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

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 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 duanyu1531 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 inhibitor chelerythrine chloride in Kc cells. In the larval salivary glands (SG), inhibition of duanyu1531 activity with the binary GAL4/UAS system reduced USP phosphorylation and down-regulated the 20E primary-response genes, E75B and Br-C, and knockdown of Rack1 had stronger inhibitory effects than overexpression of Moreover, duanyu1615 knockdown of four duanyu1531 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 was knocked down by Taken together, we conclude that USP phosphorylation at Ser35 modulates 20E signaling in Drosophila.

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