ABSTRACT
Dock3, a new member of the guanine nucleotide exchange factor family, causes cellular morphological changes by activating the small GTPase Rac1. Overexpression of Dock3 in neural cells promotes neurite outgrowth through the formation of a protein complex with Fyn and WAVE downstream of brain-derived neurotrophic factor (BDNF) signaling. Here, we report a novel Dock3-mediated BDNF pathway for neurite outgrowth. We show that Dock3 forms a complex with Elmo and activated RhoG downstream of BDNF-TrkB signaling and induces neurite outgrowth via Rac1 activation in PC12 cells. We also show the importance of Dock3 phosphorylation in Rac1 activation and show two key events that are necessary for efficient Dock3 phosphorylation: membrane recruitment of Dock3 and interaction of Dock3 with Elmo. These results suggest that Dock3 plays important roles downstream of BDNF signaling in the central nervous system where it stimulates actin polymerization by multiple pathways.
Subject(s)
Carrier Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurites/metabolism , Receptor, trkB/metabolism , Signal Transduction , Ternary Complex Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Brain-Derived Neurotrophic Factor/pharmacology , COS Cells , Carrier Proteins/genetics , Cell Membrane/genetics , Cell Membrane/metabolism , Chlorocebus aethiops , Enzyme Activation , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Guanine Nucleotide Exchange Factors , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Neurites/drug effects , Neurites/physiology , Neuropeptides/genetics , Neuropeptides/metabolism , PC12 Cells , Phosphorylation , Protein Interaction Mapping , Protein Transport , Rats , Receptor, trkB/genetics , Ternary Complex Factors/genetics , Transfection , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein , rho GTP-Binding ProteinsABSTRACT
Dock3, a new member of the guanine nucleotide exchange factors, causes cellular morphological changes by activating the small GTPase Rac1. Overexpression of Dock3 in neural cells promotes axonal outgrowth downstream of brain-derived neurotrophic factor (BDNF) signaling. We previously showed that Dock3 forms a complex with Fyn and WASP (Wiskott-Aldrich syndrome protein) family verprolin-homologous (WAVE) proteins at the plasma membrane, and subsequent Rac1 activation promotes actin polymerization. Here we show that Dock3 binds to and inactivates glycogen synthase kinase-3ß (GSK-3ß) at the plasma membrane, thereby increasing the nonphosphorylated active form of collapsin response mediator protein-2 (CRMP-2), which promotes axon branching and microtubule assembly. Exogenously applied BDNF induced the phosphorylation of GSK-3ß and dephosphorylation of CRMP-2 in hippocampal neurons. Moreover, increased phosphorylation of GSK-3ß was detected in the regenerating axons of transgenic mice overexpressing Dock3 after optic nerve injury. These results suggest that Dock3 plays important roles downstream of BDNF signaling in the CNS, where it regulates cell polarity and promotes axonal outgrowth by stimulating dual pathways: actin polymerization and microtubule assembly.
Subject(s)
Brain-Derived Neurotrophic Factor/physiology , Carrier Proteins/metabolism , Glycogen Synthase Kinase 3/metabolism , Growth Cones/metabolism , Hippocampus/metabolism , Microtubules/metabolism , Nerve Tissue Proteins/metabolism , Animals , Brain-Derived Neurotrophic Factor/pharmacology , COS Cells , Chlorocebus aethiops , Glycogen Synthase Kinase 3 beta , Growth Cones/drug effects , Growth Cones/ultrastructure , Guanine Nucleotide Exchange Factors , HEK293 Cells , Hippocampus/cytology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microtubules/drug effects , Microtubules/ultrastructureABSTRACT
While many Plusiinae species commonly secrete (Z)-7-dodecenyl acetate (Z7-12:OAc) as a key pheromone component, female moths of the rice looper (Plusia festucae) exceptionally utilize (Z)-5-dodecenyl acetate (Z5-12:OAc) to communicate with their partners. GC-MS analysis of methyl esters derived from fatty acids included in the pheromone gland of P. festucae showed a series of esters monounsaturated at the ω7-position, i.e., (Z)-5-dodecenoate, (Z)-7-tetradecenoate, (Z)-9-hexadecenoate (Z9-16:Me), and (Z)-11-octadecenoate (Z11-18:Me). By topical application of D(3)-labled palmitic acid (16:Acid) and stearic acid (18:Acid) to the pheromone glands, similar amounts of D(3)-Z5-12:OAc were detected. The glands treated with D(13)-labeled monoenoic acids (Z9-16:Acid and Z11-18:Acid), which were custom-made by utilizing an acetylene coupling reaction with D(13)-1-bromohexane, also produced similar amounts of D(13)-Z5-12:OAc. These results suggested that Z5-12:OAc was biosynthesized by ω7-desaturase with low substrate specificity, which could introduce a double bond at the 9-position of a 16:Acid derivative and the 11-position of an 18:Acid derivative. Additional experiments with the glands pretreated with an inhibitor of chain elongation supported this speculation. Furthermore, a comparative study with another Plusiinae species (Chrysodeixis eriosoma) secreting Z7-12:OAc indicated that the ß-oxidation systems of P. festucae and C. eriosoma were different.
ABSTRACT
The full-length cDNA sequence of a new pheromone-binding protein (AscrPBP2) was determined from a geometrid moth, Ascotis selenaria cretacea, which secreted a Type II sex pheromone, and an antiserum against its recombinant protein overexpressed in Escherichia coli was prepared. In addition to this antiserum against AscrPBP2, antibodies against AscrPBP1 and general odorant-binding proteins of Bombyx mori were used in Western blotting experiments to analyze the proteins in the antennae of several lepidopteran species secreting Type II sex pheromone components.
Subject(s)
Insect Proteins/metabolism , Sex Attractants/metabolism , Amino Acid Sequence , Animals , Antibodies/immunology , Base Sequence , Bombyx , DNA Primers , DNA, Complementary , Insect Proteins/chemistry , Insect Proteins/genetics , Insect Proteins/immunology , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Information on the olfactory system in antennae of Geometridae moths is very limited, and odorant-binding proteins (OBPs) working as transporters of lipophilic odors have not been identified. In the first investigation on this family of insects, we examined antennal OBPs of the Japanese giant looper, Ascotis selenaria cretacea. RT-PCR experiments using several pairs of degenerate primers designed from known cDNA sequences encoding lepidopteran OBPs successfully amplified partial sequences of two pheromone-binding proteins (PBPs), named AscrPBP1 and AscrPBP2 in reference to their corresponding nucleotide sequence homologies with other PBPs. Using 5'- and 3'-rapid amplification of cDNA end strategies, a cDNA clone for AscrPBP1 encoding a protein of 141 amino acids was isolated. Western blotting with the antiserum against recombinant AscrPBP1 overexpressed in Escherichia coli showed that the AscrPBP1 gene was more strongly expressed in male antennae than in female antennae. Furthermore, natural AscrPBP1was isolated by immunoprecipitation with the antiserum, and its binding ability was evaluated by using synthetic sex pheromonal compounds with a C(19) chain. The result indicated that AscrPBP1 bound not only the pheromone components, 3,6,9-nonadecatriene and its 3,4-epoxy derivative, but also unnatural 6,7- and 9,10-epoxy derivatives. While no general odorant-binding proteins (GOBPs) were amplified in the RT-PCR experiments, two antisera prepared from GOBP1 and GOBP2 of Bombyx mori suggested the occurrence of at least two GOBPs in the A. s. cretacea antennae.
