Studies Of The Cyclin-dependent Kinase10, Phospholipase C Gamma1and Heat Shock Protein90in Insect20-hydroxyecdysone Signaling

BackgroundInsects are ideal model systems for studying the molecular mechanisms that regulate developmental transitions in multicellular organisms, largely because their individual developmental stages are clearly punctuated by molting and metamorphosis. These transition events are under strict endocrine control and have been of interest to entomologists for close to a century, since the classical study of Stefan Kopec on the metamorphosis of the gypsy moth, Lymantria dispar. Due to the rapid development of molecular biology technology in recent years, the molecular mechanism of insect molting and metamorphosis is uncovered partly. Data to date show that insect development transitions are strictly controlled by endocrine system. Ecdysone (20E) and juvenile hormone (JH) play the main function in the processes.Questions and significances20E regulates larval molting in the presence of JH, but promotes metamorphosis in the absence of JH at the stage of larval-pupal transition. Therefore,20E signaling interacts with JH signaling to control insect development, while the mechanism remains unclear. In the20E pathway,20E induces the formation of EcR/USP and the cofactors recruitment to assemble activated transcriptional complex for gene expression. More and more studies show that there is a cell membrane-mediated non-genomic pathway in addition to the classical nuclear receptor pathway in20E signaling. But, it is still far from enough mechanism research. In the JH pathway, the identification of JH intracellular receptor (Methoprene-tolerant, Met) results in the increased studies in JH signaling, while the mechanism of the crosstalk between20E and JH signalings remains unclear. Human cyclin-dependent kinase10(CDK10) can strongly interact with heat shock protein90(Hsp90), but the significance is uncovered. Meantime, by interacting with EcR, Hsp90regulates the DNA binding activating of EcR and the20E signaling. In Rat cells, insulin induces the rapid phosphorylation of CDK10. These results suggest that CDK10may take part in20E signaling by interacting with Hsp90. Meantime, we want to know whether20E could induce CDK10phosphorylation to regulate the non-genomic action in20E pathway. In Bombyx and Drosophila,20E induces calcium signaling through G-protein-coupled-receptor (GPCR). In Drosophila,20E regulates USP phosphorylation dependent on PKC activation. However, the link between the20E-induced calcium signaling and the PKC activation remains unknown.In fact, phospholipase C gamma1can regulate GPCR-triggered calcium to activate PKC. Therefore, whether the connection between the20E-induced membrane signal and the nuclear receptor signal relies on PLCG1needs an exhaustive study. In20E signaling, Hsp90and Hsc70interact with EcR and do not appear at the promoter-bound transcription complex in Drosophila. However, Hsp90does not depart from mineralcorticoid receptor and regulates the transcription sites, suggesting the existence in transcription complex. At the same time, Hsc70plays a key role in the regulation of receptor cycling and degradation. Therefore, whether Hsp90and Hsc70participate in the20E-induced transcriptional complex is still unclear and need further investigation.Given the20E-and JH-regulated precision network of insect development, the investigation of mechanisms of the20E and JH signalings not only provide pest control strategy, but also are beneficial to the study of human endocrine mechanisms.Results1.20E regulated the association of ecdysone nuclear receptor at promoter by inducing CDK10phosphorylation via a non-genomic pathwayThe downregulation of CDK10by RNA interference (RNAi) in larvae and the epidermal cell line delayed development and suppressed20E-induced gene transcription. The rapid phosphorylation of CDK10was induced by20E, whereas it was repressed by the inhibitors of G-protein-coupled-receptors, phospholipase C, and Ca2+channels. Phosphorylated CDK10exhibited increased interactions with heat shock proteins (Hsps) Hsc70and Hsp90and then promoted the interactions between Hsps and ecdysone receptor EcRB1and the binding of Hsps-EcRB1complex to the20E response element for the regulation of gene transcription. CDK10depletion suppressed the formation of the Hsps-EcRB1complex at HR3promoter. These results suggest that20E induces CDK10phosphorylation via a non-genomic pathway to regulate gene transcription in the nucleus.2. Phospholipase C gamma1connects the cell membrane pathway to the nuclear receptor pathway in20E signalingThe depletion of PLCG1by RNAi blocked20E-enhanced pupation; caused larvae death and pupation defects, and repressed20E-induced gene expression.20E induced the tyrosine phosphorylation of PLCG1and its migration toward the plasma membrane. The Src-familay kinase inhibitor PP2and the depletions of ErGPCR and Gaq inhibited PLCG1tyrosine phosphorylation. PLCG1participated in the20E-induced Ca2+influx. The inhibition of GPCR, PLC, inositol1,4,5-trisphosphate receptor, and calcium channels repressed the20E-induced Ca2+influx. Through calcium signaling, PLCG1mediated the transcriptional activation driven by the ecdysone response element (EcRE). Through PLCG1and calcium signaling,20E regulated PKC phosphorylation of USP1at Ser21to determine its EcRE binding activity. These results suggest that20E activates PLCG1via ErGPCR and Src-family kinases to regulate Ca2+influx and PKC phosphorylation of USP1in order to subsequently modulate gene transcription.3. Heat shock protein90regulated the crosstalk between20E and JH pathway by phosphorylation variation and different proteins interactions20E and JH induced Hsp90expression and its nuclear localization by using hormone injection in larvae and immunocytochemistry in HaEpi cells. Silencing of Hsp90by RNAi reduced the20E-or JH-induced gene expression.20E induced the interaction between Hsp90and the20E receptor USP1, and JH III or methoprene induced the interaction between Hsp90and the JH candidate receptor Metl. Both JH III and methoprene induced protein kinase C (PKC)-mediated phosphorylation of Hsp90; however,20E maintained non-phosphorylation of Hsp90. These results show that Hsp90participates in the crosstalk between the20E and the JH signaling pathways for gene expression by altering phosphorylation status and forming different transcriptional complexes.Conclusion and significanceThe innovation points of this research are described as following.(1) We firstly demonstrate that the function of Hsp90in JH pathway, and its key role in the crosstalk between20E and JH signaling.(2) We found20E induces the rapid phosphorylation of CDK10to regulate the Hsp90-and Hsc70-mediated association of nuclear receptor through an ErGPCR-PLCG1-Ca2+pathway.(3) This is the first report that Hsp90and Hsc70exist in the20E transcriptional complex.(4) We verify that PLCG1activates PKC to phosphorylate USP1through ErGPCR and calcium signaling.(5) We identify the USP1phosphorylation site Ser21in H. armigera, and demonstrate it’s essential for DNA binding. This work establishes a link between20E-induced non-genomic pathway and genomic pathway, and provides a new insight to understand the crosstalk between20E and JH signaling pathways.