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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2016 April 1; 291(14): 7488–7504.
Published online 2016 February 4. doi:  10.1074/jbc.M115.698134
PMCID: PMC4817179

β-Arrestins Negatively Regulate the Toll Pathway in Shrimp by Preventing Dorsal Translocation and Inhibiting Dorsal Transcriptional Activity*

Abstract

The Toll signaling pathway plays an important role in the innate immunity of Drosophila melanogaster and mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense against Staphylococcus aureus by regulating expression of antimicrobial peptides in shrimp. We then found that β-arrestins negatively regulate Toll signaling in two different ways. β-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of β-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. β-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser276 that impairs Dorsal transcriptional activity. Our study suggests that β-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity.

Keywords: arrestin, extracellular-signal-regulated kinase (ERK), innate immunity, Staphylococcus aureus (S. aureus), toll-like receptor (TLR)

Introduction

The immune system is composed of humoral and cellular immunity. Toll and Toll-like receptor pathways play important roles in humoral and cellular immune responses in Drosophila (1, 2). Subsequent studies have revealed the central roles of mammalian Toll-like receptors in innate immunity (TLRs)2 (3). Gram-positive bacterial or fungal infection activates the Toll pathway, which leads to production of several antimicrobial peptides (AMPs) that kill infective pathogens in Drosophila melanogaster (4). The Toll pathway is also involved in the hematopoiesis, encapsulation, and killing of parasites (5). The core components of the Toll pathway in Drosophila include the cytokine-like ligand Spätzle, the receptor Toll, the intracellular adaptor MyD88, the kinases Tube and Pelle, and transcription factors, such as Dorsal and Dorsal-related immunity factor (Dif). After activation of the Toll pathway, Dorsal or Dif translocates into the nucleus, leading to activation of several AMP genes (2, 6, 7).

The Toll pathway is regulated by multiple factors at different levels. For example, Pellino, a Pelle/IL-1R-associated kinase (IRAK) interacting protein, acts as a positive regulator of the Toll pathway (8). Drosophila Pellino mutants have impaired drosomycin expression and reduced survival against Gram-positive bacteria. As all Pellino proteins contain a RING domain, it is speculated that Drosophila Pellino may ubiquitinate Pelle in a similar fashion to mammalian Pellinos' polyubiquitination of IRAK1 (9). G protein-coupled receptor kinase 2 (Gprk2) was identified as a regulator of the Toll pathway (10). Gprk2 interacts with Cactus in S2 cells but is not involved in Cactus degradation; the detailed mechanism remains to be investigated. A recent study showed that specific calcineurin isoforms are also involved in Drosophila Toll immune signaling as positive regulators (11).

The strength and duration of the activation of the Toll signaling pathway must be tightly controlled, because overactivation of Toll or TLRs can be dangerous to the host. The Toll signaling pathway is negatively regulated by different molecules that target each of the key molecules in Toll signaling through various mechanisms to prevent or terminate excessive immune responses. In Drosophila, the Cactus, a homolog of IκB in mammals, is the main cytoplasmic inhibitor of Dorsal (a homolog of mammalian NF-κB) (12). The activation of the Toll pathway is also negatively regulated by serpins (13). Kurtz, a β-arrestin in Drosophila, negatively controls Toll signaling and systemic inflammation at the level of sumoylation (14). A very recent report demonstrates that Drosophila Pellino functions as a negative regulator by targeting MyD88 for ubiquitination and degradation in Toll-mediated signaling (15).

The arrestin family in mammals includes four members as follows: two visual β-arrestins (βarrs), which are expressed in the rod and cone photoreceptor cells of the retina, respectively, and two ubiquitously expressed β-arrestins (βarrs 1 and 2) (16). As adaptor proteins, βarrs are critical for mediating endocytosis of G protein-coupled receptors (GPCRs). In addition, βarrs function in the desensitization and endocytosis of different cell surface receptors (17). βarrs are also scaffold proteins, linking GPCRs to other signaling proteins, such as the Src family kinases and members of the mitogen-activated protein kinase (MAPK) cascade. βarrs are involved in the regulation of multiple signaling pathways (18). Previous studies suggested that the role of βarrs in cell signaling is much broader and that βarrs regulate signaling molecules by modulating phosphorylation, ubiquitination, and/or subcellular distribution of their binding partners. βarrs appear to interact with TRAF6 and IκBα in the TLR signaling pathway and inhibit NF-κB activity (19,21). βarr1 functions as a positive regulator of CD4+ T cell survival and autoimmunity (22). βarrs also inhibit cell apoptosis by inhibiting pro-apoptotic extracellular signal-regulated protein kinases (ERK1/2) and p38 MAPKs and anti-apoptotic Akt signaling pathways in mouse embryonic fibroblasts (23). In Drosophila, Kurtz inhibits MAPK and Toll signaling during development (24). Another study connected Kurtz activity to sumoylation and found that Kurtz negatively controls Toll signaling and systemic inflammation at the level of sumoylation in Drosophila, although the mechanism was not clear (14). Therefore, mammalian βarrs and Drosophila Kurtz both down-regulate NF-κB signaling, but in different ways. It would be interesting to determine whether βarrs in other invertebrates regulate Toll/NF-κB signaling pathways like mammals or Drosophila.

