IkappaB kinase is an essential component of the Tpl2 signaling pathway

Abstract

IkappaB kinase (IKK), a key regulator of immune and inflammatory responses, is known as an effector kinase mediating activation of the transcription factor NF-kappaB. Whether IKK also participates in other signaling events is not known. Here we show that IKK serves as an essential component of a signaling pathway that involves activation of the Tpl2 kinase and its downstream targets, MEK1 and ERK. Inhibition of IKKbeta in macrophages eliminates Tpl2 activation and ERK phosphorylation induced by lipopolysaccharide and tumor necrosis factor alpha. Using IKK-deficient murine fibroblasts, we further demonstrate that IKKbeta, but not IKKalpha, is required for Tpl2 activation. Moreover, this novel function of IKKbeta appears to involve phosphorylation and degradation of the Tpl2 inhibitor NF-kappaB1/p105. These findings suggest that IKKbeta exerts its immune-regulatory functions by targeting different downstream signaling pathways.

FIG. 1.

Inhibitory effect of an IKKβ inhibitor on LPS-stimulated NF-κB activation and production of inflammatory mediators. (A) Immunoblot analysis of IκBα and the control tubulin in BMDM. The cells were preincubated with the indicated amounts of PS1145 for 90 min followed by stimulation with LPS (2.5 μg/ml) and TNF-α (10 ng/ml) for 20 min. Cytoplasmic extracts were subjected to immunoblot using anti-IκBα and anti-tubulin antibodies (39). (B) Nuclear extracts isolated from the BMDM described for panel A were subjected to EMSA to detect NF-κB DNA binding activity. (C) Inhibition of IKKβ, but not IKKα, by PS1145. Recombinant IKKα (10 ng) and IKKβ (5 ng) were incubated for 15 min (at 25°C) with either the solvent DMSO (−) or the IKK inhibitor PS1145 (10 μM), and their catalytic activity was analyzed by using GST-IκBα (1-54) as a substrate. (D) Inhibition of TNF-α production by PS1145. Macrophages were preincubated with the indicated amounts of PS1145 followed by stimulation with LPS for 6 h. The amounts of TNF-α secreted to the media were determined by ELISA and were expressed as pictograms per 10⁶ cells. (E) Inhibition of COX-2 induction by PS1145. Macrophages were preincubated with (+) or without (−) PS1145 for 90 min, and the cells were either not treated (NT) or were stimulated for 2 h with LPS. COX-2 expression was analyzed by immunoblot.

FIG. 2.

IKK inhibition specifically impairs LPS-stimulated ERK activation. (A) LPS-stimulated activation of ERK but not other MAPKs is sensitive to PS1145. Macrophages were preincubated with increasing doses of PS1145 followed by LPS stimulation (15 min). The phosphorylated (P-) and total MAPKs (ERK1 and ERK2, JNKs, and p38) and the loading control tubulin were analyzed by immunoblot as previously described (39). (B) Effect of PS1145 on ERK activation induced by TNF-α and PMA. The cells were preincubated for 90 min with (+) or without (−) PS1145 followed by stimulation with TNF-α (10 ng/ml) or PMA (25 ng/ml) for 15 min. ERK1 and ERK2 activation was analyzed by immunoblotting with a phosphospecific anti-ERK antibody. (C) Inhibition of ERK but not other MAPKs by another IKK inhibitor, 15dPGJ2. The cells were preincubated for 70 min with (+) or without (−) 15dPGJ2 (4 μM) and then were left either not treated (NT) or stimulated with LPS for 15 min. Immunoblotting was performed as described for panel A.

FIG. 3.

