TANK potentiates tumor necrosis factor receptor-associated factor-mediated c-Jun N-terminal kinase/stress-activated protein kinase activation through the germinal center kinase pathway

Abstract

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are mediators of many members of the TNF receptor superfamily and can activate both the nuclear factor kappaB (NF-kappaB) and stress-activated protein kinase (SAPK; also known as c-Jun N-terminal kinase) signal transduction pathways. We previously described the involvement of a TRAF-interacting molecule, TRAF-associated NF-kappaB activator (TANK), in TRAF2-mediated NF-kappaB activation. Here we show that TANK synergized with TRAF2, TRAF5, and TRAF6 but not with TRAF3 in SAPK activation. TRAF2 and TANK individually formed weak interactions with germinal center kinase (GCK)-related kinase (GCKR). However, when coexpressed, they formed a strong complex with GCKR, thereby providing a potential mechanism for TRAF and TANK synergy in GCKR-mediated SAPK activation, which is important in TNF family receptor signaling. Our results also suggest that TANK can form potential intermolecular as well as intramolecular interactions between its amino terminus and carboxyl terminus. This study suggests that TANK is a regulatory molecule controlling the threshold of NF-kappaB and SAPK activities in response to activation of TNF receptors. In addition, CD40 activated endogenous GCKR in primary B cells, implicating GCK family proteins in CD40-mediated B-cell functions.

FIG. 1

Synergy between TANK and TRAF proteins in activating SAPK. (A) TANK alone has no effect on SAPK activity. Human kidney 293T cells were transiently transfected with 0, 1, 3, or 10 μg of pEBG-TANK. Each plate received 2 μg of HA-tagged JNK1 expression plasmid, while total DNA amount was maintained with empty vector. Cell lysate preparations and in vitro SAPK assays were performed as described in Materials and Methods. The gel bands represent GST-Jun proteins phosphorylated by SAPK in the cell lysates (top). Fold activation of SAPK is shown below the phosphorylated bands and represents fold activation versus 0 μg of transfected TANK. Similar expression of HA-JNK is shown as a control (middle). Increasing expression of TANK is shown by immunoblotting of lysates (bottom). (B) TRAF2-induced SAPK activity is enhanced by coexpression of TANK. Human kidney 293T cells were transiently transfected with 3 μg of pEBG-TANK and 3 μg of pEBB-TRAF3 or pEBB-TRAF2 with 0, 1, 3, or 10 μg of pEBG-TANK. The gel bands represent GST-Jun proteins phosphorylated by SAPK in the cell lysates (top). Fold activation is shown below the phosphorylated bands and represents fold activation of a specific TRAF with increasing amounts of TANK versus the activation of the specific TRAF alone, which is set at 1. Similar expression of HA-JNK is shown as a control (bottom). TRAFs are consistently expressed at similar levels for each particular TRAF (data not shown). (C) TRAF5- and TRAF6-induced SAPK activity is enhanced by coexpression of TANK. In vitro SAPK assays were performed as described for panel B, except that 1 μg of pEBB-TRAF5 or pEBB-TRAF6 was used with 0, 1, 3, or 10 μg of pEBG-TANK expression plasmids.

FIG. 2

Inhibition of SAPK activation by TANK-C. TANK-C and TRAF2-C inhibit SAPK activation by CD40 plus CD40L, TRAF2, and TRAF2 plus TANK. Human kidney 293T cells were transfected with 3 μg of pBABE-CD40 plus 3 μg of pBABE-CD40L, 6 μg pEBB-TRAF2, or 3 μg of pEBB-TRAF2 plus 3 μg of pEBG-TANK, together with 5 μg of either pEBG vector, pEBG-TANK-C, or pEBB-TRAF2-C in the presence of 2 μg of HA-tagged JNK1 expression plasmid. Cell lysate preparations and in vitro SAPK assays were performed as described in Materials and Methods. The gel bands represent GST-Jun proteins phosphorylated by SAPK in the cell lysates (top); similar expression of HA-JNK is shown as a control (bottom).

FIG. 3

Self-association and potential intramolecular interaction of TANK. TANK can associate with itself, with interactions observed between the amino termini and between the amino and carboxyl termini. Various plasmids expressing Flag-TANK, Flag-TANK-N, GST-TANK, GST-TANK-N, GST-TANK-C, HA-TANK-C, HA-Fyn, and GST-Btk were transfected in combination, as indicated, into 293T cells. Total DNA amounts were maintained at 10 μg, using empty vector. Cell lysates were immunoprecipitated (IP) with an anti-Flag (lanes 1 to 4 and 8) or anti-HA (lanes 5 to 7) monoclonal antibody. Coprecipitated proteins were detected by immunoblotting with an anti-GST polyclonal antibody. Cutouts of the interacting bands are shown (top); aliquots of cell extracts were immunoblotted with the appropriate antibodies to confirm expression of proteins (middle and bottom). Coexpression of HA-Fyn and GST-TANK and of Flag-TANK and GST-Btk served as negative controls.

