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Regulation of the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF)2 by TRAF1 reveals mechanisms of TRAF2 signaling.

Arron JR, Pewzner-Jung Y, Walsh MC, Kobayashi T, Choi Y - J. Exp. Med. (2002)

Bottom Line: TRAF1(-/-) dendritic cells show attenuated responses to secondary stimulation by TRAF2-dependent factors and increased stimulus-dependent TRAF2 degradation.Replacement of the RING finger of TRAF2 with a raft-targeting signal restores JNK activation and association with the cyto-skeletal protein Filamin, but not NF-kappaB activation.These findings offer insights into the mechanism of TRAF2 signaling and identify a physiological role for TRAF1 as a regulator of the subcellular localization of TRAF2.

View Article: PubMed Central - PubMed

Affiliation: Tri-Institutional MD-PhD Program, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
Tumor necrosis factor receptor-associated factor (TRAF)2 is a critical adaptor molecule for tumor necrosis factor (TNF) receptors in inflammatory and immune signaling. Upon receptor engagement, TRAF2 is recruited to CD40 and translocates to lipid rafts in a RING finger-dependent process, which enables the activation of downstream signaling cascades including c-Jun NH(2)-terminal kinase (JNK) and nuclear factor (NF)-kappaB. Although TRAF1 can displace TRAF2 and CD40 from raft fractions, it promotes the ability of TRAF2 activate signaling over a sustained period of time. Removal of the RING finger of TRAF2 prevents its translocation into detergent-insoluble complexes and renders it dominant negative for signaling. TRAF1(-/-) dendritic cells show attenuated responses to secondary stimulation by TRAF2-dependent factors and increased stimulus-dependent TRAF2 degradation. Replacement of the RING finger of TRAF2 with a raft-targeting signal restores JNK activation and association with the cyto-skeletal protein Filamin, but not NF-kappaB activation. These findings offer insights into the mechanism of TRAF2 signaling and identify a physiological role for TRAF1 as a regulator of the subcellular localization of TRAF2.

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TRAF1 regulates the detergent solubility of TRAF2. (A) 293T HEK cells were transfected in 6-well plates with the indicated amounts of CD40, TRAF1, and TRAF2. Total DNA content was maintained constant at 1 μg by the addition of empty vector. Cells were lysed in 0.75% Triton X-100, and soluble (S) and insoluble (I) fractions were immunoblotted as indicated. After probing with TRAF2 antibodies (C-20), blots were stripped and reprobed with anti-Flag M2 to detect TRAF1 and CD40. (B) As in panel A, but without transfection of CD40. hTNF-α (10 ng/ml) was added to the culture medium 6 h before harvesting. (C) As in panel A. TRAF2 was transfected in the amounts indicated. (D) As in panel A, but with 0.1 μg of TRAF2 or an NH2-terminal truncation mutant removing the first 87 residues (comprising the RING finger) of TRAF2 (T2Δ87). 0.5 μg of TRAF1 was transfected where indicated (+). (E) As in panel A. (F) 293T cells were transfected with 1.5 μg of TRAF2 or T2Δ87, 2.5 μg of TRAF1, and 1.0 μg of CD40 where indicated. Cells were treated with CD40L 6 h before harvesting then subjected to sucrose gradient density centrifugation as described in Materials and Methods and immunoblotted as indicated.
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fig1: TRAF1 regulates the detergent solubility of TRAF2. (A) 293T HEK cells were transfected in 6-well plates with the indicated amounts of CD40, TRAF1, and TRAF2. Total DNA content was maintained constant at 1 μg by the addition of empty vector. Cells were lysed in 0.75% Triton X-100, and soluble (S) and insoluble (I) fractions were immunoblotted as indicated. After probing with TRAF2 antibodies (C-20), blots were stripped and reprobed with anti-Flag M2 to detect TRAF1 and CD40. (B) As in panel A, but without transfection of CD40. hTNF-α (10 ng/ml) was added to the culture medium 6 h before harvesting. (C) As in panel A. TRAF2 was transfected in the amounts indicated. (D) As in panel A, but with 0.1 μg of TRAF2 or an NH2-terminal truncation mutant removing the first 87 residues (comprising the RING finger) of TRAF2 (T2Δ87). 0.5 μg of TRAF1 was transfected where indicated (+). (E) As in panel A. (F) 293T cells were transfected with 1.5 μg of TRAF2 or T2Δ87, 2.5 μg of TRAF1, and 1.0 μg of CD40 where indicated. Cells were treated with CD40L 6 h before harvesting then subjected to sucrose gradient density centrifugation as described in Materials and Methods and immunoblotted as indicated.

