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.
J Proteome Res. 2012 Dec 7;11(12):6187-96. doi: 10.1021/pr3008804. Epub 2012 Nov 16.
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.
甾体激素 20-羟基蜕皮激素（20E）的核受体复合物是由 EcR 和 USP 组成的异二聚体。我们之前在果蝇中的研究表明，PKC 通过磷酸化 EcR-USP 来调节 20E 信号。然而，EcR 和 USP 中的确切磷酸化位点尚未确定。使用 LC-MS/MS 分析，我们首先鉴定出 USP 的 Ser35 是 PKC 的磷酸化位点。在 S2 细胞中，将 USP 的 Ser35 突变为 Ala35 不仅消除了 USP 的磷酸化，还减弱了 20E 诱导的荧光素酶活性，类似于在 Kc 细胞中用 PKC 特异性抑制剂 Chelerythrine Chloride 处理。在幼虫唾液腺（SG）中，使用二元 GAL4/UAS 系统抑制 PKC 活性降低了 USP 的磷酸化，并下调了 20E 的初级反应基因 E75B 和 Br-C，而 Rack1 的 RNAi 敲低比 PKCi 的过表达具有更强的抑制作用。此外，SG 中表达的四个 PKC 同工型基因的 RNAi 敲低对 USP 磷酸化和 E75B 和 Br-C 的表达表现出多种抑制作用，当 aPKC 被 RNAi 敲低时，抑制作用最强。综上所述，我们得出结论，PKC 介导的 USP 在 Ser35 的磷酸化调节果蝇中的 20E 信号。