In shrimp, the key molecules of the Toll pathway have been identified, such as Spätzle, Toll receptors, MyD88, Pelle, Cactus, and Dorsal (25,31), and reviewed briefly for the signal pathway by Li and Xiang (32). Toll-interacting protein (Tollip) from Litopenaeus vannamei was reported to negatively regulate the shrimp antimicrobial peptide gene, penaeidin-4 (PEN4) (33). ERK was identified and involved in defense against White spot syndrome virus invasion in Fenneropenaeus chinensis (34). However, the function and regulation of the Toll signaling are seldom studied. In our work, we found that the bacterial clearance, shrimp survival rate, and expression of antimicrobial peptides in kuruma shrimp Marsupenaeus japonicus were declined significantly after RNA interference (RNAi) of Dorsal, the transcription factor of the pathway, and subsequently infected with Staphylococcus aureus. These results demonstrated that the Toll pathway plays a central role in host defense against S. aureus by regulating AMPs expression. Then the regulation of the signaling was analyzed. We found that two βarrs (designated as Mj-βarr1 and Mj-βarr2) interacted with Mj-Cactus and Mj-ERK. Knockdown of βarrs enhanced Dorsal translocation into the nucleus to induce expression of AMPs, although knockdown of ERK showed the opposite results. The possible mechanism was further studied. βarrs interacted with Cactus to form a βarr·Cactus·Dorsal complex, which retained Dorsal in the cytoplasm of cells. βarrs also interacted with non-phosphorylated ERK to inhibit its phosphorylation and affected Dorsal phosphorylation at Ser276. Therefore, βarrs negatively regulate the Toll signaling pathway by forming a βarr·Cactus·Dorsal complex to prevent Dorsal translocation and impair Dorsal phosphorylation by inhibiting ERK phosphorylation. To the best of our knowledge, this is the first work to demonstrate that Toll signaling plays a central role in shrimp against Gram-positive bacteria, and βarrs regulate the Toll pathway in two different ways.

Experimental Procedures

Bacterial Challenge and Sample Collection

Kuruma shrimp M. japonicus (6–8 g each) were purchased from a fish market in Jinan, Shandong Province, China, and cultured for 1 day in laboratory aquarium tanks with aerated seawater at 22 °C for acclimation to the new environment. The 2 × 107 cfu of S. aureus (Shandong University Organism Culture Collection) was injected into the abdomen of shrimp. The control group was injected with PBS (140 mm NaCl, 10 mm sodium phosphate, pH 7.4). Hemocytes, heart, hepatopancreas, gill, stomach, and intestine were collected from at least three shrimp. For the hemocytes collection, the hemolymph was extracted with a syringe preloaded with 1 ml of anticoagulant buffer (0.45 m NaCl, 10 mm KCl, 10 mm EDTA, and 10 mm HEPES, pH 7.45) and immediately centrifuged at 800 × g for 8 min at 4 °C, and the hemocytes were suspended in PBS. The hemocytes and other tissues were used for RNA or protein extraction. Total RNA was extracted using the TRIzol reagent (Cwbio, Beijing, China).

cDNA Cloning and Sequence Analysis

The full-length cDNA sequences of Mj-Dorsal, Mjarr1, Mjarr2, and Mj-ERK were obtained from hemocyte and intestine transcriptome sequencing of M. japonicus. The open reading frame of Mj-Cactus was also obtained by transcriptome sequencing. Similarity analysis was conducted using BLASTx (www.ncbi.nlm.nih.gov). The corresponding cDNA was conceptually translated, and the deduced proteins were predicted using ExPASy. The domain architecture prediction of the proteins was performed using SMART. MEGA5 was used for phylogenetic analysis.

Tissue Distribution and Expression Pattern Analysis by Reverse Transcription-PCR (RT-PCR) and Western Blotting

RT-PCR was used to assess the tissue distribution of Mjarr1, Mjarr2, Mj-Dorsal, Mj-ERK, and Mj-Cactus using the primers shown in Table 1. Protein samples obtained from shrimp organs were separated by 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. The membrane was blocked for 1–2 h with 3% nonfat milk in Tris-buffered saline (10 mm Tris-HCl, pH 8.0, 150 mm NaCl) and incubated with 1:200 diluted antiserum against the proteins of interest (Mj-βarrs, Mj-ERK, or Mj-Dorsal) in TBS with 3% nonfat milk for 3 h. After washing three times in TBS, alkaline phosphatase-conjugated goat anti-rabbit IgG (1:10,000 diluted in TBS) was added. After incubation with the membrane for 3 h, unbound IgG was washed away. The membrane dipped into the reaction system was visualized by 4-chloro-1-naphthol oxidation in the dark for 5 min. Mj-βarr1, Mj-βarr2, Mj-ERK, and Mj-Cactus antisera were prepared in our laboratory using recombinant proteins. Antibodies recognizing the phosphorylated forms of ERK were purchased from Abcam, and NF-κB p65 (Ser276) antibody was purchased from ABGENT (San Diego, CA).

TABLE 1
Sequences of the primers used in this study

Recombinant Expression and Antiserum Preparation

DNA encoding Mj-βarr1, Mj-βarr2, Mj-Dorsal, Mj-ERK, and Mj-Cactus were amplified with different primers (Table 1), and recombinant plasmids were constructed. The recombinant proteins of Mj-βarr1 and Mj-βarr2 and Mj-Dorsal with His tag (pET32a(+) vector), Mj-βarr1, Mj-βarr2, Mj-ERK, and Mj-Cactus with GST tag (pGEX4T-1) were expressed in Escherichia coli Rosetta. E. coli cells with different plasmids were cultured until the A600 of the bacterial culture reached 0.5, and isopropyl thiogalactoside (0.5 mm) was added to induce protein expression at 37 °C for 4 h. The recombinant proteins were purified by affinity chromatography using His-Bind resin (Ni2+-resin; Novagen, Darmstadt, Germany) or GST-resin (GenScript, Nanjing, China). The antiserum preparation was performed as described previously (35).

Quantitative Real Time PCR

The cDNA templates were diluted 50-fold in nuclease-free water for qRT-PCR analysis. SYBR Premix Ex Taq (TaKaRa, Dalian, China) was used in a real time thermal cycler (Bio-Rad) with a total volume of 10 μl containing 5 μl of 2× Premix Ex Taq, 1 μl of the 1:100 diluted cDNA, and 2 μl (1 μm) each of the forward primer and the reverse primer. qRT-PCR was performed with the following conditions: 94 °C for 3 min, 40 cycles of 94 °C for 10 s, and 60 °C for 1 min, and a final dissociation protocol from 65 to 95 °C. Three parallel experiments were conducted to increase the credibility of this study. We used the 2−ΔΔCt method to calculate the mRNA relative expression (36). The obtained data were subjected to statistical analysis and unpaired sample t test.

