Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.

The transcription factor Broad Complex (BR-C) is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB) domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1), a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, duanyu1615 against the endogenous gene as well as phosphorylation-deficient mutation of the predicted duanyu1531 phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 duanyu1615 and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the duanyu1531 phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting duanyu1531 to close and phosphorylate target proteins, we suggest that the phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.

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