Identification and characterization of DEDDL, a human-specific isoform of DEDD

Abstract

Death effector domain (DED) containing molecules are usually involved in the intracellular apoptosis cascade as executioners or regulators. One of these molecules, DEDD, was identified as a final target of the CD95 signaling pathway by which it would be transferred into the nucleolus to inhibit RNA polymerase I-dependent transcription. Here we describe a longer isoform of DEDD, DEDDL, produced by alternatively splicing, as an immune cell-specific DED-containing molecule. It is only expressed in human T lymphocytes and dendritic cells (DCs), and the mRNA expression in DCs was elevated upon inductive maturation. In cell lines MCF-7 and Jurkat, the overexpression of DEDDL could induce apoptosis more potently than that of DEDD. That DEDDL could bind FADD and cFLIP more potently than DEDD in vivo was revealed by cotransfection and immunoprecipitation. This may explain why DEDDL is a more potent apoptosis inducer, because DED-containing proteins usually induce apoptosis through DED binding. Finally, why DEDD and DEDDL are unstable in the overexpression and other studies may be explained by the finding that they are potential substrates of active caspases.

Figure 1

Identification of DEDDl, an alternatively spliced form of DEDD. (A) Partial sequence of DEDDl protein deduced form exon 3 and exon 4. The prolonged part of DEDDl is underlined and the putative ITIM is shaded. (B) Alignment of the genome sequence from exon 3 to exon 4 from human (NCBI, AL591806.16, 177237–176402) and mouse (NCBI, AC087229.26, 85551–86323). The exons are underlined and the prolonged part of DEDDl is double underlined.

Figure 2

mRNA expression of DEDDl in immune cells. (A) Hematopoietic cell lines and immune cells were arranged in the following order: lane 1, NB4; lane 2, HL-60; land 3, U937; lane 4, K562; lane 5, Raji; lane 6, Daudi; lane 7, HuT78; lane 8, Molt-4; lane 9, DC; lane 10, DC-KLH. (B) Lane 1, DC; lanes 2–4, DC treated with LPS for 1, 8, and 48 h; lanes 5–7, DC treated with TNF-α for 1, 8, and 48 h. (C) Lane 1, T lymphocytes; lanes 2–4, T lymphocytes treated with PHA for 1 and 8 h and 4 days; lanes 5–7, T lymphocytes treated with rIL-2 for 1 and 8 h and 4 days.

Figure 3

DEDDl is a more potent apoptosis inducer than DEDD. MCF-7 cells were transfected with DEDDl or DEDD genes and treated with TNF-α for indicated times. The percentage of apoptosis cells indicated were counted by PI-positive (I) plus Rh123 low staining (II) cells.

Figure 4

Comparison of the apoptosis induction between Jurkat T cells transfected with DEDDl or DEDD.

Figure 5

Cellular localization of DEDDl. Cells were transfected with Myc-tagged DEDDl and subjected to immunofluorescence staining with 9B11 monoclonal anti-Myc antibody followed with FITC-labeled secondary antibody. (A) Transfected 293T cells. (B) TNF-α-treated transfected 293T cells. (C) Tranfected MCF-7 cells. (D) TNF-α-treated transfected MCF-7 cells.

Figure 6

Interaction of DEDDl with other DED-containing proteins. (A) DEDDl binds with FADD and cFLIP much stronger than does DEDD, especially with cFLIP. 293T cells were transiently transfected with various combinations of plasmids encoding FLAG-DEDDl, FLAG-DEDD, Myc-FADD, and Myc-cFLIP. Cell lysates were prepared and immunoprecipitations were performed using anti-Myc monoclonal antibodies (9B11), followed by Western blot analysis using anti-FLAG M2 monoclonal antibody. To show the difference clearly, the same blot was exposed to film for a short period and long period as indicated. (B) DEDDl and DEDD cannot bind caspase-8 in vivo. 293T cells were transfected with DEDDl or DEDD combined with caspase-8 with or without the presence of crmA. Cell lysates were immunprecipitated by using the anti-caspase-8 monoclonal antibody and the following immunoblotting was perfomed with anti-Myc antibody. (C) DEDDl and DEDD bind each other. (D) Caspase-8 can degrade DEDDl and DEDD.