Juvenile hormone (JH) and 20-hydroxyecdysone (20E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant (Met) and germ-cell expressed (Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases. Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C (PKC) which phosphorylated ultraspiracle (USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20E through its nuclear receptor complex EcR-USP. The usp^S35A mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20E signaling that delayed developmental timing. The usp^S35A mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.

保幼激素 (JH) 和 20-羟基蜕皮激素 (20E) 协调调节昆虫的发育和变态。已在果蝇 Drosophila melanogaster 中鉴定出两种 JH 细胞内受体，甲氧普林耐受 (Met) 和生殖细胞表达 (Gce) 。为了在不受 JH 细胞内信号传导干扰的情况下研究 JH 膜信号通路，我们对Met gce的磷酸化蛋白质组谱进行了表征在慢性和急性期均存在或不存在 JH 的双突变体。通过潜在的受体酪氨酸激酶和磷脂酶 C 途径发挥作用，JH 膜信号激活蛋白激酶 C (PKC) 在 Ser35 处磷酸化超气孔 (USP)，PKC 磷酸化位点是 20E 通过其核受体复合物 EcR-USP 发挥最大作用所需的位点. 在usp ^S35A突变体中，通过基因组编辑将第 35 位的 Ser 替换为 Ala，表明负责蜕皮激素生物合成的 Halloween 基因表达减少，从而减弱了延迟发育时间的 20E 信号。usp ^S35A _突变体还表现出较低的约克夏活性，从而减小了体型。总之，JH 膜信号在 Ser35 位点磷酸化 USP，从而增强调节正常果蝇发育的 20E 作用。这项研究有助于更好地理解复杂的 JH 信号网络。