文章目录

1. Introduction

2. Materials and methods

2.1. Fly strains

2.2. Generation of the uspS35Amutant allele

2.3. Generation of clones

2.4. Sample preparations for phosphoproteomics

2.5. Protein extraction and quantitation

2.6. Proteins digestion and phosphopeptide enrichment

2.7. LC-MS/MS analysis

2.8. Database search and identi?cation of phosphosites

2.9. Alignment and phylogenetic analyses of homologous USP sequences

2.10. Western blotting

2.11.Quantitative real-time PCR

2.12.Developmental timing,20E feeding,pupal body size,body weight,ovary size,and egg laying assessments

2.13.Immunohistochemistry

2.14.Statistics

3. Results

3.1. In vivo analysis of protein phosphorylation chronically increased by JH

3.2. In vitro analysis of protein phosphorylation rapidly induced by JH through the RTK pathway

3.3. JH membrane signaling induces USP Ser35 phosphorylation through the RTK-PLC-PKC pathway

3.4. USP Ser35 phosphorylation is required for proper developmental timing and body size

3.5. USP Ser35 phosphorylation potentiates 20E action by affecting Halloween gene expression

3.6. USP Ser35 phosphorylation has little effects on IIS

3.7. USP Ser35 phosphorylation signi?cantly affects Yorkie activity

3.8. JH membrane signaling phosphorylates USP and thus regulates?y normal development

4. Discussion and conclusion

Con?ict of interest

Author contributions

Appendix A.Supplementary materials

文章摘要:保幼激素（juvenile hormone, JH）与蜕皮激素（20-hydroxyecdysone, 20E）协同调控昆虫的发育与变态.果蝇中JH有两个胞内受体, Met和Gce.为了在完全排除JH胞内信号干扰的前提下探究JH膜信号,本文作者结合果蝇遗传学和磷酸化蛋白组学,对Met Gce双突变体在长期和短期JH存在或缺失的情况下做了大量的磷酸化蛋白组学分析,并鉴定出USP（20E受体复合体EcR-USP的重要组分）Ser35为JH膜信号诱导的磷酸化位点.后续实验证明JH膜信号通过激活PLC-PKC以诱导USP Ser35磷酸化.使用CRISPR-Cas9基因编辑技术,将USP Ser35替换成无磷酸化活性的Ala35,得到了USPS35A突变品系.研究发现USPS35A造成前胸腺ecdysone合成减弱,从而系统性引起全身20E信号下降,导致发育时间延迟.这些研究成果为阐明JH膜受体及其下游的磷酸化调控网络奠定了重要基础.

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