Tumor necrosis factor receptor-associated factor 6 (TRAF6) stimulates extracellular signal-regulated kinase (ERK) activity in CD40 signaling along a ras-independent pathway

Abstract

CD40 activates nuclear factor kappa B (NF kappa B) and the mitogen-activated protein kinase (MAPK) subfamily, including extracellular signal-regulated kinase (ERK). The CD40 cytoplasmic tail interacts with tumor necrosis factor receptor-associated factor (TRAF)2, TRAF3, TRAF5, and TRAF6. These TRAF proteins, with the exception of TRAF3, are required for NF kappa B activation. Here we report that transient expression of TRAF6 stimulated both ERK and NF kappa B activity in the 293 cell line. Coexpression of the dominant-negative H-Ras did not affect TRAF6-mediated ERK activity, suggesting that TRAF6 may activate ERK along a Ras-independent pathway. The deletion mutant of TRAF6 lacking the NH2-terminal domain acted as a dominant-negative mutant to suppress ERK activation by full-length CD40 and suppress prominently ERK activation by a deletion mutant of CD40 only containing the binding site for TRAF6 in the cytoplasmic tail (CD40 delta 246). Transient expression of the dominant-negative H-Ras significantly suppressed ERK activation by full-length CD40, but marginally suppressed ERK activation by CD40 delta 246, compatible with the possibility that TRAF6 is a major transducer of ERK activation by CD40 delta 246, whose activity is mediated by a Ras-independent pathway. These results suggest that CD40 activates ERK by both a Ras-dependent pathway and a Ras-independent pathway in which TRAF6 could be involved.

Figure 1

Transient expression of TRAF6-stimulated ERK activity in 293 cells. (A) Cells (10⁶) were transiently transfected with expression plasmid of TRAF proteins (5 μg each) together with GST-ERK2. GST-ERK2 fusion protein was precipitated by use of glutathione Sepharose 4B from cell lysates 48 h after transfection and subjected to in vitro kinase assay using MBP as a substrate. Arrow, phosphorylated MBP in the autoradiogram. (B) Cells (10⁶) were transiently transfected with 100 ng of ERK-dependent activator pGAL4MycN and 500 ng of the reporter pGAL4tkLuc (a) or 20 ng of the NFκB-dependent luciferase reporter pkBtkLuc (b), together with empty vector, full-length TRAF6 (3 or 5 μg), or the TRAF-C of TRAF6 (3 or 5 μg). Total amount of DNA was adjusted with empty vector at 10 μg. Luciferase activities were measured 48 h after transfection and normalized by β-gal expression. The extent of stimulation (relative activation) was calculated by comparison between luciferase activity given by the cells cotransfected with each TRAF expression plasmid and that of cells transfected with empty vector. (C) Cells (10⁶) were transiently cotransfected with either TRAF2, TRAF3, or TRAF5 (5 μg each) and ERK– (a) or NFκB–dependent luciferase reporter (b). Cell lysates were subjected to luciferase assay 48 h after transfection. The results shown in B and C were obtained from parallel experiments. For comparison, the extent of stimulation in ERK and NFκB by transient expression of TRAF6 (5 μg) in B is also shown in C. The mean value of two independent experiments (column) was calculated from a mean value of a single experiment in triplicate (closed circles).

Figure 4

ERK activation by cross-linking of full-length, but not that by CD40Δ246, was prominently suppressed by dominant negative mutant of Ras. Cells (10⁶) were cotransfected with ERK– (A) or NFκB–dependent reporter (B) and either 1 μg of full-length CD40 (a) or CD40Δ246 (b), together with 5 μg of N17Ras, DNRaf, and MEK^DN. Total amount of DNA was adjusted with empty vector at 10 μg. Cells were stimulated with anti-CD40 mAb 24 h after transfection. After 24 h incubation, cell lysates were subjected to luciferase assay. Relative activation of ERK and NFκB in each transfection was calculated as described above. The mean value of two to three independent experiments (column) was calculated from a mean value of a single experiment in triplicate (closed circle).

Figure 2

Inhibition of TRAF6-mediated ERK activity by dominant-negative mutant of Raf and MEK1, but not by Ras. Cells (10⁶) were cotransfected with ERK-dependent activator and reporter (A) or 500 ng of GST-ERK2 (B) and full-length TRAF6 (5 μg), together with 5 μg of N17Ras (DN Ras), DNRaf, or MEK1^DN (DN MEK). Cell lysates were prepared 48 h after transfection and subjected to luciferase assay (A) or in vitro kinase assay (B). The mean value of relative activity of ERK- dependent luciferase reporter (column) in four independent experiments was calculated from a mean value of a single experiment in triplicate (closed circles). Arrowhead, phosphorylated MBP in the autoradiogram.

Figure 3

Transient expression of TRAF-C of TRAF6 caused inhibition of ERK and NFκB activity mediated by cross-linking of CD40. Cells (10⁶) were transiently cotransfected with ERK-dependent activator and reporter (A), GST-ERK2 fusion construct (B), or NFκB-dependent reporter (C) and either l μg of full-length CD40 (a) or CD40Δ246 (b), together with 1, 3, or 5 μg of TRAF-C domain of TRAF6. Total amount of DNA was adjusted with empty vector at 10 μg. Cells were stimulated with anti-CD40 mAb 24 h after transfection. After 24 h of incubation, ERK activity was measured by luciferase assay (A) or in vitro kinase assay (B). Arrowhead, phosphorylated MBP in the autoradiogram is indicated by an arrowhead. NFκB activity was measured by luciferase assay (C). Relative activation of ERK and NFκB in 293 cells at each transfection was calculated as described above. The mean value of two independent experiments (column) was calculated from a mean value of a single experiment in triplicate (closed circles).