The Drosophila atypical protein kinase C-ref(2)p complex constitutes a conserved module for signaling in the toll pathway

Abstract

Recent results showed the critical role of the mammalian p62-atypical protein kinase C (aPKC) complex in the activation of NF-kappaB in response to different stimuli. Here we demonstrate using the RNA interference technique on Schneider cells that the Drosophila aPKC (DaPKC) is required for the stimulation of the Toll-signaling pathway, which activates the NF-kappaB homologues Dif and Dorsal. However, DaPKC does not appear to be important for the other Drosophila NF-kappaB signaling cascade, which activates the NF-kappaB homologue Relish in response to lipopolysaccharides. Interestingly, DaPKC functions downstream of the nuclear translocation of Dorsal or Dif, controlling the transcriptional activity of the Drosomycin promoter. We also show that the Drosophila Ref(2)P protein is the homologue of mammalian p62 as it binds to DaPKC, its overexpression is sufficient to activate the Drosomycin but not the Attacin promoter, and its depletion severely impairs Toll signaling. Collectively, these results demonstrate the conservation of the p62-aPKC complex for the control of innate immunity signal transduction in Drosophila melanogaster.

FIG. 1.

DaPKC is essential for Drosomycin transcription. (A) S2tpll cells were transfected with DaPKC dsRNA (RNAi) or left untreated (Mock), after which they were stimulated with either Cu²⁺ (left panel) or LPS (right panel) and the transcriptional activation of Drosomycin (left panel) or Diptericin (right panel) was determined by RT-PCR. Dactin mRNA levels were also determined as a control for RNA loading. The levels of DaPKC were determined in parallel cell extracts by immunoblotting. The results shown are representative of results from at least two other experiments. (B) In another experiment, S2tpll cells that were transfected with DaPKC dsRNA were transfected with either the plasmid control, C, or an expression vector for human ιPKC, after which they were stimulated with Cu²⁺ and the transcriptional activation of Drosomycin was determined by RT-PCR. Dactin mRNA levels were also determined as a control for RNA loading. The lower panel, which shows the results of immunoblotting with an antibody that cross-reacts with both aPKCs, demonstrates the effective depletion of DaPKC and the expression of human ιPKC. Note that ιPKC runs faster than DaPKC in this gel.

FIG. 2.

DaPKC is not required for Cactus or Relish degradation. S2tpll cells were transfected with DaPKC dsRNA (RNAi) or not transfected (Mock) and stimulated as described above, after which the levels of Cactus (left panel), Relish (right panel), and DaPKC (both panels) were determined by immunoblotting with the corresponding antibodies. The results shown are representative of results from at least three other experiments.

FIG. 3.

DaPKC is not required for the nuclear translocation of Dorsal or Dif. S2tpll cells were transfected (B) or not transfected (A) with a Flag-tagged Dif expression vector along with a Cactus plasmid with (RNAi) or without (Mock) DaPKC dsRNA, after which they were stimulated or not stimulated with Cu²⁺, and the levels of nuclear Dorsal (Dorsal-nuc) (A), cytosolic Dorsal (Dorsal-cyt) (A), nuclear Flag-Dif (Dif-nuc) (B), cytosolic Flag-Dif (Dif-cyt) (B), and total DaPKC (A and B) were determined by immunoblotting. The results shown are representative of results from at least three other experiments.

FIG. 4.

DaPKC is required for the activation of the Drosomycin promoter. S2tpll cells were (RNAi) or were not (Mock) transfected with DaPKC dsRNA along with either Drosomycin or Attacin luciferase reporter vectors, after which cells were stimulated (black bars) with Cu²⁺ (for the Drosomycin promoter) or LPS (for the Attacin promoter) or were left unstimulated (white bars), and the luciferase activities of cell extracts were determined. The results shown are representative of results from at least three other experiments. A representative immunoblot is shown for endogenous DaPKC.

FIG. 5.

Sequence and structure conservation between Ref(2)P and p62. (A) Schematic representation of Ref(2)P and p62 sequences. OPCA is the sequence with homology to the AID domain, ZnF (ZZ) is the atypical ZZ zinc finger domain, and UBA is the ubiquitin-associated domain. (B) Sequence alignments of the OPCA, ZnF, and UBA domains of Ref(2)P and p62 were done with the Clustal program.

FIG. 6.

Ref(2)P expression activates the Drosomycin promoter. S2 cells were transfected with the Drosomycin (black bars) or the Attacin (white bars) luciferase reporter as described above, along with increasing amounts (1, 3, and 10 μg) of a Ref(2)P expression vector, and the luciferase (Luc.) activities of cell extracts were determined. Results are the means ± standard deviations (SD) of results from three independent experiments with duplicate incubations. A representative immunoblot for Ref(2)P that was subjected to the corresponding anti-tag antibody is shown below the graph.

