Ecdysone Via Activating Calcium/Calmodulin-dependent Protein Kinase ⅡSignaling Pathway And Protein Kinase A Signaling Pathway To Regulate Gene Expression

Background and scientific questions20-Hydroxyecdysone (20E) plays a key role in insect molting and metamorphosis. In the larvae, the increase of ecdysone induces the molting; and at the period of larval metamorphosis, the increase of ecdysone induces the initiation of metamorphosis. 20E is considered entering cells directely initiate gene expression, so called genomic pathway in past studies. Resent studies reveal that 20E via activating ecdysone-responsible G-protein-coupled receptor (ErGPCRs) induces an increase of intracellular Ca2+ concentration and activates protein kinase C (PKC) pathway, so called nongenomic pathway. With the increase of intracellular Ca2+, many complexes are formed by Ca2+ association with a variety of intracellular calcium binding proteins for playing important roles, such as calmodulin (CaM). CaM binds to 4 calciums to form a Ca2+/CaM complex, and the complex can bind and activate autophosphorylation of the calcium/calmodulin dependent protein kinase II (CaMKII), which phosphorylates transcription factors for genes transcription in nucleaus. However, the function and mechanism of CaMKII in 20E-induced Ca2+ signaling is unclear. In Drosophila melanogaster, the dopamine/molting hormone receptor (DmDopEcR), which binds to 20E, induces an increase of intracellular cAMP concentration. The rapid increase in cAMP in response to 20E whithin 30 seconds is observed in the anterior silk glands of Bombyx mori. These evidences suggest that 20E induces cAMP nongenomic signaling pathway. The cAMP binds to regulatory subunit (PKAR) of cAMP-dependent protein kinase A (PKA) to result in the disassociation of PKA catalytic subunit (PKAC) in PKA. The free PKAC enters into nucleaus and induces the phosphorylation of adenosine cyclophosphate response element binding protein (CREB), which binds to the adenosine cyclophosphate response element (CRE) for increasing or decreasing the genes expression. However, the function and mechanism of PKAC in 20E-regulated insect metamorphosis is uncear.Threfore, this work investigated the function and mechanism of CaMKII in 20E-induced Ca2+ signaling, and the function and mechanism of PKAC in 20E-regulated insect metamorphosis.Results1.20E via inducing CaMKII phosphorylation regulates the formation of EcRBl/USP1 heterodimer for gene expressionThe expression of CaMKII is increased during molting and metamorphosis in H. armigera. Knockdown of CaMKII by RNAi in larvae results in decrease of the expression of 20E-response gene, and disrupts the pupation. 20E induces CaMKII phosphorylation in threonine at the amino acid 290, and then the activated CaMKII enters into nucleus. The GPCR inhibitor suramin, phospholipase C (PLC) inhibitor U73122 and inositol 1,4,5-trisphosphate (IP3R) inhibitor xestospongin C (XeC) repress the 20E-induced increase of intracellular Ca2+ and depress the phosphorylation of CaMKII. The knockdown of ErGPCR1, ErGPCR2 and G protein alpha q (Gaq) also depress the phosphorylation of CaMKII. The phosphorylated-CaMKII induces histone deacetylase 3 (HDAC3) phosphorylation and nucler export, which maintains USP1 lysine acetylation at amino acid 303. The lysine acetylation of USP1 is necessary for the interaction of USP1 with EcRB1 and their binding to the ecdysone response element (EcRE). The result of chromatin immunoprecipitation assay (ChIP) indicates the lysine acetylation of USP1 is necessary for its binding to EcRE. Results suggest that 20E via ErGPCRs-, Gaq-, PLC- and calcium signaling activates CaMKII phosphorylation and nuclear translocation, thereby regulates USP1 lysine acetylation to form an EcRB1/USP1 complex for the ininiation of 20E-response genes transcription.2.20E via activating PKA singnaling induces CREB phosphorylation for enhancing the genomic singnaling pathway.Knockdown of PKA catalytic subunit 1 (PKACI) in larvae prevents pupation and represses the expression of 20E-response genes. GPCR inhibitor suramin, cAMP production inhibitor bupivacaine hydrochloride and knockdown of ErGPCR2 repress the 20E-induced PKAC1 phosphorylation. Phosphorylated-PKAC1 directly phosphorylates the serine of CREB at amino acid 143, which is regulated by 20E-induced nongenomic signaling pathway.20E enhances the expression of red fluorescent protein (RFP) by pIEx-4-RFP-His plasmid, which conatains CRE and EcRE. GPCR inhibitor suramin, PKA inhibitor H89 and knockdown of ErGPCRl and ErGPCR2 repress the RFP expression. Deleting the CRE in the plasmid decreases the expression of RFP induced by 20E. CREB binds to CRE under 20E-induction, which can be prevented by knockdown of PKACl Results suggest that 20E via ErGPCR2 induces PKACl phosphorylation, which directly phosphorylates CREB for its binding to CRE to enhance genes expression.Conclusions and scientific significances20E via GPCRs induces CaMKII phosphorylation at T290 and nuclear translocation. The phosphorylated-CaMKII induces HDAC3 phosphorylation and nuclear export, which maintains the lysine acetylation of USP1. The lysine acetylation of USP1 is necessary for the interaction of USP1 with EcRB1 and their binding to the ecdysone response element (EcRE) for the ininiation of genes expression.20E via ErGPCR2 induces PKAC1 phosphorylation, which directly phosphorylates CREB at S143. The phosphorylated-CREB binds to CRE to enhance the expression of 20E-response genes. This study resolves the downstream cascade transduction from the increase of intracellular Ca2+ and the activiation of PKAC1 triggered by 20E, and proves the existence of 20E nongenomic signal pathway. These evidences enrich the knowledge of 20E signaling, exploit a new field in the studies of insects’development, and provide new gene targets for pest control.