FIG. 4

Inhibition of SAPK activation with dominant-negative forms of GCKR, MEKK1, and SEK1 (DN-GCKR, DN-MEKK1, and DN-SEK1). (A) Dominant-negative GCKR inhibits TRAF2-mediated SAPK activation. Human 293T cells were transiently transfected with 6 μg of pEBB, 6 μg of pEBB-TRAF2, 3 μg of pEBB-TRAF2 plus 3 μg of pEBG-TANK, or 3 μg of pBABE-CD40 plus 3 μg of pBABE-CD40L in the presence of 5 μg of either pcDNA3 or pcDNA3-DN-GCKR. The cells in lanes 3 and 4 were treated with 10 ng of human TNF-α per ml for 10 min before lysing. Cell lysate preparations and in vitro SAPK assays were performed as described in Materials and Methods. The gel bands represent GST-Jun proteins phosphorylated by SAPK in the cell lysates (top rows in all panels); similar expression of HA-JNK is shown as a control (bottom rows in all panels). (B) Dominant-negative MEKK1 and SEK1 but not dominant-negative Rac1 and cdc42 inhibit TRAF2- and TANK-mediated SAPK activation. For transient transfections, 3 μg of pEBB-TRAF2 and 3 μg of pEBG-TANK, together with 3 μg of pEBG vector, pEBG-TANK-C, pEBG-DN-MEKK1, pEBG-SEK1, pEBG-DN-Rac1, or pEBG-DN-cdc42 were used. (C) MEKK1 is downstream of GCKR. For transient transfections, 6 μg of pEBG, 3 μg of pEBG-MEKK1, 3 μg of pEBG-MEKK1 plus 3 μg of pcDNA3-DN-GCKR, 3 μg of pcDNA3-GCKR, or 3 μg of pEBG-DN-MEKK1 plus 3 μg of pcDNA3-GCKR were used.

FIG. 5

Activation of endogenous GCKR in human tonsil B cells. (A) GCKR activity is enhanced by stimulation of CD40 in tonsil B cells. Lysates from human tonsil B cells were immunoprecipitated with an antiserum to GCKR and assayed for the ability to phosphorylate MBP at 0, 5, 10, and 15 min after CD40 stimulation with anti-CD40 monoclonal antibody G28.5 (top); expression of endogenous GCKR is shown by immunoblotting with antiserum to GCKR (bottom). (B) Kinetics of JNK1 activation are similar to those of GCKR activation by CD40 stimulation in Ramos B cells. Activation of JNK1 by CD40 in Ramos B cells was performed at 0, 5, 15, 30, and 60 min after stimulation of cells by soluble CD8-gp39. Lysates were immunoprecipitated with a polyclonal anti-JNK1 antibody and assayed for the ability to phosphorylate GST c-Jun(1-79) (top); expression of endogenous GCKR is shown by immunoblotting with a polyclonal anti-JNK antibody (bottom). (C) Expression of endogenous GCKR was examined in the indicated cell types. GCKR was detected by immunoblotting with antiserum to GCKR and migrates at about 95 kDa.

FIG. 6

Synergistic interaction of TRAF2 and TANK with GCKR. Individual TRAF2 and TANK interactions with GCKR are enhanced by coexpression of TRAF2 and TANK. Human 293T cells were transfected with pEBB-HA-TRAF2, pEBB-GST-TANK, or pcDNA3-Flag-GCKR, either alone or in combination, as indicated. (A) Total DNA amounts were maintained at 10 μg, using empty vector. Cell lysates were immunoprecipitated (IP) with an anti-HA antibody and the coimmunoprecipitated complexes were analyzed by immunoblotting with an anti-Flag antibody to demonstrate TRAF2 and GCKR interactions (B). Lysates were precipitated with an anti-GST antibody, and the coimmunoprecipitated complexes were immunoblotted with an anti-Flag antibody to show TANK and GCKR interactions (C). TRAF2 and TANK interactions are shown by immunoprecipitation of lysates with an anti-HA antibody and subsequent immunoblotting with an anti-GST antibody (D). Expression levels of input proteins were obtained by immunoblotting lysates with appropriate antibodies. The arrow indicates a nonspecific protein found in 293T cells that cross-reacts with the anti-HA monoclonal antibody.

FIG. 7

Involvement of GLK in TRAF- and TANK-mediated SAPK activation. GLK-induced SAPK activity is inhibited by dominant-negative MEKK1 and SEK1 (DN-MEKK1 and DN-SEK1), while dominant-negative GLK (DN-GLK) inhibits SAPK activation by CD40 and TRAF2 plus TANK but not by MEKK1. Human 293T cells were transfected with 2 μg of plasmid expressing HA-JNK1 and 3 μg of pEBB vector (lane 1), pcDNA3-GLK (lane 2), pcDNA3-GLK plus pEBB-DN-MEKK1 (lane 3), pcDNA3-GLK plus pEBB-DN-SEK1 (lane 4), pBABE-CD40 plus pBABE-CD40L (lane 5), pBABE-CD40 and pBABE-CD40L plus pcDNA3-DN-GLK (lane 6), pEBB-TRAF2 plus pEBG-TANK (lane 7), pEBB-TRAF2 and pEBG-TANK plus pcDNA3-DN-GLK (lane 8), DN-MEKK1 (lane 9), or DN-MEKK1 plus pcDNA3-DN-GLK (lane 10). Total DNA amounts were kept at 9 μg, with empty vector. Cell lysate preparations and in vitro SAPK assays were performed as described in Materials and Methods. The gel bands represent GST-Jun proteins phosphorylated by SAPK in the cell lysates (top); similar expression of HA-JNK is shown as a control (bottom).