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.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Down-Regulation , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Transcription, Genetic , Animals , Bombyx , Chromatography, Liquid , Cyclic AMP-Dependent Protein Kinases/isolation & purification , Phosphorylation , Tandem Mass Spectrometry , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Aurora B kinase, a member of serine/threonine kinase family, is the catalytic subunit of the chromosomal passenger complex and is essential for chromosome alignment, chromosome segregation, and cytokinesis during mitosis. Here, we cloned the full-length cDNA sequence of silkworm Aurora B (BmAurB) gene and predicted that BmAurB protein contains a conserved S_TKc domain. Phylogenetic analysis between BmAurB and other Aurora kinases indicates that Aurora kinases may have evolved after separation between mammalian and insect, and prior to radiation of either mammalian or insects. RT-PCR examination revealed that the expression of the BmAurB gene was high in mitotic cycling gonads, moderate in mitotic cycling brain, and undetectable in endocycling silk gland during silkworm larval development. RNAi or inhibitor-mediated inhibition of the BmAurB gene in silkworm ovary-derived BmN4-SID1 cells disrupted cell cycle progression during mitosis and induced an accumulation of polyploid cells, cell cycle arrest at G2/M phase, chromosome misalignment, chromosome bridge, and bi-nucleation. Taken together, our results suggest that the BmAurB gene is required for cell cycle progression in silkworm.
