Abstract
The nuclear receptor complex of the steroid hormone, 20-hydroxyecdysone (20E), is a heterodimer composed of EcR and USP. Our previous studies in Drosophila suggest that PKC modulates 20E signaling by phosphorylating EcR-USP. However, the exact phosphorylation sites in EcR and USP have not been identified. Using LC-MS/MS analysis, we first identified Ser35 of USP as a PKC phosphorylation site. Mutation of USP Ser35 to Ala35 in S2 cells not only eliminated USP phosphorylation, but also attenuated the 20E-induced luciferase activity, mimicking the treatment with a PKC-specific inhibitor chelerythrine chloride in Kc cells. In the larval salivary glands (SG), inhibition of PKC activity with the binary GAL4/UAS system reduced USP phosphorylation and down-regulated the 20E primary-response genes, E75B and Br-C, and RNAi knockdown of Rack1 had stronger inhibitory effects than overexpression of PKCi. Moreover, RNAi knockdown of four PKC isozyme genes expressed in the SG exhibited a variety of inhibitory effects on USP phosphorylation and expression of E75B and Br-C, with the strongest inhibitory effects occurring when aPKC was knocked down by RNAi. Taken together, we conclude that PKC-mediated USP phosphorylation at Ser35 modulates 20E signaling in Drosophila.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Benzophenanthridines / pharmacology
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Binding Sites
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Blotting, Western
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Cells, Cultured
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Crosses, Genetic
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Drosophila / genetics
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Drosophila / metabolism*
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Drosophila Proteins / genetics
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Drosophila Proteins / metabolism*
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Ecdysterone / metabolism*
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Enzyme Activation
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Gene Knockdown Techniques
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Genes, Insect
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Luciferases / metabolism
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Molecular Sequence Data
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Mutation
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Phosphorylation
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Plasmids / genetics
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Plasmids / metabolism
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Protein Kinase C / antagonists & inhibitors
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Protein Kinase C / genetics
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Protein Kinase C / metabolism*
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RNA Interference
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Receptors for Activated C Kinase
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Receptors, Cytoplasmic and Nuclear / genetics
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Receptors, Cytoplasmic and Nuclear / metabolism
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Receptors, Steroid / metabolism
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Serine / metabolism*
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Signal Transduction*
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Tandem Mass Spectrometry
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Transfection
Substances
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Benzophenanthridines
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DNA-Binding Proteins
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Drosophila Proteins
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RACK1 protein, Drosophila
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Receptors for Activated C Kinase
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Receptors, Cytoplasmic and Nuclear
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Receptors, Steroid
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Transcription Factors
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USP protein, Drosophila
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ecdysone receptor
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Serine
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Ecdysterone
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chelerythrine
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Luciferases
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Protein Kinase C