Abstract
Ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle protein (USP), are nuclear receptors that mediate the action of the insect molting hormone 20-hydroxyecdysone (20E). There is evidence that the activity of both receptors is affected by phosphorylation. Using a proteomic approach, we have shown that protein kinase C (PKC) activity is necessary for mediating 20E-induced expression of 14 specific proteins, including three previously reported 20E responsive proteins, and is also responsible for the intracellular localization of EcR and USP in larval salivary glands of Drosophila melanogaster. The 20E-dependent expression of the proteins was verified using real-time PCR and/or Western blot analysis. For some genes, inhibition of PKC activity reduced 20E-dependent transcriptional activity rapidly, raising the possibility that these are direct gene targets of EcR and USP. The data further indicate that PKC-mediated phosphorylation is also required for genes regulated indirectly by 20E-induced changes in the larval salivary gland.
Highlights
Insect steroid hormones, mainly 20-hydroxyecdysone (20E), trigger and coordinate the molting and metamorphosis of insects via ecdysone receptor (EcR) and its heterodimer ultraspiracle protein (USP).[1,2] Both EcR and USP are members of the nuclear receptor superfamily and are ligand-activated transcription factors
Temporal response studies revealed that both EcR and USP signals started to decline when the salivary gland (SG) were incubated with 100 μM protein kinase C (PKC) inhibitor for 3 h and became barely detectable when the SGs were incubated with the PKC inhibitor for 6 h or longer
Using chelerythrine chloride (CC), we showed that inhibition of PKC by its specific inhibitor resulted in the decline of EcR and USP signals in the nucleus of salivary glands in a dose- and temporal-dependent manner (Figure 2)
Summary
Mainly 20-hydroxyecdysone (20E), trigger and coordinate the molting and metamorphosis of insects via ecdysone receptor (EcR) and its heterodimer ultraspiracle protein (USP).[1,2] Both EcR and USP are members of the nuclear receptor superfamily and are ligand-activated transcription factors. The activities of nuclear receptors are subjected to regulation by transcriptional/translational mechanisms and by posttranslational mechanisms such as phosphorylation. Studies of transcription factors in vertebrates have shown that phosphorylation can play roles in nuclear translocation, DNA binding, interactions with other proteins, and transactivation.[3,4]. In insects, both EcR and USP have been demonstrated to be phosphoproteins and EcR and USP phosphorylation are regulated by 20E.5-8. Phosphorylation of EcR and USP has been shown to play roles in mediating the ligand- and perhaps DNA-binding activity in the prothoracic glands of M. sexta.
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