Targeting of TAK1 by the NF-kappa B protein Relish regulates the JNK-mediated immune response in Drosophila

Abstract

The molecular circuitry underlying innate immunity is constructed of multiple, evolutionarily conserved signaling modules with distinct regulatory targets. The MAP kinases and the IKK-NF-kappa B molecules play important roles in the initiation of immune effector responses. We have found that the Drosophila NF-kappa B protein Relish plays a crucial role in limiting the duration of JNK activation and output in response to Gram-negative infections. Relish activation is linked to proteasomal degradation of TAK1, the upstream MAP kinase kinase kinase required for JNK activation. Degradation of TAK1 leads to a rapid termination of JNK signaling, resulting in a transient JNK-dependent response that precedes the sustained induction of Relish-dependent innate immune loci. Because the IKK-NF-kappa B module also negatively regulates JNK activation in mammals, thereby controlling inflammation-induced apoptosis, the regulatory cross-talk between the JNK and NF-kappa B pathways appears to be broadly conserved.

Figure 1.

Requirement for JNK in the transient response gene induction by LPS. (A) LPS-induced transient response gene expression in S2 cells. S2 cells were stimulated with LPS for the length of time indicated on the abscissa. Total RNA was prepared and analyzed using the Affymetrix Drosophila gene chip. Values on the ordinate indicate the relative mRNA level. (B) LPS-induced sustained response gene expression in S2 cells. (C) S2 cells were mock-treated or treated with jnk dsRNA for 3 d and then left unstimulated or stimulated with LPS for 1 h. Total RNA isolated from those cells was analyzed by RNase protection to assay the expression of the genes listed on the left. In this and subsequent panels, attacin refers to the attacin A gene. (D) After treating S2 cells with dsRNA and LPS as in C, the puckered and attacin mRNA levels in S2 cells were measured by real-time PCR analysis. The results shown are averages of three determinations.

Figure 2.

Prolonged expression of JNK targets in flies and S2 cells that have a defect in Relish activation. (A) Wild-type and relish mutant adult flies were infected by wounding with a needle dipped in E. cloacae. Total RNA was prepared at the indicated time points after infection and subjected to real-time PCR analysis. (B) S2 cells were mock-treated or treated with relish dsRNAs for 3 d and then left unstimulated or stimulated with LPS. Total RNA was prepared and analyzed as in A. (C) Wild-type adult flies and imd, tak1, and ird5 mutant flies were analyzed as in A. (D) RNAi in S2 cells and LPS stimulation were carried out as in B. Total RNA was analyzed by RNase protection assay to measure the expression of the genes listed on the left.

Figure 3.

Sustained activation of JNK signaling in S2 cells lacking Relish activation. (A) S2 cells were treated with dsRNAs specific to kenny, ird5, or relish for 3 d and then unstimulated or stimulated with LPS for the length of time indicated. Cell lysates were prepared and analyzed by immunoblotting with antibodies specific to the phosphorylated forms of JNK. (B) S2 cells were treated with 0.1% (v/v) dimethylsulfoxide (DMSO), 1 μg/mL actinomycin D (Act D), or 25 μg/mL cycloheximide (CHX) for 1 h and then unstimulated or stimulated with LPS for the length of time indicated. Cell lysates were prepared and analyzed as in A with antibodies specific to the phosphorylated forms of JNK (Phos-JNK) and Relish. For Relish, only the full-length (FL) form is shown. (C) S2 cells were mock-treated or were treated with ird5, kenny, or dredd dsRNAs and analyzed as in Figure 2B.

Figure 4.

Inhibition of the JNK signaling module by Relish activation prior to LPS stimulation. (A) Structure of Relish protein and its Rel-homology domain (RHD) derivative. (B) S2 cells stably transfected with this RHD construct (S2RHD) and control cells transfected with the backbone vector construct (S2MK) were untreated or treated with 0.7 mM CuSO₄ for 12 h. Cell lysates were prepared and analyzed by immunoblotting with antibodies specific to the RHD of Relish (Relish^RHD). (C) S2MK and S2RHD cells were unstimulated or stimulated with CuSO₄. Total RNA was prepared after the length of time indicated and analyzed by RNase protection assay. (D) S2MK and S2RHD cells were treated with CuSO₄ for 12 h and then unstimulated or stimulated with LPS for the length of time indicated. Cell lysates were prepared and analyzed by immunoblotting with antibodies specific to phos-JNK, Relish^RHD, and Cactus. (E) S2MK and S2RHD cells were transfected with a plasmid that expresses Flag-DJNK and then treated with CuSO₄ and stimulated with LPS as in D. Flag-DJNK proteins were immunoprecipitated from cell lysates and subjected to in vitro kinase assay using GST-DJun as a substrate. (F) S2MK and S2RHD cells were treated with CuSO₄ and stimulated with LPS. Total RNA isolated at the indicated time point was analyzed by RNase protection assay.

Figure 5.

Involvement of proteasomal degradation in Relish-dependent JNK inhibition. (A) S2MK and S2RHD cells were transfected with Flag-DJNK expression plasmid and then treated with CuSO₄. S2RHD cells were untreated or further treated with 50 μM MG-132 for 2 h. After stimulating with LPS, cell lysates were prepared and analyzed by immunoblotting with antibodies specific to phos-JNK (upper panels) and Flag (lower panels). (B) S2 cells were treated with DMSO, 50 μM MG-132, or 5 μM lactacystin for 2 h and then unstimulated or stimulated with LPS for the length of time indicated. Phos-JNK was analyzed by immunoblotting. (C) S2 cells were treated with dsRNAs specific to rpn6 or csn5 for 3 d and then unstimulated or stimulated with LPS for the length of time indicated. Cell lysates were prepared and analyzed by immunoblotting with the antibodies indicated on the left.

Figure 6.

Destabilization of Tak1 protein by Relish activation. (A) S2MK and S2RHD cells were treated with CuSO₄ and then unstimulated or stimulated with 0.4 M NaCl for the length of time indicated. Phospho-JNK and FL Relish were analyzed by immunoblotting. (B) S2MK and S2RHD cells treated with CuSO₄ were unstimulated or stimulated with LPS. FL Relish and its C-terminal domain (CTD) fragment generated by endoproteolytic processing were analyzed by immunoblotting. (C) S2 cells were treated with LPS for the length of time indicated. Cytoplasmic and nuclear protein fractions were prepared and analyzed by immunoblotting. (D) S2MK and S2RHD cells were transfected with HA-TAK1 and Flag-DJNK expression plasmids and then treated with CuSO₄. Protein lysates were prepared at the indicated time point and analyzed by immunoblotting. (E) S2MK and S2RHD cells were transfected with the HA-TAK1 expression plasmid and then treated with CuSO₄ for 8 h. Cells were then pulse-labeled with [³⁵S]methionine for 1 h and chased with nonradioactive medium for the time points indicated. HA-Tak1 protein was immunoprecipitated from protein lysates and analyzed by SDS-PAGE and autoradiography.

Figure 7.

Model of branched Imd pathway.