Interleukin-1 and TRAF6-dependent activation of TAK1 in the absence of TAB2 and TAB3

Abstract

Interleukin-1 (IL-1) signaling induces the formation of Lys63-linked ubiquitin (K63-Ub) chains, which are thought to activate the 'master' protein kinase TGFβ-activated kinase 1 (TAK1) by interacting with its TAK1-binding 2 (TAB2) and TAB3 subunits. Here, we report that IL-1β can also activate the TAB1-TAK1 heterodimer present in TAB2/TAB3 double knockout (DKO) IL-1 receptor-expressing cells. The IL-1β-dependent activation of the TAB1-TAK1 heterodimer in TAB2/3 DKO cells is required for the expression and E3 ligase activity of tumor necrosis factor receptor-associated factor 6 (TRAF6) and is reduced by the small interfering RNA (siRNA) knockdown of ubiquitin conjugating 13 (Ubc13), an E2-conjugating enzyme that directs the formation of K63-Ub chains. IL-1β signaling was restored to TAB1/2/3 triple KO cells by the re-expression of either TAB1 or TAB2, but not by an ubiquitin binding-defective mutant of TAB2. We conclude that IL-1β can induce the activation of TAK1 in two ways, only one of which requires the binding of K63-Ub chains to TAB2/3. The early IL-1β-stimulated, TAK1-dependent activation of p38α mitogen-activated protein (MAP) kinase and the canonical IκB kinase (IKK) complex, as well as the NF-κB-dependent transcription of immediate early genes, was similar in TAB2/3 DKO cells and TAB2/3-expressing cells. However, in contrast with TAB2/3-expressing cells, IL-1β signaling was transient in TAB2/3 DKO cells, and the activation of c-Jun N-terminal kinase 1 (JNK1), JNK2 and p38γ was greatly reduced at all times. These observations indicate a role for TAB2/3 in directing the TAK1-dependent activation of MAP kinase kinases that switch on JNK1/2 and p38γ MAP kinases. These observations and the transient activation of the TAB1-TAK1 heterodimer may explain why IL-1β-dependent IL-8 mRNA formation was abolished in TAB2/3 DKO cells.

Keywords: TAB1; TAB2; TAK1; TRAF6; interleukin-1; ubiquitin.

Figure 1.. IL-1β signaling in TAB2/3 DKO cells.

(A) Generation of IL-1R* cells lacking TAB2 and TAB3 or all three TAB subunits. TAK1 was immunoprecipitated from the extracts of WT IL-1R* cells (lane 1), two different clones (4 and 11) of cells devoid of TAB2 and TAB3 (lanes 2 and 3) and two different clones (A4 and H17) lacking expression of all three TAB components. Immunoprecipitates were subjected to SDS–PAGE, and immunoblotting as in the Methods with antibodies recognizing TAK1 or each TAB protein. (B) TAB2/3 DKO cells (clone 4 from A) were stimulated with 5 ng/ml IL-1β for the times indicated, and TAK1 immunoprecipitated from the extracts and processed as in A. (C and D) WT or TAB2/3 DKO IL-1R* cells (clone 4 from A) were stimulated with IL-1β for up to 1 h (C) or 2 h (D) as in B, and cell extracts analyzed by SDS–PAGE and immunoblotting with phospho-specific antibodies recognizing phosphorylated (p) serine (pS), threonine (pT) and tyrosine (pY) residues in the activation loops of TAK1, IKKα/β, p105/NF-κB1, JNK1/2, p38α/p38γ and ERK1/ERK2 MAP kinases, or with antibodies recognizing all forms of TAK1, p38α and GAPDH.

Figure 2.. IL-1β-dependent gene transcription in TAB2/3 DKO IL-1R* cells.

(A–C) Cells were stimulated with IL-1β as in Figure 1, and at the times indicated, the mRNA encoding IκBα (A), A20 (B) and IL-8 (C) was measured by qRT-PCR relative to 18S ribosomal mRNA and normalized to the level found in WT cells stimulated with IL-1β. The results are presented as arithmetic mean ± SEM for three independent experiments, each performed in triplicate. (D) As in C, except that IL-8 secreted into the culture medium was measured by ELISA.

