PKC-mediated USP phosphorylation at Ser35 modulates 20-hydroxyecdysone signaling in Drosophila

Sheng Wang; Jiawan Wang; Yaning Sun; Qisheng Song; Sheng Li

doi:10.1021/pr3008804

PKC-mediated USP phosphorylation at Ser35 modulates 20-hydroxyecdysone signaling in Drosophila

J Proteome Res. 2012 Dec 7;11(12):6187-96. doi: 10.1021/pr3008804. Epub 2012 Nov 16.

Authors

Sheng Wang¹, Jiawan Wang, Yaning Sun, Qisheng Song, Sheng Li

Affiliation

¹ 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.

PMID: 23136906
DOI: 10.1021/pr3008804

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

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Benzophenanthridines / pharmacology
Binding Sites
Blotting, Western
Cells, Cultured
Crosses, Genetic
DNA-Binding Proteins / genetics
DNA-Binding Proteins / metabolism*
Drosophila / genetics
Drosophila / metabolism*
Drosophila Proteins / genetics
Drosophila Proteins / metabolism*
Ecdysterone / metabolism*
Enzyme Activation
Gene Knockdown Techniques
Genes, Insect
Luciferases / metabolism
Molecular Sequence Data
Mutation
Phosphorylation
Plasmids / genetics
Plasmids / metabolism
Protein Kinase C / antagonists & inhibitors
Protein Kinase C / genetics
Protein Kinase C / metabolism*
RNA Interference
Receptors for Activated C Kinase
Receptors, Cytoplasmic and Nuclear / genetics
Receptors, Cytoplasmic and Nuclear / metabolism
Receptors, Steroid / metabolism
Serine / metabolism*
Signal Transduction*
Tandem Mass Spectrometry
Transcription Factors / genetics
Transcription Factors / metabolism*
Transfection

Substances

Benzophenanthridines
DNA-Binding Proteins
Drosophila Proteins
RACK1 protein, Drosophila
Receptors for Activated C Kinase
Receptors, Cytoplasmic and Nuclear
Receptors, Steroid
Transcription Factors
USP protein, Drosophila
ecdysone receptor
Serine
Ecdysterone
chelerythrine
Luciferases
Protein Kinase C