankTM EST database for sequence homologus to previously known DED-containing proteins, using the tBLASTN program. One human EST clone (AA090354) encoding a partial open reading frame with significant homology to the C-terminal domain of DEDD protein was identified. Based on its sequence, PCR primers were generated and 5'-RACE was performed with a human PBL library. This resulted in cloning of the entire open reading frame of this new protein, which was subsequently named FLAME-3 based on its overall sequence and structural homology with DEDD/FLAME-2. The human FLAME-3 (Figure 1A) is 46.6% identical to DEDD and its domain structure is very similar to that of DEDD (Figure 1B). FLAME-3 has an N-terminal death effector domain (DED, residues 25 ± 102), which is very similar to the DED Figure 1 Sequence alignment and domain structure of FLAME-3. (A)Co- domains present in other DED proteins such as DEDD, linear alignment of the predicted amino acid sequences of human and mouse FADD, caspase-8, caspase-10 and c-FLIP (FLAME-1) FLAME-3 proteins. Non-identical residues in the two sequences are shaded (Figure 1C). Following the DED, FLAME-3 contains three black. (B) Bar diagrams representing the domain structure of human FLAME-3 central nuclear localization sequences (NLSs, residues and DEDD proteins. The three NLSs in FLAME-3 (residues 104 ± 109, 131 ± 104 ± 109, 131 ± 134 and 153 ± 174) and a C-terminal 134 and 153 ± 174) and two NLSs in DEDD (residues 104 ± 107 and 164 ± 181) are indicated. (C) Amino acid sequence alignment of the DED of FLAME-3 with domain (CTD, residues 175 ± 326), which has no significant DEDs found in human DEDD, FADD, caspase-8, caspase-10 and c-FLIP. homology to any known protein except DEDD. Black shading indicates identical residues The mouse FLAME-3 was also identified by searching the GenBankTM EST database and a cDNA encoding its entire open reading frame was obtained by overlapping Northern blot analysis of multiple human tissues PCR. The human and mouse FLAME-3 proteins share 91% detected two FLAME-3 mRNA species of 1.6 and 2.0 identity with each other (Figure 1A). Interestingly, most of kilobase pairs (kb) in all tissues examined (Figure 2A). the amino acid substitutions in the human and mouse These two mRNA species could result from the use of sequences are present in the linker region, which contain alternative polyadenylation sites and/or alternative splicing the NLSs between the DED and CTD. The lack of of the FLAME-3 pre-mRNA. The highest level of expression significant substitutions in the sequences of the DED and of the FLAME-3 mRNA was found in peripheral blood CTD suggests that the overall structures of these domains leukocytes, spleen and testes and lower expression levels are critical for the function of FLAME-3. were detected in all other tissues tested. The high A BLAST search of the NCBI high-throughput genome expression of FLAME-3 mRNA in peripheral blood sequences revealed that the human FLAME-3 gene is leukocytes, spleen and testes is reminiscent of that of the located on chromosome 19. DEDD mRNA,22 suggesting that the two genes are under Cell Death and Differentiation DEDD and FLAME-3 interact with the TFIIIC transcriptional complex YZhanet al 441 DEDD nor GST ± FLAME-3 fusion proteins did interact with FADD or caspase-10 (Figure 3B). However, both fusion proteins interacted with c-FLIP and weakly with caspase-8.