Figure 3.. TRAF6 E3 ligase activity is required for the IL-1β-dependent activation of the TAB1–TAK1 heterodimer in TAB2/3 DKO cells.

(A) TAB2/3 DKO (clone 4 from Figure 1A) and TAB2/TAB3/TRAF6 TKO IL-1R* cells were stimulated with IL-1β for the times indicated, and cell extracts subjected to SDS–PAGE and immunoblotting with the antibodies used in Figure 1 and an antibody recognizing IRAK4 phosphorylated at Thr245 and Ser346. (B) As in A, except that WT TRAF6 or the indicated E3-ligase-inactive mutants of TRAF6 were re-expressed in the TAB2/TAB3/TRAF6 TKO IL-1R* cells. (C) WT and TAB2/3 DKO cells were incubated for 72 h with siRNA against Ubc13 and stimulated for 10 min with 5 ng/ml IL-1β. Other details are as in A.

Figure 4.. IL-1 signaling is restored to TAB1/2/3 TKO cells by the re-expression of TAB1.

(A) The experiment was carried out as in Figure 3A, except that TAB1/2/3 TKO cells (clone A4 from Figure 1A) and WT IL-1R* cells were used. (B) As in A, except that HA-TAB1 was re-expressed in the TAB1/2/3 TKO IL-1R* cells where indicated and the cells stimulated for 10 min with 5 ng/ml IL-1β. Extracts from WT cells (20 µg of protein) and TAB1/2/3 TKO IL-1R* cells (40 µg of protein) were then processed as in A (top five panels). In the bottom two panels, TAK1 was immunoprecipitated from the extracts and immunoblotted with anti-TAB1 and anti-TAK1 to confirm that the re-expressed TAB1 had recombined with TAK1.

Figure 5.. IL-1β signaling in TAB1 KO IL-1R* cells.

(A) Generation of two clones of TAB1 KO IL-1R* cells. Other details are as in Figure 1A. (B) WT IL-1R* cells and TAB1 KO cells (clone 44) were stimulated for up to 2 h with IL-1β . Cell extracts were denatured in SDS, subjected to SDS–PAGE and immunoblotted with the antibodies indicated. (C–E) As in B, except that RNA was extracted from the cells at the times indicated and the formation of IκBα (C), A20 (D) and IL-8 (E) mRNA was measured by qRT-PCR relative to 18S ribosomal mRNA and normalized to the level found in WT cells stimulated with IL-1β. The results are presented as arithmetic mean ± SEM for three independent experiments, each performed in triplicate. (F) As in E, except that IL-8 secreted into the culture medium was measured by ELISA.

Figure 6.. IL-1 signaling in different TAB- and TRAF6-deficient cells.

(A) Generation of TAB1/TRAF6 DKO cells from TAB1 KO cells (clone 44 from Figure 5A). (B) WT TRAF6 and the E3 ligase-inactive TRAF6[L74H] and TRAF6[120-522] mutants were re-expressed in TAB1/TRAF6 DKO cells (clone 1 from A). These cells, TAB1 KO cells (clone 44 from Figure 5A) and TAB1/TRAF6 DKO cells not re-transfected with TRAF6, were then stimulated with IL-1β for the times indicated. Other details are as in A. (C) HA-TAB2 or the K63-Ub-binding-defective HA-TAB2[T674/F675A] mutant were re-expressed in TAB1/2/3 TKO IL-1R* cells (clone A4 from Figure 1A). These cells, TAB1/2/3 TKO cells not re-transfected with HA-TAB2 and WT IL-1R* cells, were stimulated for 10 min with 5 ng/ml IL-1β. Cell extracts (20 µg of protein-WT cells or 40 µg of protein-TAB1/2/3 TKO cells) were subjected to SDS–PAGE and immunoblotted with the antibodies indicated.