title: PKC-mediated USP phosphorylation at Ser35 modulates 20-hydroxyecdysone signaling in Drosophila.
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.
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Wang
authors.forename: Jiawan
authors.initials: J
authors.lastname: Wang
authors.forename: Yaning
authors.initials: Y
authors.lastname: Sun
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Song
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053119:Benzophenanthridines / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured; D003433:Crosses, Genetic; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004441:Ecdysterone / Q000378:metabolism*; D004789:Enzyme Activation; D055785:Gene Knockdown Techniques; D017344:Genes, Insect; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D000075370:Receptors for Activated C Kinase; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D053119:Benzophenanthridines; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C524464:RACK1 protein, Drosophila; D000075370:Receptors for Activated C Kinase; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors; C066030:USP protein, Drosophila; C055469:ecdysone receptor; D012694:Serine; D004441:Ecdysterone; C016299:chelerythrine; D008156:Luciferases; D011493:Protein Kinase C