4-1BB and Ox40 are members of a tumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bind TNF receptor-associated factors and activate nuclear factor kappaB

Abstract

Members of the tumor necrosis factor (TNF)-nerve growth factor (NGF) receptor family have been shown to be important costimulatory molecules for cellular activation. 4-1BB and Ox40 are two recently described members of this protein family which are expressed primarily on activated T cells. To gain insight into the signaling pathways employed by these factors, yeast two-hybrid library screens were performed with the cytoplasmic domains of 4-1BB and Ox40 as baits. TNF receptor-associated factor 2 (TRAF2) was identified as an interacting protein in both screens. The ability of both 4-1BB and Ox40 to interact with TRAF2 was confirmed in mammalian cells by coimmunoprecipitation studies. When the binding of the receptors to other TRAF proteins was investigated, 4-1BB and Ox40 displayed distinct binding patterns. While 4-1BB bound TRAF2 and TRAF1, Ox40 interacted with TRAF3 and TRAF2. Using deletion and alanine scanning analysis, we defined the elements in the cytoplasmic domains of both receptors that mediate these interactions. The 4-1BB receptor was found to have two independent stretches of acidic residues that can mediate association of the TRAF molecules. In contrast, a single TRAF binding domain was identified in the cytoplasmic tail of Ox40. The cytoplasmic domains of both receptors were shown to activate nuclear factor kappaB in a TRAF-dependent manner. Taken together, our results indicate that 4-1BB and Ox40 bind TRAF proteins to initiate a signaling cascade leading to activation of nuclear factor kappaB.

FIG. 1

TRAF2 binds to m4-1BBCP and mOx40CP in HEK293 cells. (A) Representation of the chimeric constructs used for the immunoprecipitation and luciferase experiments. The extracellular and transmembrane domains of murine CD28 were fused in frame to the entire cytoplasmic tail (aa 213 to 257) or a deletion mutant (aa 213 to 246) lacking the C-terminal 11 aa of murine 4-1BB or to the entire cytoplasmic domain of Ox40 (aa 237 to 272). (B) Coimmunoprecipitation of TRAF2 with m4-1BBCP and mOx40CP. The chimeric proteins were immunoprecipitated with an anti-CD28 serum after transfection of HEK293 cells. Immunoprecipitates were separated on sodium dodecyl sulfate–10% polyacrylamide gels under reducing conditions and analyzed by Western blotting with an anti-TRAF2 serum. The left panel shows the immunoprecipitates (IP); the right panel shows 5% of the cell lysates of 3 × 10⁶ cells used for immunoprecipitation. The transfected receptor constructs are indicated above the lanes. Positions of truncated human TRAF2 protein and of immunoglobulin heavy and light chains (asterisks) are indicated.

FIG. 2

Directed yeast two-hybrid analyses using cytoplasmic domains of murine 4-1BB and Ox40 as baits. The TRAF molecules used for these experiments were cloned in frame to the GAL4 transactivation domain of pACT, and the designations of the resulting constructs are indicated. All filters were incubated for 12 h at room temperature to determine β-galactosidase activity. (A) Representative filters of the assays performed with m4-1BBCP and indicated mutants of this molecule as bait. (B) Representative filters of the yeast two-hybrid experiments done with the entire cytoplasmic tail of Ox40 (mOx40CP) and the indicated mutants as baits.

FIG. 3

TRAF binding domains in the cytoplasmic tails of 4-1BB and Ox40 are conserved between species. (A) Alignment of the protein sequences of the cytoplasmic domains of 4-1BB and Ox40. The amino acid residues shown to be important for interaction of the TRAF molecules with either receptor are indicated by grey boxes. The tables summarize identical and similar amino acids in the cytoplasmic tails of the different species determined by BLAST analyses (4), showing percentages of similarity (top) and identity (bottom) between species of the amino acids in the grey boxes. The numbers in parentheses indicate the overall identity or similarity of the cytoplasmic tails between the various species. (B) Summary of yeast two-hybrid experiments. At least three experiments were performed for all analyses as described in the legend to Fig. 2. Shown is the level of β-galactosidase expression after 12 h at room temperature. +++, very strong signals; ++, strong signals; +, weak but significant interactions, −, no signal; n.d., not determined.

FIG. 4

4-1BB induces NF-κB activation. (A) HEK293 cells were transfected with the chimeric CD28 constructs of a full-length CD28 expression vector, β-galactosidase expression constructs, and luciferase reporter constructs containing either two canonical NF-κB sites or a minimal promoter. Cells were harvested 25 h after transfection and analyzed for luciferase activity. The relative luciferase units were standardized to the β-galactosidase expression levels. The induction of the luciferase activity was calculated by dividing the relative luciferase units obtained after transfection of the reporter plasmid with the 2×NF-κB promoter by the relative luciferase units obtained after transfection of the reporter plasmid with the minimal promoter. The transfected DNAs are indicated. The error bars represent the standard deviations of triplicate transfections. Shown is one representative experiment of three independent transfection experiments. (B) HEK293 cells were transfected with a chimeric receptor construct of 4-1BB alone or cotransfected with full-length TRAF2 or a deletion mutant lacking most of the N-terminal ring finger (TRAF2DN) and assayed for NF-κB induction as described for panel A. Black bars represent the reporter construct with a minimal promoter; grey bars indicate transfections with the NF-κB reporter plasmid. The data are representative of three independent experiments.

FIG. 5

TRAF proteins regulate Ox40-mediated NF-κB activation. HEK293 cells were transfected with DNAs as indicated. The inductions of NF-κB were calculated as described in the legend to Fig. 4A. The reporter construct with a minimal promoter is indicated by black bars; grey bars represent transfections with the NF-κB reporter plasmid. (A) HEK293 cells were transfected with a full-length CD28 expression vector or chimeric CD28 fusion proteins, and the induction of NF-κB was determined as described in the legend to Fig. 4A. (B) A CD28-Ox40CP fusion protein was expressed alone or in combination with full-length TRAF2 or TRAF3. Induction of NF-κB was calculated as described in the legend to Fig. 4A. Shown is the average induction of NF-κB obtained in three independent experiments that were all done as triplicate transfections. The error bars show the standard error of the mean for all transfections. (C) A CD28-fusion protein of Ox40CP was transfected alone or in combination with either the dominant negative TRAF2DN, full-length TRAF3, or an N-terminal deletion mutant of TRAF3 lacking aa 1 to 381 (TRAF3ΔN). The error bars represent the standard deviations of triplicate transfections. The experiment shown is representative of at least three independent transfection experiments.