FIG. 7.

Ref(2)P is required for the activation of the Drosomycin promoter. S2tpll cells were (RNAi) or were not (Mock) transfected with Ref(2)P dsRNA along with either Drosomycin or Attacin luciferase reporter vectors, after which cells were stimulated (black bars) with Cu²⁺ (for the Drosomycin promoter) or LPS (for the Attacin promoter) or were left unstimulated (white bars), and the luciferase (Luc.) activities of cell extracts were determined. Results are the means ± SD of results from three independent experiments with duplicate incubations. A representative RT-PCR gel for Ref(2)P is shown below the graph.

FIG. 8.

Ref(2)P is essential for Drosomycin transcription. (A) S2tpll cells were transfected with Ref(2)P dsRNA (RNAi) or were not transfected (Mock), after which they were stimulated with either Cu²⁺ (left panel) or LPS (right panel), and the levels of transcriptional activation of Drosomycin (left panel) and Diptericin (right panel) were determined by RT-PCR. The levels of Ref(2)P RNA were determined in parallel cell extracts by RT-PCR. (B) S2tpll cells were transfected or not transfected with Ref(2)P dsRNA, after which they were stimulated or not stimulated with Cu²⁺, and the localization of DaPKC in Triton-soluble membrane fractions was determined by immunoblot analysis. The results shown are representative of results from at least two other experiments.

FIG. 9.

Ref(2)P interacts with aPKC in vivo. (A) Cultures of 293 cells were transfected with either an empty plasmid or an HA-tagged Ref(2)P expression vector, after which cell extracts (Ext) were immunoprecipitated with an irrelevant (−) or an anti-aPKC (+) antibody (Ab) and the immunoprecipitates (IP) were analyzed with an anti-HA antibody. (B) S2 cells were transfected with a Myc-tagged Ref(2)P expression plasmid along with either an empty vector or a DaPKC expression plasmid. Cell extracts were immunoprecipitated with an anti-aPKC antibody, and the immunoprecipitates were analyzed with an anti-Myc antibody. (C) Cultures of 293 cells were transfected with a Myc-tagged Ref(2)P expression plasmid along with either an empty vector or a Flag-tagged DTRAF2 expression plasmid. Cell extracts were immunoprecipitated with an anti-Flag antibody, and the immunoprecipitates were analyzed with an anti-Myc antibody. Aliquots corresponding to 1/10 of the amount of extracts (Ext) used for the immunoprecipitation were loaded into the gels and analyzed by immunoblotting with the corresponding antibodies. The results shown are representative of results from at least two other experiments. WB, Western blot.

FIG. 10.

. Ref(2)P and DTRAF2 cooperate to activate the Drosomycin promoter. S2 cells were transfected with the Drosomycin luciferase reporter as described above, along with increasing amounts (1, 3, and 10 μg) of DTRAF2. (A) In addition, S2 cells were transfected with 1 μg of DTRAF2 expression vector either alone or with increasing amounts (1, 2, and 5 μg) of Ref(2)P expression plasmid (B). Results are the means ± SD of results from three independent experiments with duplicate incubations. Representative immunoblots for Ref(2)P and DTRAF2 performed with the corresponding anti-tag antibodies are shown below the graph.

FIG. 11.

ζPKC phosphorylates Dif in vitro. Recombinant pure GST-Dif or GST was incubated with pure recombinant ζPKC, and the phosphorylation was determined as described in Materials and Methods. The results shown are representative of results from two other experiments.

FIG. 12.

Innate immunity pathways in Drosophila. Triggering the antifungal pathway leads to the activation of Drosomycin transcription. This cascade is initiated when Spätzle binds Toll, recruiting the adapters DMyD88, Pelle, Tube, and possibly DTRAF2. The latter interacts with Ref(2)P, which also binds DaPKC. Both Ref(2)P and DaPKC are essential for Toll-induced Drosomocyn expression, but they are not required for Cactus degradation. Instead, we propose that they stimulate Dif and/or Dorsal transcriptional activity. A parallel pathway is triggered by gram-negative bacteria, which leads to the activation of Diptericin and Attacin transcription. This cascade signals through the receptor peptidoglycan recognition protein (PGRP)-LC and the adapter Imd and activates a Drosophila IKK complex. The D. melanogaster IKK complex regulates Relish endoproteolytic activation, via the caspase Dredd, and thus controls Diptericin expression. In parentheses are shown the names of the mammalian homologues of Pelle, DTRAF2, Ref(2)P, Dorsal, Dif, Imd, and Relish.