IKK is required for Tpl2 activation. (A) Induction of MEK1 phosphorylation by LPS, but not by PMA, is sensitive to the IKK inhibitor. Macrophages were preincubated with (+) or without (−) PS1145 followed by stimulation for 15 min with LPS or PMA (25 ng/ml). Activated (upper panel) and total (lower panel) MEK1 proteins were detected by immunoblot using anti-phospho-MEK1 (P-MEK1) and anti-MEK1, respectively. (B) Macrophages were either not treated (NT) or were stimulated for 15 min with LPS following 90 min of preincubation with PS1145, carrier control DMSO, or medium (None). Tpl2 was isolated by immunoprecipitation and was subjected to kinase assays (KA) with GST-MEK1 as substrate (upper panel). The Tpl2 protein was monitored by immunoblot using a C-terminal-specific anti-Tpl2 antibody (anti-Cot M20; Santa Cruz). (C) Macrophages were preincubated with (+) or without (−) PS1145 followed by stimulation with TNF-α (10 ng/ml) for 15 min. In vitro kinase assays (top panel) and phosphospecific immunoblotting (second panel) were performed to measure the catalytic activity of Tpl2 and in vivo phosphorylation of MEK1, respectively. The expression levels of total MEK1 and the loading control tubulin were monitored by immunoblot (lower two panels). (D) Recombinant IKKβ purified from insect cells (top panel), recombinant Tpl2 purified from an in vitro translation system (middle panel), and recombinant MEK1 purified as a GST fusion protein from bacteria (bottom panel) were preincubated for 15 min (at 25°C) with increasing concentrations of PS1145 and then were subjected to kinase assays using GST-IκBa(1-54) (top panel), GST-MEK1 (middle panel), and ERK2 (bottom panel) as substrates, respectively.

FIG. 4.

Activation of IKK precedes the activation of Tpl2 and ERK phosphorylation. BMDM were stimulated with either LPS or TNF-α for the indicated time periods. IKK and Tpl2 were isolated by immunoprecipitation using anti-IKKγ and anti-Tpl2 (anti-Cot M20), respectively, and were subjected to kinase assays (KA) as described in the legend to Fig. 3D. ERK phosphorylation was analyzed by immunoblotting (IB) using the phosphospecific anti-ERK antibody.

FIG. 5.

IKKβ and IKKγ, but not IKKα, are essential for Tpl2 activation. MEFs derived from wild-type (WT) mice or the indicated IKK knockout mice were stimulated for the indicated times with TNF-α (50 ng/ml) followed by protein kinase assays (KA) to detect Tpl2 kinase activity (upper panel). The level of Tpl2 was monitored by immunoblot (IB) (lower panel).

FIG. 6.

IKKβ and IKKγ are required for signal-induced release of Tpl2 from p105. (A) Macrophages were preincubated with (+) or without (−) PS1145 followed by LPS stimulation for the indicated times. P105 and its associated Tpl2 were isolated by immunoprecipitation (IP) using a C-terminal-specific anti-p105 antibody (29). The precipitated p105 (top panel) and Tpl2 isoforms (panel 2) were detected by immunoblot (IB) using anti-p105 and horseradish peroxidase-conjugated anti-Tpl2, respectively. The total amount of Tpl2 (panel 3) and the control tubulin (Tub) (bottom panel) in the cell lysates was monitored by direct immunoblot. (B) MEFs derived from wild-type (WT) mice or the indicated IKK knockout mice were either not treated (−) or were stimulated for 15 min with TNF-α (50 ng/ml) followed by coIP assays to detect the level of p105 (top panel) and p105-associated Tpl2 (panel 2). The total amounts of Tpl2 (panel 3) and tubulin (panel 2) in the cell lysates were monitored by direct immunoblotting (bottom panel). (C) Densitometry quantitation of protein bands presented in the first two gels of panel A. The lanes indicated below the graphs are the same as those indicated in panel A. (D) Densitometry quantitation of protein bands presented in the first two gels of panel B. The lanes indicated below the graphs correspond to the lanes of panel B.

FIG. 7.

A time course analysis to correlate Tpl2 activation with p105 degradation and Tpl2_L release. (A) BMDM were stimulated with LPS for the indicated time periods and then were subjected to Tpl2 kinase assay (KA) (top panel) as described in the legend to Fig. 3B. In parallel, the p105/Tpl2 complexes were isolated by immunoprecipitation (IP) using anti-p105C antibody followed by immunoblot (IB) detection of precipitated p105 (panel 2) and Tpl2 (panel 3) using specific antibodies. The expression levels of the Tpl2 isoforms (panel 4) and tubulin (bottom panel) in the cell lysates were analyzed by direct immunoblot. (B) Densitometry analysis of the protein bands from panels 2, 3, and 4 of panel A. The lanes indicated below the figure correspond to the lanes in panel A.

