Abstract
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 in vitro and ex vivo 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.
Highlights
Previous studies have shown that the insect BR-C protein, which is involved in the 20E signaling pathway, may be phosphorylated [5, 11]
We further examined by using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)qPCR whether protein kinase A (PKA) phosphorylation-induced transcriptional activity inhibition of silkworm BR-C was caused by decreasing its DNA-binding ability
The results demonstrated that the nucleoproteins extracted from BmE cells overexpressing either intact BR-C or Ser-186 was mutated to Ala (S186A) mutation can bind to the biotin-labeled probes covering cis-regulatory element (CRE) for BR-C in silkworm WCP10 promoter in a dose-dependent manner, and this binding was competitively suppressed by the unlabeled probe (Fig. 6A)
Summary
Previous studies have shown that the insect BR-C protein, which is involved in the 20E signaling pathway, may be phosphorylated [5, 11]. Given that the Ser-186 residue was identified as a phosphorylation site for silkworm BR-C by LC-MS/MS analysis, we designed a phospho-BR-C antibody against this site on silkworm BR-C To further validate this phosphorylation site, we performed a dephosphorylation assay in vitro. Western blot analysis revealed that the BR-C phosphorylation level was sharply decreased and was undetectable compared with that of the control (Fig. 2A), whereas the amount of total BR-C proteins was elevated. Using previously reported protocols [16, 17], we treated the silkworm BmE cells overexpressing BR-C with two PKA activators, namely, cAMP and forskolin, and two inhibitors, namely, H89 and KT5720, and BR-C phosphorylation levels were examined by Western blot assays with the p-BR-C antibody against the Ser-186 site. Similar changes in BR-C phosphorylation levels in response to PKA activators or inhibitors were observed in human HEK293FT cells (Fig. 3, C and D). Given that silkworm BR-C is a member of the nuclear receptor family of transcription factors, we examined whether PKA-mediated BR-C phosphorylation at Ser-186 is required for
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