Juvenile Hormone-stimulated Vitellogenin Receptor Phosphorylation And Migration To Cell Membrane

During insect vitellogenesis,the ovaries absorb and accumulate vitellogenin(Vg)for oocyte maturation and embryo development.Vitellogenin receptor(VgR)located on the surface of oocyte membrane,transports Vg into oocyte by endocytosis.Previous studies have reported that juvenile hormone(JH)regulates VgR-mediated Vg uptake via Protein kinase C(PKC),but the regulatory mechanism is unclear,particularly how VgR responds to the JH-PKC signaling pathway to mediate Vg uptake.The migratory locust,Locusta migratoria is a worldwide insect pest.Its vitellogenesis is governed by juvenile hormone.It is also one of the model insects in juvenile hormone regulated insect reproduction.We therefore used the locust as a model system,through the approaches of molecular and cellular biology including but not limited to RNAi,Western blot,Co-IP,immunohistochemistry,to explore the molecular mechanisms of JH regulation in VgR-mediated Vg uptake.Initially,we screened seven PKC isoforms from the ovarian transcriptome of migratory locusts and performed RNAi for each of them.Interestingly,VgR phosphorylation was significantly down-regulated by silencing PKC delta,PKC alpha,PKC epsilon,and PKC iota.The results indicate that PKC delta,PKC alpha,PKC epsilon,and PKC iota mediate VgR phosphorylation regulated by juvenile hormone.Subsequent studies using subcellular localization technology revealed that PKC iota was induced and moved near the cell membrane in response to JH analog,suggesting that PKC iota may regulate JH-induced VgR phosphorylation.Co-IP experiments showed that the interaction between PKC iota and VgR was significantly enhanced with JH analog treatment,indicating that PKC iota directly activated VgR phosphorylation under juvenile hormone induction.VgR subcellular localization was analyzed by immunohistochemical techniques.JH analog induced VgR from oocyte cytoplasm to membrane,suggesting that juvenile hormone promotes VgR recycling.But treatment with CC,a specific inhibitor of PKC or PKC iota RNAi inhibited JH-stimulated VgR recycling.Comprehensive analyses by LC/MS/MS and site-directed mutation demonstrated that JH and PKC iota-mediated VgR phosphorylation was present at the serine residue 1361.Collectively,the data indicate that juvenile hormone catalyzes VgR phosphorylation by activating PKC iota,consequently promoting VgR recycling.Further studies on aLepidopteran insect,the cotton bollworm Helicoverpa armigera and a Blattarian species,the cockroach Periplaneta Americana suggest that JH and PKC iota-mediated VgR phosphorylation and recycling were conserved in these insect species.In summary,we found that JH activated a signaling pathway including PKC iota to phosphorylate VgR and stimulate its migration from oocyte cytoplasm to membrane.On the oocyte membrane,Vg bound to phosphorylated VgR,consequently accumulating in maturing oocytes via VgR-mediated endocytosis.The results advance the view of VgR-mediated ovarian Vg uptake in insects.The results also provide new insight into our understanding of JH regulation and membrane signaling pathway in insect reproduction.