RNA Interference Assay

The cDNA fragments amplified by primers Fi and Ri (Table 1) linked to the T7 promoter (Table 1) were used as templates for the synthesis of dsRNA. The cDNA fragment of GFP used for dsGFP synthesis was amplified using primers GFP-Fi and GFP-Ri (Table 1).

The assay for dsRNA synthesis was performed in accordance with previous reports (35). The dsRNA (40 μg) of Mj-MEK, Mjarr1, Mjarr2, Mj-ERK, or Mj-Dorsal was injected into the abdominal segment of each shrimp. To enhance the RNAi effect, a second injection was performed 12 h after the first injection. The dsGFP was used as the control. The intestine was collected from the shrimp 24 h after the second injection, and total RNA was extracted and detected by RT-PCR using primers RT-F and RT-R (Table 1) to check the efficiency of RNAi. The experiments were repeated three times.

After setting up the RNAi assay, S. aureus was injected into the gene-silenced shrimp. The expression levels of AMPs and the tumor necrosis factor (TNF) superfamily (TNFSF) gene regulated by the Toll pathway (37) in hemocytes and intestine and gill, and the bacterial clearance and the survival rate (see the following method) of the shrimp were analyzed for Mj-Dorsal RNAi shrimp. The translocation of Mj-Dorsal in hemocytes and intestine, the AMPs expression in hemocytes, intestine, or gill, the bacterial clearance, and the survival rate were detected for Mjarr1 and Mj-ERK RNAi shrimp. The AMPs expression was analyzed by quantitative real time reverse transcription PCR (qRT-PCR) with the AMPs primers (Table 1). β-Actin was used as the control. Data show the mean ± S.D. from three independent repeats. The p value was calculated by Student's t test for paired samples, and significant or most significant differences were accepted at p < 0.05 or p < 0.01.

Nuclear and Cytoplasmic Protein Extraction

To confirm Mj-Dorsal and Mj-ERK translocation into the nucleus, nuclear and cytoplasmic protein extraction assays were performed. Shrimp intestine and gill were cut into pieces with small scissors, washed with PBS, and then centrifuged at 1000 × g for 7 min. The precipitate was suspended in 100 μl of buffer A (10 mm HEPES, pH 7.5, 10 mm KCl, 0.2 mm EDTA, 3 mm MgCl2, 1 mm DTT, 1 mm PMSF, 1% inhibitor mixture (Merck, Germany), 1% Nonidet P-40) and incubated on ice for 10 min. The tissue solution was then centrifuged at 1000 × g for 10 min. The obtained supernatant contained cytoplasmic proteins, and the precipitate was resuspended in the buffer A, incubated for 10 min on ice to wash off the remaining cytoplasm, and then centrifuged at 1000 × g for 10 min again. Buffer B (100 μl) (20 mm HEPES, 400 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm PMSF, 1% mixture) was added to the precipitate and vortexed for 30 min at 4 °C. The solution was centrifuged at 14,000 × g for 10 min to obtain the nuclear proteins. Nuclear and cytoplasmic proteins were then subjected to Western blotting.

Bacteria Clearance and Survival Rate Assays

The shrimp (6–8 g each) were divided into four groups as following: three groups for RNAi of target genes and one group for GFP-RNAi control. After knockdown of Mj-Dorsal by RNAi, shrimp were injected with S. aureus (2 × 107 CFU) at 24 h after the second dsRNA injection. At 6 h after S. aureus injection, S. aureus (2 × 107 CFU) was injected into shrimp again, and the hemolymph was extracted at 1 h after the second S. aureus injection. The hemolymph was then diluted and cultured on LB agar plates overnight at 37 °C. The number of bacterial colonies was counted. The same methods were performed for Mjarr1 and Mj-ERK RNAi and bacterial clearance.

Survival Rate Assay

After Mj-Dorsal was knocked down by RNAi, the shrimp were injected with S. aureus (2 × 107 cfu). The dead shrimp were monitored and counted every day. Data show the mean ± S.D. from three independent repeats. The p value was calculated by Student's t test for paired samples, and significant or most significant differences were accepted at p < 0.05 or p < 0.01, respectively.

Pulldown Assay

The recombinant proteins (30 μg) were added to 20 μl of charged nickel-nitrilotriacetic acid beads (for His-tagged proteins) or glutathione resin (for GST-tagged proteins) and incubated at room temperature for 2 h with slight rotation. The mixture (resin and binding proteins) was washed three times by centrifugation at 500 × g for 3 min to remove the unbound proteins. The test protein, without a His tag or GST tag, was added into the mixture containing the nickel-nitrilotriacetic acid beads or glutathione resin and the tagged protein and gently rotated at room temperature for 2 h. After washing three times, the mixture was analyzed by SDS-PAGE. We also used recombinant proteins to pull down the natural proteins from shrimp. Shrimp gills were homogenized with lysis buffer (150 mm NaCl, 1.0% Nonidet P-40, 0.1% SDS, 50 mm Tris-containing protease inhibitor mixture (Abcam)) and then centrifuged at 14,000 × g for 12 min. The supernatant (1000 μl) was added into the GST resin with recombinant proteins and incubated at room temperature for 2 h. The resin was washed three times by centrifugation at 500 × g for 3 min and then analyzed using SDS-PAGE. The proteins in the gel were transferred onto a nitrocellulose membrane, followed by blocking with 3% nonfat milk dissolved in Tris-buffered saline (10 mm Tris-HCl, 150 mm NaCl, pH 7.5) and incubated with 1:100 diluted antiserum in TBS with 3% nonfat milk for 2 h. Horseradish peroxidase goat anti-rabbit IgG (1:10,000 diluted in TBS) was then added. After incubation with the membrane for 2 h, unbound IgG was washed off. The membrane was dipped into the reaction system (9 ml of TBS, with 1 ml of 4-chlorine naphthol and 6 μl of H2O2) in the dark for 5 min to visualize the target protein.