Mentions: Recently, several groups have shown that CD40 engagement results in translocation of TRAF2 to detergent-resistant membranes (13–17). As TRAF1 can hetero-oligomerize with TRAF2 and interact with the TRAF2 binding site of CD40, we investigated the effect of TRAF1 on the solubility of TRAF2 in nonionic detergent (0.75% Triton X-100). We cotransfected HEK 293T cells with constant amounts of plasmids driving the expression of CD40 and TRAF2, while titrating the amount of TRAF1. In the absence of TRAF1, a majority of TRAF2 was found in the insoluble fraction, whereas the addition of TRAF1 resulted in a dose-dependent redistribution of TRAF2 to the soluble fraction (Fig. 1 A). TNF-α stimulation for the last 6 h before harvesting of cells transfected with TRAF2 results in a similar distribution of TRAF2 to the insoluble fraction, which is reversed by increasing doses of transfected TRAF1 (Fig. 1 B). As overexpression of TRAF2 can activate signaling independent of receptor engagement, we examined the solubility of overexpressed TRAF2 over a range of concentrations. At low concentrations (similar to those used in Fig. 1, A and B), TRAF2 is predominantly soluble, consistent with the inability of low concentrations of TRAF2 to self-aggregate and activate signaling. At higher concentrations of TRAF2 consistent with the ability to independently activate signaling, an increasing fraction of TRAF2 is insoluble (Fig. 1 C).


Regulation of the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF)2 by TRAF1 reveals mechanisms of TRAF2 signaling.

Arron JR, Pewzner-Jung Y, Walsh MC, Kobayashi T, Choi Y - J. Exp. Med. (2002)

