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. 1997 Nov 1;11(21):2810-21.
doi: 10.1101/gad.11.21.2810.

CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival

C S Duckett  1 , C B Thompson
Affiliations

CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival

C S Duckett et al. Genes Dev. .

Abstract

CD30 is a cell-surface receptor that can augment lymphocyte activation and survival through its ability to induce the transcription factor NF-kappaB. CD30, however, has also been implicated in the induction of apoptotic cell death of lymphocytes. Here we show that one of the effects of CD30 signal transduction is to render cells sensitive to apoptosis induced by the type 1 tumor necrosis factor receptor (TNFR1). This sensitization is dependent on the TRAF-binding sites within the CD30 cytoplasmic domain. One of the proteins that binds to these sites is TRAF2, a signal transduction molecule that is also utilized by TNFR1 to mediate the activation of several downstream kinases and transcription factors. During CD30 signal transduction, we found that binding of TRAF2 to the cytoplasmic domain of CD30 results in the rapid depletion of TRAF2 and the associated protein TRAF1 by proteolysis. These data suggest a model in which CD30 limits its own ability to transduce cell survival signals through signal-coupled depletion of TRAF2. Depletion of intracellular TRAF2 and its coassociated proteins also increased the sensitivity of the cell to undergoing apoptosis during activation of death-inducing receptors such as TNFR1. Consistent with this hypothesis, expression of a dominant-negative form of TRAF2 was found to potentiate TNFR1-mediated death. These studies provide a potential mechanism through which CD30, as well as other TRAF-binding members of the TNFR superfamily, can negatively regulate cell survival.