Immunocytochemical Assays

The hemolymph was collected from three shrimp using a syringe preloaded with 1 ml of anticoagulant and then fixed by adding 4% paraformaldehyde. The hemocytes were isolated by centrifugation (700 × g for 4 min at 4 °C), washed with PBS, and incubated in 0.2% Triton X-100 at 37 °C (5 min) twice. After blocking with 3% bovine serum albumin (BSA) for 30 min at 37 °C, hemocytes were incubated overnight with anti-Mj-Dorsal serum (1:100 in blocking buffer) at 4 °C. After washing with PBS, the hemocytes were then incubated with 3% BSA for 10 min, after which the second antibody, goat anti-rabbit Alexa Fluor 488 (1:1,000 dilution in 3% BSA), was added. The reaction was kept in the dark for 1 h at 37 °C and then washed with PBS. Hemocytes were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, AnaSpec Inc., San Jose, CA) for 10 min at room temperature and washed again. Hemocytes were observed under a fluorescence microscope (Olympus BX51).

Co-immunoprecipitation (Co-IP)

Sixty milligrams of CNBr-activated Sepharose 4B (Amersham Biosciences) was swollen in 500 μl of HCl (1 mm) and 100 μl of medium, and then washed twice with 1 mm HCl. The antibodies of interested were incubated with CNBr-activated Sepharose 4B with rotation at 25 °C for 1 h. The resin was washed five times with coupling buffer and then equilibrated in 0.1 m Tris-HCl, pH 8.0, at 25 °C for 2 h. Then, after four rounds of alternate washing with acetic acid buffer (0.1 m sodium acetate, 0.5 m NaCl, pH 4.0) and Tris buffer (0.1 m Tris-HCl, 0.5 m NaCl, pH 8.0), the resin was equilibrated in Tris buffer. The supernatant (1000 μl) from normal and challenged shrimp hemocytes, intestine, or gill was prepared and then mixed with 100 μl of resin and gently rotated overnight at 4 °C. After five washes with Tris buffer, the native protein was eluted using elution buffer (0.1 m glycine, pH 2.5) and neutralized with neutralization buffer (1 m Tris-HCl, pH 8.0). The purified proteins were confirmed by Western blotting.

PD98059 Inhibitor Assay in Shrimp

PD98059, a mitogen-activated protein kinase kinase MEK1/2 inhibitor, was purchased from Cell Signaling Technology (Danvers, MA). PD98059 (4 μm) was injected into shrimp (8–10 g), and the same volume of DMSO injection was used as the control. S. aureus was injected after 1 h of inhibitor injection. Subsequently, shrimp intestines were collected 1 h after bacterial injection for Western blotting. Antibodies against Mj-ERK and Mj-Dorsal were used in the analysis. The total RNAs from intestine were extracted from shrimp 6 h after S. aureus injection to detect AMPs expression.

Results

Toll Pathway Is Activated by S. aureus Challenge and Plays a Central Role in Defense against S. aureus in Shrimp

To study the function and regulation of the Toll pathway in shrimp during bacterial infection, we first ascertained the activation of the Toll pathway by translocation of Mj-Dorsal (GenBankTM accession number KU160503) and expression of AMPs after bacterial infection. The Gram-positive bacterium S. aureus was used to activate the Toll pathway. After bacterial activation of the Toll pathway in shrimp, the protein level of Mj-Dorsal increased in hemocytes, intestine, and gill (Fig. 1, A–C). Mj-Dorsal was translocated from the cytoplasm into the nucleus in hemocytes, intestine, and gill at 1 h after S. aureus challenge (Fig. 1, D and F). The expression of AMPs, ALF-C2 (GenBankTM accession number KU160498), CruI-1 (GenBankTM accession number KU160502), and another effector TNFSF (GenBankTM accession number KU160505) but not ALF-D1 (GenBankTM accession number KU160499) regulated by the Toll pathway also, was increased significantly (Fig. 1, G, J and M). After knockdown of Mj-Dorsal by RNA interference and the subsequent challenge by S. aureus, the expression of the effectors ALF-C2, CruI-1, and TNFSF in hemocytes (Fig. 1H), intestine (Fig. 1K), and gill (Fig. 1N) was significantly decreased compared with the control (Fig. 1, I, L, and O). These results indicated that expression and translocation of Mj-Dorsal and expression of the Toll pathway effectors (AMP genes) were significantly increased in hemocytes, intestine, and gill after S. aureus infection and suggest that systemic immune responses occurred also in the intestine and gill in addition to hemocytes. Therefore, we mainly used intestine or gill in the following studies for the convenience of sample collection.

FIGURE 1.
Toll pathway plays a central role in host defense against S. aureus in shrimp. A–C, upper panels show Dorsal expressions in the hemocytes (A), intestine (B), and gill (C) of shrimp challenged with S. aureus, as analyzed by Western blotting; bottom ...

To evaluate whether the Toll pathway plays a central role in host defense against S. aureus, the bacterial clearance and survival of shrimp were conducted after shrimp were injected with dsRNA of Mj-Dorsal and subsequently infected with S. aureus. dsGFP injection followed by S. aureus infection was used as the control. The results showed that after RNAi of Mj-Dorsal, S. aureus clearance was significantly reduced compared with the control group (Fig. 1P), and the survival rate of Mj-Dorsal-RNAi shrimp infected with S. aureus was significantly lower than that of the control from day 3 to day 5 post-infection (Fig. 1Q). These data indicated that the Toll signaling may play a central role in defense against Gram-positive bacteria in shrimp.

