Krüppel Homolog 1 Phosphorylation And Its Function In Transducing Juvenile Hormone Signal

Juvenile hormone(JH),the sesquiterpenoid hormone secreted from insect corpora allata,controls insect development,metamorphosis,and reproduction.Identification and analysis of JH nuclear receptor,Methoprene-tolerant(Met)has accelerated the understanding of JH action,but the molecular mechanisms of JH regulation have not been fully understood.The migratory locust,Locusta migratoria is a wordwide agricultural pest with high fecundity.In many insects including L.migratoria,vitellogenesis,ovarian development and oocyte maturation are stimulated by JH.Therefore,the migratory locust is an ideal model to investigate the molecular mechanisms of JH action on insect reproduction.Krüppel homolog 1(Kr-h1),an early JH response gene,plays a key role in maintaining larval or nymphal status and in promoting adult reproduction.The transcription of Kr-h1 is activated by the JH-Met/Tai receptor complex.Kr-h1 also acts as a transcription factor to regulate the transcription of its target genes.Previous studies in our laboratory have indicated that Kr-h1 is likely to be phosphorylated,but the signaling pathway involved in the phosphorylation of Kr-h1 and the function of phosphorylated Kr-h1 should be elucidated.In this dissertation study,we used the migratory locust as the model system to explore Kr-h1 phosphorylation and its biological functions by the utilization of molecular biology,cell biology and genetic approaches.We aimed to:(1)identify Kr-h1 phosphorylation site as well as the protein kinase that activates Kr-h1 phosphorylation;(2)determine the dependence of Kr-h1 phosphorylation on JH and identify JH-dependent signaling transduction pathway of Kr-h1 phosphorylation;(3)elucidate the transcriptional regulation of phosphorylated Kr-h1 on target genes and its role in insect metamorphosis and reproduction.We initially identified the phosphorylation site of Kr-h1 at Ser154 by mutagenesis,Western blot and IP.This phosphorylation site was further confirmed by raising the specific antibody against phosphorylated Kr-h1,followed by λPP phosphatase treatment,dsRNA mediated RNAi and quantification of phosphorylated Kr-h1.Western blot and IP analysis of PLC inhibitor(U73122)-,PKC inhibitor(NPC)-,or PKC-α dsRNA-injected larvae and adults demonstrated that Kr-h1 was phosphorylated by PKC-α.The levels of phosphorylated Kr-h1 increased in the vitellogenic stage,which correlated with the elevated titer of JH.When endogenous JH was deprived by precocene treatment,Kr-h1 phosphorylation was inhibited.Additional application of JH analog(JHA)induced Kr-h1 phosphorylation.These observations indicate that Kr-h1 phosphorylation depends on JH.Dual luciferase reporter assay demonstrated that phosphorylated Kr-h1 mediated a significantly higher transinhibition of adult specifier gene E93 in the larval stage,but a significantly higher transactivation of reproduction-related gene RL36 in the adult phase.Interestingly,the higher transinhibition of E93 by phosphorylated Kr-h1 was conserved in the fruitfly Drosophila melanogaster,the red flour beetle Tribolium castaneum,and the silkworm Bombyx mori.Data in this dissertation research indicate that JH induces Kr-h1 phosphorylation at Ser154 via a signaling transduction pathway including PKC-α.Phosphorylated Kr-h1 appears to more active in transducing the anti-metamorphic and vitellogenic JH signal,which is conserved across insect species.This dissertation is the first report about Kr-h1 phosphorylation as well as its function in transducing JH signal.The results provide new insights into our understanding of JH non-genomic action.