Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.
Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.
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DOI：
10.1371/journal.pone.0109111
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Xia Q
中科院分区：
文献类型：
--
作者：
Cheng D;Qian W;Wang Y;Meng M;Wei L;Li Z;Kang L;Peng J;Xia Q
转录因子 Broad Complex (BR-C) 是昆虫中的早期蜕皮激素反应基因，包含两种类型的结构域：两个用于激活基因转录的锌指结构域和用于蛋白质-蛋白质相互作用的 Bric-a-brac/Tramtrack/Broad 复合体 (BTB) 结构域。尽管锌指介导的基因转录机制已得到充分研究，但与 BR-C 的 BTB 结构域相互作用的伙伴至今尚未阐明。在这里，我们使用家蚕 BR-C 的 BTB 结构域作为诱饵进行了酵母双杂交筛选，并鉴定了活化 C 激酶 1 (RACK1) 的受体（一种支架/锚定蛋白）作为能够与 BR-C 相互作用的新伙伴。 BR-C 和 RACK1 之间的相互作用通过远蛋白印迹和 Pull-down 测定进一步得到证实。重要的是，通过针对内源性 RACK1 基因的 RNAi 或 BTB 结构域的删除，破坏了这种相互作用，从而消除了 BmN4 细胞中 BR-C 的核输入。此外，针对内源性 PKC 基因的 RNAi 以及 BR-C 中 Ser373 或 Thr406 处预测 PKC 磷酸化位点的磷酸化缺陷突变，对 RACK1 RNAi 进行了表型复制，并改变了 BR-C 的核定位。然而，当 BTB 结构域被删除时，Ser373 或 Thr406 的磷酸化模拟物对 BR-C 的核输入没有影响。此外，突变 Ser373 和 Thr406 处的 PKC 磷酸化位点或删除 BTB 结构域可显着降低 BR-C 靶基因的转录激活。鉴于 RACK1 对于招募 PKC 关闭和磷酸化靶蛋白是必需的，我们认为 PKC 介导的磷酸化和 BR-C 的核输入是由其与 RACK1 的相互作用决定的。这一新发现将有助于进一步破译 BR-C 蛋白在昆虫发育过程中的作用机制。
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.
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