Mj-βarr1, Mj-βarr2, and Mj-ERK Participate in Regulation of the Toll Pathway

The full-length cDNA sequences of Mj-βarr1 (GenBankTM accession number KU160500), Mj-βarr2 (GenBankTM accession number KU160501), and Mj-ERK (GenBankTM accession number KU160504) were obtained by transcriptome sequencing of M. japonicus. To analyze whether Mj-βarr1, Mj-βarr2, and Mj-ERK participate in regulation of the Toll signaling pathway, translocation of Mj-Dorsal and expression of the Toll pathway effectors were detected after RNAi knockdown of βarrs and ERK, followed by bacterial challenge. The results showed that in the βarr-silenced shrimp, most of Mj-Dorsal was detected in the nucleus of intestine cells by Western blotting (Fig. 2, A and A1), and the expression of effector transcripts, such as ALF-C2, CruI-1, and TNFSF mRNAs, was significantly up-regulated (Fig. 2A2). The Mj-Dorsal translocation was also detected in hemocytes in βarr-silenced shrimp following bacterial challenge (Fig. 2, A3 and A4). The results showed that in the normal (untreated) shrimp, Mj-Dorsal could be observed in the cytoplasm and nucleus of hemocytes, but most of the Mj-Dorsal was located in the cytoplasm (Fig. 2A4). In the dsGFP-injected shrimp, most of Mj-Dorsal was translocated into the nucleus, and only a small amount of Mj-Dorsal could be detected in the cytoplasm after S. aureus challenge (Fig. 2A4). After βarr knockdown and bacterial challenge, Mj-Dorsal was only detected in the nuclei of hemocytes (Fig. 2A4). These results suggested that βarrs may inhibit the expression of the Toll pathway effector genes by preventing Mj-Dorsal from translocation into the nucleus. On the contrary, most Mj-Dorsal was detected in the cytoplasm but not in the nuclei of intestine cells (Fig. 2B1) after Mj-ERK was knocked down (Fig. 2B) followed by S. aureus challenge. This result indicated that Mj-Dorsal could not be translocated into the nucleus after bacterial challenge in the Mj-ERK knockdown shrimp, and the expression of effectors was also significantly decreased (Fig. 2, B1 and B2) at 6 h after bacterial injection. In the hemocyte immunocytochemical assay, almost no Mj-Dorsal could be detected in the nucleus after Mj-ERK knockdown (Fig. 2, B3 and B4). These results suggested that Mj-ERK promoted the nuclear translocation of Mj-Dorsal and increased the expression of the AMPs. Taken together, our results suggested that Mj-βarrs negatively regulate the Toll pathway, and Mj-ERK positively regulates the pathway.

FIGURE 2.
Knockdown of Mjarrs or Mj-ERK affects Toll signaling in shrimp. A, efficiency of RNAi for Mjarr1 and Mjarr2 in shrimp intestine analyzed at protein (upper) and RNA (lower) levels. dsGFP injection was used as the control. ...

To further confirm that Mj-βarrs and Mj-ERK participate in regulating the Toll pathway, the bacteria clearance and survival rate of Mjarr1-RNAi or Mj-ERK-RNAi shrimp were also analyzed. In the Mjarr1-RNAi shrimp injected with S. aureus, bacteria clearance was enhanced, although in the Mj-ERK-RNAi shrimp bacteria clearance was reduced compared with the control group (Fig. 2, A5 and B5). The survival rate of the Mjarr1-RNAi shrimp injected with S. aureus was about 55% at day 5 post-infection, which was significantly higher than that (20%) of the GFP-RNAi control group (Fig. 2A6). However, the survival rate of Mj-ERK-RNAi shrimp injected with S. aureus was about 40% at day 3 post-infection, which was significantly lower than that (75%) of dsGFP shrimp (Fig. 2B6). These data indicated that Mjarr1 and Mj-ERK play important roles in the antibacterial immunity of shrimp.

Mj-βarrs Interact with Mj-Cactus to Form a βarr·Cactus·Dorsal Complex

To understand the regulatory mechanism of Mj-βarrs in the Toll pathway, the interactions of Mj-βarrs with Mj-Cactus and Mj-Dorsal were analyzed by Co-IP assays. We first determined the amount of Mj-Cactus after S. aureus challenge, and the results showed that the amount of Mj-Cactus was decreased from 10 to 60 min in hemocytes, intestine, and gill of the shrimp, and only small amounts of Mj-Cactus were detected in hemocytes at the 60-min post-bacterial challenge (Fig. 3A). These results suggested that Mj-Cactus was degraded after activation of Toll pathway. The antibody against Mj-βarr1 was coupled to CNBr-activated Sepharose 4B for binding of natural Mj-Cactus and Mj-Dorsal from unchallenged and bacterially challenged shrimp. The results showed that in the unchallenged shrimp, Mj-Cactus and Mj-Dorsal could be co-immunoprecipitated with Mj-βarr1, but in the bacterially challenged shrimp, no Mj-Cactus nor Mj-Dorsal was co-immunoprecipitated (Fig. 3, B and B1). It is known that after activation of the Toll pathway by S. aureus, Cactus is degraded to release Dorsal, which then translocates into the nucleus. Therefore, the above results suggest that Mj-βarrs may interact with Mj-Cactus and/or Mj-Dorsal. When Mj-Cactus antibody was used for the Co-IP assay, Mj-βarr1, Mj-βarr2, and Mj-Dorsal were co-precipitated in the unchallenged shrimp, but only a little Mj-Dorsal was co-precipitated in the bacterially challenged shrimp, and almost no Mj-βarrs were detected in gill (Fig. 3, C and C1). The results suggested that Mj-Cactus was not completely degraded in gill of shrimp challenged by bacteria. From Fig. 3A, we can see that most of Mj-Cactus was degraded at 60 min post-bacterial challenge; we used hemocytes to do the co-immunoprecipitation assay, and the results showed that Mj-βarr1, Mj-βarr2, and Mj-Dorsal were not co-immunoprecipitated in the bacterially challenged shrimp (Fig. 3, D and D1). These results suggested that Mj-Cactus interacted with Mj-Dorsal and Mj-βarrs, and almost no interaction of Mj-Cactus and Mj-βarrs was detected at the early stage of bacterial infection in S. aureus challenged shrimp, probably because the upstream signal pathway was activated, and the Mj-Cactus would be degraded to lead to the dissociation of Mj-βarrs with Mj-Cactus. The Mj-Dorsal antibody was also used for Co-IP assays, and the results showed that Mj-βarrs and Mj-Cactus were detected in the unchallenged shrimp, and no Mj-βarrs and Mj-Cactus were detected in the challenged shrimp (Fig. 3, E and E1). Taken together, our results suggested that Mj-βarrs, Mj-Cactus, and Mj-Dorsal could form a heterotrimeric complex, and the complex would disassociate followed by Mj-Cactus degradation.