FIG. 8.

LPS-stimulated Tpl2/ERK signaling is sensitive to proteasome inhibitors. (A) Macrophages were either not treated (NT) or were stimulated for 15 min with LPS following preincubation (60 min) with medium (None), carrier control (DMSO), or the proteasome inhibitors MG132 and lactacysteine (LC). Tpl2 kinase activity (upper panel) and protein level (lower panel) were determined by kinase assays (KA) and immunoblotting (IB). (B) Cells were either not treated (NT) or were stimulated for 15 min with LPS following preincubation (30 min) with (+) or without (−) MG132. Activated (upper panels) and total (lower panels) MAPKs were detected by immunoblot using corresponding phosphospecific (P-) and pan-antibodies. As a loading control, an immunoblot with antitubulin was included. (C) Macrophages were either not treated (NT) or were stimulated for 15 min with PMA following preincubation with MG132 (30 min). Activated (upper panel) and total (lower panel) ERK proteins were detected by immunoblot using anti-phospho-ERK (P-ERK) and anti-ERK, respectively. (D) Macrophages were preincubated without (−) or with MG132 (MG) or lactacysteine (LC) and then left either not treated (NT) or stimulated with LPS for 25 min. The release of Tpl2_L from p105 was determined by coIP (top and middle panels) as described in the legend to Fig. 6. The expression level of Tpl2 isoforms in the cell lysates was determined by direct immunoblot (bottom panel).

FIG. 9.

The IKK phosphorylation site of p105 is essential for mediating Tpl2 activation in macrophages. (A) Site-specific phosphorylation of p105 by LPS-stimulated IKK in macrophages. Macrophages were stimulated with the indicated inducers. IKK complex was isolated by immunoprecipitation (IP) using anti-IKKγ followed by kinase assays (KA) (top panel) using GST-p105C or GST-p105C harboring serine-to-alanine mutations at the IKK phosphorylation site of p105 (p105C SSS/AAA). Following autoradiography, the kinase filter was subjected to immunoblotting (IB) using anti-p105C (middle panel) and anti-IKKα/β (H-470; Santa Cruz) (bottom panel) to detect the levels of substrates and IKKβ. (B) nfκb1^−/− macrophages were infected with retroviruses encoding green fluorescence protein (GFP), wild-type p105, or a p105 mutant harboring serine-to-alanine mutations at the IKK phosphorylation site (p105SSS/AAA). The exogenous p105 proteins (upper panel) and rescued Tpl2 isoforms (lower panel) were detected by immunoblot using N-terminal-specific anti-p105 and C-terminal-specific anti-Tpl2 antibodies, respectively. (C) The retrovirus-infected nfκb1^−/− macrophages were either not treated (−) or were stimulated for 15 min with LPS followed by immunoblot to analyze the degradation of p105 (top panel), activation of ERK1 and ERK2 (middle panel), and total ERK1 and ERK2 (bottom panel). (D) Tpl2_L cannot be released from a phosphorylation-defective p105 mutant. Raw264 murine macrophage cells were infected with retroviruses encoding HA-tagged wild-type p105 or a phosphorylation-defective p105 mutant (p105SSS/AAA). Bulk infected cells were either not treated (−) or were stimulated (+) with LPS for 15 min followed by isolation of the HA-tagged p105/Tpl2 complexes by using anti-HA antibody. The exogenous p105 and coprecipitated Tpl2 proteins were detected by immunoblotting using anti-HA and horseradish peroxidase-conjugated anti-Tpl2 (anti-Cot M20), respectively. The use of horseradish peroxidase-conjugated anti-Tpl2 was essential to avoid interference from the immunoglobulin heavy chain. A tubulin immunoblot was included as a loading control (bottom panel). (E) Densitometry quantitation of protein bands from the first two gels of panel D. The lanes indicated below the graphs correspond to the lanes in panel D.