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Figures

Figure 1
Figure 1
Potentiation of TNFR1-mediated cell death by constitutively activated chimeric CD30. (A) 293 cells were transfected with a vector (100 ng) encoding GFP together with expression vectors (1 μg) encoding a fusion between CD28 and CD30, a mutated CD28–CD30 vector lacking the carboxy-terminal TRAF-binding sites (ΔTraf), or a control CD28 mutant lacking a cytoplasmic tail (ΔTail), as indicated. Twelve hours following transfection, cells were stimulated with human TNF-α (200 U/ml) or a medium control for a further 24 hr, and viability of GFP-expressing cells was determined by fluorescence microscopy as described in Materials and Methods. (B) CD30-enhanced death is blocked by apoptosis inhibitors. 293 cells were transfected either with the CD28 ΔTail control vector (Control) or with the CD28–CD30 chimera (CD30), together with the indicated expression vectors. Transfectants were stimulated with recombinant TNF-α and viability determined as described above and in Materials and Methods.
Figure 1
Figure 1
Potentiation of TNFR1-mediated cell death by constitutively activated chimeric CD30. (A) 293 cells were transfected with a vector (100 ng) encoding GFP together with expression vectors (1 μg) encoding a fusion between CD28 and CD30, a mutated CD28–CD30 vector lacking the carboxy-terminal TRAF-binding sites (ΔTraf), or a control CD28 mutant lacking a cytoplasmic tail (ΔTail), as indicated. Twelve hours following transfection, cells were stimulated with human TNF-α (200 U/ml) or a medium control for a further 24 hr, and viability of GFP-expressing cells was determined by fluorescence microscopy as described in Materials and Methods. (B) CD30-enhanced death is blocked by apoptosis inhibitors. 293 cells were transfected either with the CD28 ΔTail control vector (Control) or with the CD28–CD30 chimera (CD30), together with the indicated expression vectors. Transfectants were stimulated with recombinant TNF-α and viability determined as described above and in Materials and Methods.
Figure 2
Figure 2
TRAF2 is destabilized by CD30. 293 cells were cotransfected with TRAF2 and CD28 chimera expression vectors as indicated. (A) Cells were lysed 18 hr following transfection, standardized for protein levels, and TRAF2 levels were detected by immunoblot analysis as described in Materials and Methods. (B) Total RNA was prepared from an equivalent aliquot of cells used for A, and TRAF2 mRNA was evaluated by Northern analysis with a TRAF2 cDNA probe. (Bottom) An ethidium bromide-stained agarose gel to standardize the RNA samples; (top) an autoradiograph of the TRAF2-probed blot.
Figure 3
Figure 3
TRAF2 destabilization can be blocked by protease inhibitors. 293 cells were transfected with TRAF2 and chimeric CD28–CD30 expression vectors as indicated. Parallel wells were stimulated 36 hr following transfection with the indicated protease inhibitors each at a final concentration of 50 μm or with a DMSO solvent control at a final concentration of 0.001%, and lysates were prepared 48 hr after transfection. Standardized extracts were examined for TRAF2 by immunoblot analysis as described in Materials and Methods.
Figure 4
Figure 4
Determinants of TRAF degradation. (A) 293 cells were transfected with expression vectors encoding the indicated proteins. Lysates were prepared 48 hr following transfection and standardized aliquots were subjected to immunoblot analysis with a polyclonal antibody to TRAF1 (top) or TRAF2 (bottom). (B) 293 cells were transfected with the indicated plasmids, including either wild-type TRAF2 (1-501) or a dominant-negative mutant (87–501) lacking a functional RING finger, as indicated. Cells were prepared 24 hr following transfection and analyzed as described in A with a TRAF2 polyclonal antibody.
Figure 4
Figure 4
Determinants of TRAF degradation. (A) 293 cells were transfected with expression vectors encoding the indicated proteins. Lysates were prepared 48 hr following transfection and standardized aliquots were subjected to immunoblot analysis with a polyclonal antibody to TRAF1 (top) or TRAF2 (bottom). (B) 293 cells were transfected with the indicated plasmids, including either wild-type TRAF2 (1-501) or a dominant-negative mutant (87–501) lacking a functional RING finger, as indicated. Cells were prepared 24 hr following transfection and analyzed as described in A with a TRAF2 polyclonal antibody.
Figure 5
Figure 5
Both TRAF-binding sites in CD30 contribute to TRAF degradation. 293 cells were cotransfected with TRAF2 and CD28 chimera expression vectors encoding wild-type CD30, tailless CD28 (ΔTail), or mutated proteins lacking both TRAF-binding motifs (Δ dom 2) or each of the individual TRAF-binding domains (Δ dom 2A or Δ dom 2B), as indicated. Cells were lysed 18 hr following transfection and standardized for protein levels, and TRAF2 levels were detected by immunoblot analysis as described in Materials and Methods.
Figure 6
Figure 6
TNFR2 can enhance TNFR1 sensitivity and induces TRAF2 degradation. (A) 293 cells were transfected with the indicated CD28 chimeric vectors (1 μg), along with a GFP vector (100 ng), exactly as described in the legend to Fig. 1. Transfectants were stimulated with TNF-α (200 U/ml) and cell viability was determined by DAPI staining and fluorescence microscopy as described in Materials and Methods. (B) 293 cells were transfected with the TRAF2 expression vector along with the indicated chimeric vectors. Twelve hours following transfection, cells were either mock treated or treated with human TNF-α (200 U/ml) as indicated. Cells were harvested 24 hr following transfection, standardized by protein content and TRAF2 was detected by immunoblot analysis as described in Materials and Methods.
Figure 6
Figure 6
TNFR2 can enhance TNFR1 sensitivity and induces TRAF2 degradation. (A) 293 cells were transfected with the indicated CD28 chimeric vectors (1 μg), along with a GFP vector (100 ng), exactly as described in the legend to Fig. 1. Transfectants were stimulated with TNF-α (200 U/ml) and cell viability was determined by DAPI staining and fluorescence microscopy as described in Materials and Methods. (B) 293 cells were transfected with the TRAF2 expression vector along with the indicated chimeric vectors. Twelve hours following transfection, cells were either mock treated or treated with human TNF-α (200 U/ml) as indicated. Cells were harvested 24 hr following transfection, standardized by protein content and TRAF2 was detected by immunoblot analysis as described in Materials and Methods.
Figure 7
Figure 7
Dominant-negative TRAF2 can potentiate TNFR1-mediated death. 293 cells were transfected with 100 ng of GFP plasmid, together with 1 μg of the indicated plasmids, and either mock treated or treated with human TNF-α (200 U/ml), and cell viability was determined as described in the legend to Fig. 1 and in Materials and Methods.
Figure 8
Figure 8
CD30 signaling abrogates NF-κB induction by TNF-α. 293 cells were transfected with 50 ng each of κB-luciferase reporter plasmid and β-galactosidase plasmid, along with 100 ng of either CD28–CD30 vector or tailless CD28 as indicated. Cells were stimulated with TNF-α 48 hr following transfection, and incubated further for 10 hr prior to harvest. The induction of NF-κB is presented as the percentage increase in NF-κB of TNF-α-treated samples over the activity of the reporter plasmid in mock-treated cells. Luciferase assays were performed as described in Materials and Methods.
Figure 9
Figure 9
Model: Control of TRAF2 levels may modulate the sensitivity of cells to TNFR1 signals (see text for details).
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