FIGURE 3.
Mj-βarrs interacts with Mj-Cactus and Mj-Dorsal, forming a βarr·Cactus·Dorsal complex. A, shrimp was injected with S. aureus, and the proteins from different tissues (hemocytes, intestine, and gill) were extracted at 10, ...

To further confirm the possibility of forming a heterotrimeric complex in vivo, the tissue distribution of Mj-βarr1, Mj-βarr2, Mj-Dorsal, and Mj-Cactus was detected at the mRNA and protein levels. The results showed that the four mRNAs and proteins were distributed in all tissues tested, including hemocytes, heart, hepatopancreas, gill, stomach, and intestine (Fig. 3F), suggesting that heterotrimeric complex of Mj-βarr-Cactus-Dorsal could be formed in shrimp.

N-terminal Domain of Mj-βarrs Interacts with the PEST Domain of Mj-Cactus

Pulldown assays were performed to confirm the above-mentioned interactions using recombinant proteins of Mj-βarrs and Mj-Cactus. Mj-βarr contains arrestin-N and arrestin-C domains, whereas Mj-Cactus contains an N-terminal domain (amino acid residues 1–168), ANK repeats domain (amino acid residues 169–395), and a PEST domain (amino acid residues 396–452) at the C terminus (Fig. 4A). The full-length βarrs and their two individual domains were expressed and purified. The full-length Mj-Cactus protein, the N-terminal domain-ANK repeats domain (amino acid residues 1–395), N-terminal domain (amino acid residues 1–168), ANK repeats domain (amino acid residues 169–395), and PEST domain (amino acid residues 396–452) were also expressed and purified. Two different pulldown assays with GST-binding resin and His-binding resin were performed.

FIGURE 4.
N terminus of Mj-βarrs interacts with the PEST domain of Mj-Cactus. A, domain architectures of Mj-βarr1 and Mj-βarr2 (left and middle panels) and Mj-Cactus (right panel). B, interaction of Mj-Cactus with Mj-βarr1, as analyzed ...

The recombinant Mj-βarrs and Mj-Cactus were used for GST and His pulldown assays. As shown in Fig. 4, B and C, Mj-βarr1 or Mj-βarr2 interacted with Mj-Cactus. To understand which domain of Mj-βarrs was responsible for the interaction with Mj-Cactus, the N- and C-terminal domains of βarrs (His-Mj-βarr1-N, His-Mj-βarr1-C and His-Mj-βarr2-N, His-Mj-βarr2-C) were used in GST and His pulldown assays. The results showed that Mj-βarrs-N, but not Mj-βarrs-C, interacted with Mj-Cactus (Fig. 4, D–G) to further study which domain of Mj-Cactus interacted with Mj-βarrs-N. The GST-tagged N-ANK and PEST domains of Mj-Cactus were used for pulldown assays. The results showed that The Mj-Cactus-N-ANK(1–395) could not interact with Mj -βarrs-N (Fig. 4, H and I); however, Mj-Cactus-PEST (Fig. 4, F and G) interacted with Mj-βarrs-N (Fig. 4, J and K). Thus, the results suggested that the N-terminal domain of Mj-βarrs interacted with the C-terminal PEST domain of Mj-Cactus.

ANK Domain of Mj-Cactus Interacts with the RHD Domain of Mj-Dorsal and βarrs, Cactus and Dorsal Form a Complex

To detect which domain of Mj-Cactus is responsible for the interaction with Mj-Dorsal, the recombinant proteins of Cactus and its individual domains with GST tags (Mj-Cactus, Mj-Cactus-N, Mj-Cactus-ANK, Mj-Cactus-N-ANK, and Mj-Cactus-PEST) (Fig. 5A) and Mj-Dorsal and its different domains with His tags (Mj-Dorsal, Mj-Dorsal-RHD, and Mj-Dorsal-IPT) (Fig. 5B) were used for pulldown assays. As shown in Fig. 5C, Mj-Cactus could interact with Mj-Dorsal, and further study showed that Mj-Cactus interacted with the RHD domain but not the IPT domain of Mj-Dorsal (Fig. 5D). Recombinant Mj-Cactus-N-ANK, Mj-Cactus-N, Mj-Cactus-ANK, and Mj-Cactus-PEST were used for pulldown assays to analyze the interaction with RHD domain of Mj-Dorsal (Fig. 5E). The results showed that only the ANK repeats domain of Mj-Cactus interacted with the RHD domain of Mj-Dorsal.

FIGURE 5.
Mj-Cactus interacts with Mj-Dorsal to form a βarr·Cactus·dorsal complex. A and B, schematic representations of the full-length protein, and the RHD and IPT domains of Mj-Dorsal (A); full-length protein, N terminus, N-ANK, ANK, ...

In summary, the arrestin-N domain of Mj-βarrs interacts with the PEST domain of Mj-Cactus. The ANK repeat domain of Mj-Cactus interacts with RHD domain of Mj-Dorsal. The βarr·Cactus·Dorsal complex is formed using Mj-Cactus as a bridge. To confirm the result, we used recombinant Mj-βarr1 or Mj-βarr2, Mj-Cactus, and Mj-Dorsal in the GST-pulldown assay, and the result showed that Mj-βarrs, Mj-Cactus, and Mj-Dorsal could form a complex (Fig. 5F) but Mj-βarrs could not interact with Mj-Dorsal (Fig. 5H).

Mj-βarrs Interacts with Non-phosphorylated Mj-ERK to Decrease Phosphorylation of Mj-ERK

In Fig. 2, B and B4, we noticed that ERK positively regulated Dorsal translocation into the nucleus and expression of AMPs. The possible mechanism was further studied. The interactions of Mj-βarrs with Mj-ERK were first studied by pull down (Fig. 6A) and Co-IP (Fig. 6B) assays. Here, we used recombinant proteins to pull down the native proteins from shrimp. The results showed that Mj-ERK interacted with native Mj-βarr1 or Mj-βarr2 (Fig. 6A) and that Mj-βarrs interacted with native non-phosphorylated Mj-ERK, but little native phosphorylated Mj-ERK was co-precipitated (Fig. 6B). To further confirm whether native phosphorylated Mj-ERK could interact with Mj-βarr1 or Mj-βarr2, phosphorylated Mj-ERK antibody was used to do the Co-IP assay. The results showed almost no native Mj-βarr1 or Mj-βarr2 was co-precipitated (Fig. 6C). Therefore, the phosphorylated Mj-ERK could not interact with Mj-βarr1 or Mj-βarr2.