TRAF1 regulates the detergent solubility of TRAF2. (A) 293T HEK cells were transfected in 6-well plates with the indicated amounts of CD40, TRAF1, and TRAF2. Total DNA content was maintained constant at 1 μg by the addition of empty vector. Cells were lysed in 0.75% Triton X-100, and soluble (S) and insoluble (I) fractions were immunoblotted as indicated. After probing with TRAF2 antibodies (C-20), blots were stripped and reprobed with anti-Flag M2 to detect TRAF1 and CD40. (B) As in panel A, but without transfection of CD40. hTNF-α (10 ng/ml) was added to the culture medium 6 h before harvesting. (C) As in panel A. TRAF2 was transfected in the amounts indicated. (D) As in panel A, but with 0.1 μg of TRAF2 or an NH2-terminal truncation mutant removing the first 87 residues (comprising the RING finger) of TRAF2 (T2Δ87). 0.5 μg of TRAF1 was transfected where indicated (+). (E) As in panel A. (F) 293T cells were transfected with 1.5 μg of TRAF2 or T2Δ87, 2.5 μg of TRAF1, and 1.0 μg of CD40 where indicated. Cells were treated with CD40L 6 h before harvesting then subjected to sucrose gradient density centrifugation as described in Materials and Methods and immunoblotted as indicated.
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fig1: TRAF1 regulates the detergent solubility of TRAF2. (A) 293T HEK cells were transfected in 6-well plates with the indicated amounts of CD40, TRAF1, and TRAF2. Total DNA content was maintained constant at 1 μg by the addition of empty vector. Cells were lysed in 0.75% Triton X-100, and soluble (S) and insoluble (I) fractions were immunoblotted as indicated. After probing with TRAF2 antibodies (C-20), blots were stripped and reprobed with anti-Flag M2 to detect TRAF1 and CD40. (B) As in panel A, but without transfection of CD40. hTNF-α (10 ng/ml) was added to the culture medium 6 h before harvesting. (C) As in panel A. TRAF2 was transfected in the amounts indicated. (D) As in panel A, but with 0.1 μg of TRAF2 or an NH2-terminal truncation mutant removing the first 87 residues (comprising the RING finger) of TRAF2 (T2Δ87). 0.5 μg of TRAF1 was transfected where indicated (+). (E) As in panel A. (F) 293T cells were transfected with 1.5 μg of TRAF2 or T2Δ87, 2.5 μg of TRAF1, and 1.0 μg of CD40 where indicated. Cells were treated with CD40L 6 h before harvesting then subjected to sucrose gradient density centrifugation as described in Materials and Methods and immunoblotted as indicated.
Mentions: Recently, several groups have shown that CD40 engagement results in translocation of TRAF2 to detergent-resistant membranes (13–17). As TRAF1 can hetero-oligomerize with TRAF2 and interact with the TRAF2 binding site of CD40, we investigated the effect of TRAF1 on the solubility of TRAF2 in nonionic detergent (0.75% Triton X-100). We cotransfected HEK 293T cells with constant amounts of plasmids driving the expression of CD40 and TRAF2, while titrating the amount of TRAF1. In the absence of TRAF1, a majority of TRAF2 was found in the insoluble fraction, whereas the addition of TRAF1 resulted in a dose-dependent redistribution of TRAF2 to the soluble fraction (Fig. 1 A). TNF-α stimulation for the last 6 h before harvesting of cells transfected with TRAF2 results in a similar distribution of TRAF2 to the insoluble fraction, which is reversed by increasing doses of transfected TRAF1 (Fig. 1 B). As overexpression of TRAF2 can activate signaling independent of receptor engagement, we examined the solubility of overexpressed TRAF2 over a range of concentrations. At low concentrations (similar to those used in Fig. 1, A and B), TRAF2 is predominantly soluble, consistent with the inability of low concentrations of TRAF2 to self-aggregate and activate signaling. At higher concentrations of TRAF2 consistent with the ability to independently activate signaling, an increasing fraction of TRAF2 is insoluble (Fig. 1 C).

Bottom Line: TRAF1(-/-) dendritic cells show attenuated responses to secondary stimulation by TRAF2-dependent factors and increased stimulus-dependent TRAF2 degradation.Replacement of the RING finger of TRAF2 with a raft-targeting signal restores JNK activation and association with the cyto-skeletal protein Filamin, but not NF-kappaB activation.These findings offer insights into the mechanism of TRAF2 signaling and identify a physiological role for TRAF1 as a regulator of the subcellular localization of TRAF2.

View Article: PubMed Central - PubMed

Affiliation: Tri-Institutional MD-PhD Program, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
Tumor necrosis factor receptor-associated factor (TRAF)2 is a critical adaptor molecule for tumor necrosis factor (TNF) receptors in inflammatory and immune signaling. Upon receptor engagement, TRAF2 is recruited to CD40 and translocates to lipid rafts in a RING finger-dependent process, which enables the activation of downstream signaling cascades including c-Jun NH(2)-terminal kinase (JNK) and nuclear factor (NF)-kappaB. Although TRAF1 can displace TRAF2 and CD40 from raft fractions, it promotes the ability of TRAF2 activate signaling over a sustained period of time. Removal of the RING finger of TRAF2 prevents its translocation into detergent-insoluble complexes and renders it dominant negative for signaling. TRAF1(-/-) dendritic cells show attenuated responses to secondary stimulation by TRAF2-dependent factors and increased stimulus-dependent TRAF2 degradation. Replacement of the RING finger of TRAF2 with a raft-targeting signal restores JNK activation and association with the cyto-skeletal protein Filamin, but not NF-kappaB activation. These findings offer insights into the mechanism of TRAF2 signaling and identify a physiological role for TRAF1 as a regulator of the subcellular localization of TRAF2.

Show MeSH
Related in: MedlinePlus