title: Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.
abstract: 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 RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi 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 PKC 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 PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated 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.
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
authors.forename: Daojun
authors.initials: D
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
authors.forename: Wenliang
authors.initials: W
authors.lastname: Qian
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
authors.forename: Yonghu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Meng
authors.affiliation: School of Life Science, Southwest University, Chongqing, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Wei
authors.affiliation: Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Fukuoka, Japan.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Kang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Peng
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
authors.forename: Qingyou
authors.initials: Q
authors.lastname: Xia
mesh_terms: D000818:Animals; D001665:Binding Sites; D012831:Bombyx / Q000378:metabolism*; D002460:Cell Line; D005786:Gene Expression Regulation; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D020798:Two-Hybrid System Techniques
chemical_list: D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D014157:Transcription Factors