FIGURE 6.
Mj-βarrs interacts with non-phosphorylated Mj-ERK and phosphorylation of Mj-ERK was increased in the Mjarrs-knockdown shrimp challenged by S. aureus at 1 h. A, GST pulldown assay demonstrated the interaction of GST-Mj-ERK with native ...

After knockdown of Mjarr1 or Mjarr2 (Fig. 6D), the phosphorylation level of Mj-ERK was detected by Western blotting. The results showed that the phosphorylation level of Mj-ERK was increased in Mjarr1 and Mjarr2 knockdown shrimp challenged with S. aureus for 1 h (Fig. 6E). These results suggested that Mj-βarrs inhibited ERK phosphorylation by interacting with non-phosphorylated Mj-ERK.

C Terminus of Mj-βarrs Interacts with Mj-ERK

To detect which domain of Mj-βarrs was responsible for the interaction with Mj-ERK, recombinant Mj-βarrs and their domains (His-Mj-βarr1, His-Mj-βarr1-N, His-Mj-βarr1-C, His-Mj-βarr2, His-Mj-βarr2-N, and His-Mj-βarr2-C) (Fig. 7, A and B) and Mj-ERK (GST-Mj-ERK) were used for pulldown assays. The results showed that Mj-βarr1 interacted with Mj-ERK (Fig. 7A1). The C-terminal domain of Mj-βarr1 (Mj-βarr1-C) (Fig. 7A2), but not the N-terminal domain of Mj-βarr1 (Fig. 7A3), was interacted with Mj-ERK. The same results were obtained for the interaction of Mj-βarr2 with Mj-ERK (Fig. 7, B–B3).

FIGURE 7.
Arrestin-C domain of Mj-βarrs interacts with Mj-ERK. A and B, schematic representation of the full-length protein, and the arrestin-N and arrestin-C domains of Mj-βarr1 (A) and Mj-βarr2 (B). A1 and B1, interaction of Mj-ERK with ...

Mj-ERK Affected the Phosphorylation of Mj-Dorsal in the Nucleus

To further study the possible mechanism of ERK in regulation of the Toll pathway, we analyzed the phosphorylation of Mj-ERK and Mj-Dorsal using antibodies to phosphorylated ERK (Abcam) or phosphorylated Mj-Dorsal. To determine whether commercial phosphorylated Dorsal antibody could recognize the phosphorylated Mj-Dorsal from shrimp, Mj-Dorsal was knocked down in shrimp and then challenged with S. aureus, and Western blot was performed to detect the Dorsal. As shown in Fig. 8A, phosphorylated Mj-Dorsal could be detected by the antibody to phosphorylated Dorsal. Then phosphorylation of Mj-Dorsal and Mj-ERK was analyzed by Western blotting. The results showed that S. aureus challenge enhanced phosphorylation of Mj-ERK and Mj-Dorsal. The phosphorylation of Mj-ERK increased rapidly at 15 min and phosphorylation at Ser276 of Mj-Dorsal increased at 30–60 min (Fig. 8B). After knockdown of Mj-ERK and challenge with S. aureus in shrimp, the phosphorylation of Mj-Dorsal obviously decreased (Fig. 8C). To confirm the result, Mj-MEK, which affects the Mj-ERK activity, was knocked down, and the phosphorylation of Mj-Dorsal and Mj-ERK was detected. The results showed phosphorylation of Mj-Dorsal and Mj-ERK was decreased in Mj-MEK-silenced shrimp (Fig. 8E). PD98059, a MEK1/2 inhibitor, was also used to inhibit ERK phosphorylation, and a similar result was obtained (Fig. 8F). We further detected the subcellular distribution of phosphorylated Mj-Dorsal by Western blotting. The distribution and phosphorylation levels of Mj-Dorsal in the nucleus decreased after PD98059 injection and challenge with S. aureus for 1 h (Fig. 8G). The expressions of effectors (MjALF-C2, CruI-1, and TNFSF) regulated by the Toll pathway in the intestine were significantly decreased after PD98059 injection and challenge with S. aureus at 6 h (Fig. 8H). Taken together, the results showed that phosphorylation of Mj-ERK induced Mj-Dorsal phosphorylation and the expression of effectors regulated by the Toll pathway. However, the interaction of Mj-βarr with Mj-ERK inhibited the phosphorylation of Mj-ERK (Fig. 6) and subsequently reduced Mj-Dorsal phosphorylation, which inhibited Toll signaling and reduced the expression of the effectors.

FIGURE 8.
Inhibitor of Mj-ERK or knockdown of Mj-ERK decreased the phosphorylation of Mj-Dorsal in nucleus. A, Western blot detected reduced the Ser276-phosphorylated Mj-Dorsal in the Mj-Dorsal knockdown shrimp challenged by S. aureus at 1 h. The lower panel is ...

Discussion

The Toll signaling pathway plays an important role in innate immunity in invertebrates (38, 39). Dorsal is a transcription factor that modulates the transcriptional activity of the downstream targets. In Drosophila, the Toll signal pathway is activated by fungi and Gram-positive bacteria to regulate expression of AMPs to defend against pathogen invasion (40, 41). In shrimp, the Toll pathway plays an important role in regulating AMP expression (42, 43). In this report, we find that the bacterial clearance and survival rate of Mj-Dorsal-silenced shrimp infected with S. aureus had declined significantly, suggesting that the Toll pathway plays a central role in host defense against S. aureus in shrimp. Toll signaling is activated by S. aureus, resulting in Mj-Dorsal nuclear translocation and an increase in its phosphorylation level. The expression of the effectors (MjALF-C2, CruI-1, and TNFSF) is regulated by the Toll pathway. Mj-βarrs interacted with Mj-Cactus and Mj-ERK to negatively regulate the Toll pathway.

