Activation of the Drosophila NF-kappaB factor Relish by rapid endoproteolytic cleavage

Abstract

The Rel/NF-kappaB transcription factor Relish plays a key role in the humoral immune response in Drosophila. We now find that activation of this innate immune response is preceded by rapid proteolytic cleavage of Relish into two parts. An N-terminal fragment, containing the DNA-binding Rel homology domain, translocates to the nucleus where it binds to the promoter of the Cecropin A1 gene and probably to the promoters of other antimicrobial peptide genes. The C-terminal IkappaB-like fragment remains in the cytoplasm. This endoproteolytic cleavage does not involve the proteasome, requires the DREDD caspase, and is different from previously described mechanisms for Rel factor activation.

Fig. 1. Relish-derived proteins. (A) Map of proteins produced from the Relish gene, including the cleavage products REL-68 and REL-49. Positions of peptides used to raise specific antibodies are indicated by asterisks. (B) Western blot analysis of crude extracts from wild-type embryos, male and female flies and the Relish mutant Rel^E20, detected with α-C. Where indicated, flies have been injected with E. cloacae for 6 h. Molecular weight markers are given in kDa.

Fig. 2. Signal-induced processing of Relish. (A and B) Western blots show time courses (in minutes) of protein extracts from either induced mbn-2 cells [(A), 25 µg protein/lane] or infected wild-type larvae [(B), ∼0.5 animal equivalent/lane]. In (A) the membrane was stripped before the second detection. Antibodies used to detect different forms of Relish are indicated to the left of each membrane. (C) Pulse–chase of in vivo labeled Relish-derived proteins. [³⁵S]methionine-labeled proteins were extracted from mbn-2 cells, either induced with LPS for 5 min or left untreated. Relish products were immunoprecipitated with the indicated rabbit antibody. The samples were separated by gel electrophoresis, the gel dried and exposed to X-ray film. (D) Immunoblot of extracts from mbn-2 cells treated with cycloheximide to inhibit protein biosynthesis prior to and during the challenge.

Fig. 3. Subcellular localization of Relish before and after an immune challenge. (A) Western blot with cytoplasmic and nuclear extracts from mbn-2 cells (50 µg protein/lane), developed with α-C, stripped and then developed with α-RHD. (B) Immunostaining of mbn-2 cells with α-RHD and α-C, untreated or 30 s after challenge. (C) Fatbody from wild-type larvae, untreated or taken 1 h after infection, immunostained with α-RHD. Insets in (B) and (C) show DAPI staining as overlays from the same sample area. (D) Gel shift assay for involvement of Relish in the κB binding activity. Mbn-2 cells were induced for 1 h and nuclear extracts were incubated with CecA1 κB oligonucleotide. Where indicated, an anti-Relish antiserum or the corresponding preimmune serum (Pi) was added (left panel). In a competition experiment we also added increasing amounts of the peptide, against which the serum was raised. Addition of 500 ng of peptide without antibody did not cause any effect.

Fig. 4. Proteasome inhibition and Relish processing. (A) Immunoblotting of protein extracts from cell cultures, treated with different concentrations of MG132 prior to an immune challenge. The membrane was first developed with α-C and then with an anti-ubiquitin antibody to visualize the accumulation of polyubiquitylated proteins, indicated by the bracket. Addition of the solvent DMSO or of the inhibitors alone did not induce Relish processing. (B) Western blot with protein extracts from wild-type and EP(X)1412 third instar larvae. Where indicated (+) the animals have been infected 45 min before extract preparation. Different forms of Relish were detected with α-C.