Abstract
The Toll signaling pathway plays an important role in the innate immunity ofDrosophila melanogasterand 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 againstStaphylococcus aureusby 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 Ser(276)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.
Highlights
The immune system is composed of humoral and cellular immunity
Previous studies suggested that the role of arrs in cell signaling is much broader and that arrs regulate signaling molecules by modulating phosphorylation, ubiquitination, and/or subcellular distribution of their binding partners. arrs appear to interact with TRAF6 and IB␣ in the Toll-like receptors in innate immunity (TLRs) signaling pathway and inhibit NF-B activity (19 –21). arr1 functions as a positive regulator of CD4ϩ T cell survival and autoimmunity [22]. arrs inhibit cell apoptosis by inhibiting pro-apoptotic extracellular signal-regulated protein kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) and anti-apoptotic Akt signaling pathways in mouse embryonic fibroblasts [23]
These results demonstrated that the Toll pathway plays a central role in host defense against S. aureus by regulating antimicrobial peptides (AMPs) expression
Summary
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 hemocytes and other tissues were used for RNA or protein extraction. CDNA Cloning and Sequence Analysis—The full-length cDNA sequences of Mj-Dorsal, Mj-arr, Mj-arr, and MjERK were obtained from hemocyte and intestine transcriptome sequencing of M. japonicus. Protein samples obtained from shrimp organs were separated by 10% SDS-PAGE and 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-arrs, Mj-ERK, or Mj-Dorsal) in TBS with 3% nonfat milk for 3 h. Mj-arr, Mj-arr, Mj-ERK, and Mj-Cactus antisera were prepared in our laboratory using recombinant proteins
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