The systemic immune response in Drosophila induced the synthesis of several families of AMPs by cells in the fat body, but AMP induction in the epidermis and epithelia of gut is regulated by the immune deficiency pathway for local immune challenges (44). In our study, we found that S. aureus challenge could induce the expression of the same kinds of AMPs in hemocytes, intestine and gill. AMP expression was not induced in these tissues of Mj-Dorsal RNAi shrimp. These results indicated that S. aureus challenge could induce the systemic immune responses in intestine and gill.

β-Arrestins were initially identified as mediators of GPCR desensitization and endocytosis (45). Nowadays, they are acknowledged to have signaling functions in a wide variety of signaling pathways and modes of regulation, including the Hedgehog, Wnt, Notch, and TGFβ pathways (46). As negative regulators, β-arrestins regulate the TLR-IL-1R signal pathway in mammals by interacting with TRAF6 and IKBα (19, 20). β-Arrestins also regulate the MAPK signaling pathway by interacting with inactive ERK to inhibit its phosphorylation (47, 48). In Drosophila, Kurtz inhibits MAPK and Toll signaling during development (24). Anjum et al. (14) demonstrated that Kurtz and SUMO protease Ulp1 work synergistically to regulate the Toll immune pathway in Drosophila. In shrimp, we found that the arrestin-N domain of Mj-βarrs interacted with the C-terminal PEST domain of Mj-Cactus; the ANK repeats domain of Mj-Cactus interacted with the RHD domain of Mj-Dorsal, and the βarr·Cactus·Dorsal complex was formed using Mj-Cactus as a bridge. The complex prevented phosphorylation and degradation of Mj-Cactus and Mj-Dorsal translocation from the cytoplasm to the nucleus. The C-terminal domain of Mj-βarrs also interacted with non-phosphorylated Mj-ERK and inhibited its phosphorylation. Phosphorylation of Mj-ERK could promote Mj-Dorsal translocation into the nucleus and the phosphorylation of Ser276 of Mj-Dorsal (47). The interaction of Mj-βarrs and Mj-ERK inhibited Mj-ERK phosphorylation and prevented Mj-Dorsal translocation and phosphorylation.

MAPK signaling has been implicated in stress and immunity in evolutionarily diverse species (45). The relationship between MAPK and Toll signaling still needs to be studied in invertebrates. ERK, as one of the three MAPKs, plays a role in regulating transcription activity of p65 in mammals (47, 48). In our study, Mj-βarrs bound to non-phosphorylated Mj-ERK and inhibited its phosphorylation by MEK. Mj-ERK phosphorylation regulated Mj-Dorsal nuclear translocation and phosphorylation of Ser276 of Mj-Dorsal and then regulated Mj-Dorsal transcriptional activity and DNA binding activity. Therefore, the interaction of Mj-βarrs with Mj-ERK indirectly inhibited Mj-Dorsal translocation and phosphorylation and limited Toll signaling activity. Uncontrolled immune responses have detrimental outcomes in organisms; therefore, the intensity and duration of immune signaling must be finely regulated. As described above, activation of the Toll pathway is under multiple layers of control, which would limit the damage caused an inappropriate immune response.

Antimicrobial responses in insects mainly consisted of a two-stage process as follows: constitutive defense, such as engulfment and melanization, acts immediately, and more than 99% of bacteria in the hemocoel can be cleared in less than an hour. The second stage is inducible defense, i.e. is the production of antimicrobial peptides, which eliminates or suppresses the remaining microbes (49). Similar immune responses occurred in shrimp. However, some pre-synthetized AMPs, such as penaeidins, stored in hemocytes could be released upon immune stimulation (50). Therefore, we performed an initial challenge and then made a second challenge against S. aureus in the bacterial clearance assay (see under “Experimental Procedures”).

In mammals, βarrs appear to interact with TRAF6 and IκBα in the TLR signaling pathway and inhibit NF-κB activity (19,21). In Drosophila, Kurtz negatively controls Toll signaling and systemic inflammation at the level of sumoylation (14). In our study, we found that β-arrestins could interact with Cactus and inhibit Toll signaling activity. The function of βarrs in shrimp is similar to that of mammals.

In summary, our study revealed that in shrimp the bacterial challenge could activate the Toll signaling pathway, and the pathway played a central role in antibacterial immunity. We also showed that that Mj-βarrs could block or limit Toll signaling by forming a heterotrimeric complex of βarr-Cactus-Dorsal to prevent Mj-Cactus phosphorylation and degradation. Mj-βarrs also inhibited phosphorylation of Mj-ERK, which affected Mj-Dorsal translocation and phosphorylation. Therefore, Mj-βarrs negatively regulate the Toll pathway by limiting the translocation and phosphorylation of Mj-Dorsal (Fig. 9). These findings for the first time demonstrate that Mj-βarrs negatively regulate the Toll signaling pathway in two different ways in shrimp.

FIGURE 9.
Model for βarrs' involvement in the Toll signaling pathway in shrimp. In shrimp, the Toll signaling pathway is activated by bacterial challenge. The transcription factor Dorsal is translocated into the nucleus and ERK is phosphorylated. Several ...

Author Contributions

J. X. W., J. J. S., and X. F. Z. conceived and designed the experiments. J. J. S., J. F. L., X. Z. S., M. C. Y., G. J. N., and D. D. performed the experiments. J. J. S. and J. X. W. analyzed the data. J. J. S., J. X. W., and X. Q. Y. wrote the paper.

Acknowledgment

We thank Shandong University Organism Culture Collection for providing the bacterium S. aureus.

*This work was supported by National Natural Science Foundation of China Grants 31130056 and 31472303 and National Basic Research Program of China 973 Program Grant 2012CB114405. The authors declare that they have no conflicts of interest with the contents of this article.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s)

2The abbreviations used are:

TLR
toll-like receptor
ANK
ankyrin
βarr
β-arrestin
AMP
antimicrobial peptide
GPCR
G protein-coupled receptor
qRT
quantitative RT
TNFSF
tumor necrosis factor superfamily.

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