The insect-specific transcription factor Broad-Complex (BR-C) is transcriptionally activated by the steroid 20-hydroxyecdysone (20E) and regulates the expression of many target genes involved in insect growth and development. However, although the transcriptional regulation of BR-C proteins has been well studied, how BR-C is regulated at post-transcription and -translation levels is poorly understood. To this end, using liquid chromatography-tandem mass spectrometry analysis, we identified residue Ser-186 as a phosphorylation site of BR-C in silkworm. Site-directed mutagenesis and treatment with specific kinase activators and inhibitors indicated that the Ser-186 residue in silkworm BR-C is phosphorylated by protein kinase A (PKA). Immunostaining assays disclosed that PKA-mediated phosphorylation of silkworm BR-C has no effect on its nuclear import. However, luciferase reporter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation revealed that the PKA phosphorylation event suppresses the transcriptional activation of silkworm BR-C target genes and that this inhibition was caused by repression of BR-C binding to its DNA targets. Of note, both and experiments disclosed that a continuous 20E signal inhibits the PKA-mediated BR-C phosphorylation and also the cAMP/PKA pathway, indicating that 20E's inhibitory effect on PKA-mediated phosphorylation of silkworm BR-C contributes to maintaining BR-C transcriptional activity. In conclusion, our findings indicate that PKA-mediated phosphorylation inhibits silkworm BR-C activity by interfering with its binding to DNA and that 20E signaling relieves PKA-mediated phosphorylation of BR-C, thereby maintaining its transcriptional activity.

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