Figure 1.
Identification of BR-C-interacting proteins through a yeast two-hybrid screen.
(A) Yeast two-hybrid screen of BR-C-interacting proteins from a silkworm silk gland cDNA library using the BTB domain of BR-C as bait. A total of 54 positive clones were found to grow on selective medium. A negative control consisting of pGBKT7-Lam with pGADT7-T and a positive control consisting of pGBKT7-53 with pGADT7-T were used to measure the efficiency of the system. (B) Yeast two-hybrid confirmation of the interaction between BR-C and its novel interacting partner RACK1. cDNA encoding full-length BR-C, the BTB domain or partial BR-C lacking the BTB domain was cloned into the pGBKT7 vector. The full-length cDNA sequence of RACK1 gene was inserted into the pGADT7 vector. The yeast two-hybrid experiment was performed to test the interaction between BR-C and RACK1.
Figure 2.
Interaction of BR-C with RACK1 in vitro.
(A) Interaction between BR-C and RACK1 by far-western blotting. GST, GST-tagged RACK1 (GST-RACK1), and SUMO-tagged BR-C (SUMO-BR-C) were expressed and purified from prokaryotic cells. The purified proteins were separated on 12% SDS-PAGE gels and transferred onto PVDF membranes, which were incubated with SUMO-BR-C or GST proteins at 4°C overnight, and analyzed by western blotting using anti-BR-C or anti-GST antibodies. (B-C) Interaction between BR-C and RACK1 by GST pull-down assay. Purified SUMO-BR-C was incubated with purified GST-RACK1 and examined by immunoblotting using an anti-BR-C antibody (B). Precleared BmN4 cell lysates containing endogenous RACK1 were incubated with purified GST-BR-C at 4°C for 6 h, and were separated via 12% SDS-PAGE gel and visualized by immunoblotting using an anti-RACK1 antibody (C).
Figure 3.
Subcellular localization of BR-C and RACK1.
RACK1 fused with Venus (Venus-RACK1) and BR-C fused with Venus (Venus-BR-C) were separately transfected into BmN4 cells. Two days post transfection, the cells were fixed and observed using confocal microscopy. The DNA was stained with DAPI.
Figure 4.
Disrupting the interaction between BR-C and RACK1 affects the nuclear import of BR-C.
(A) BTB domain deletion affects the nuclear localization of exogenously expressed BR-C in BmN4 cells. Constructs expressing full-length BR-C and partial BR-C lacking the BTB domain were fused to DsRed and transfected into BmN4 cells, and the cells were observed by confocal microscopy two days after transfection. (B) RNAi against endogenous RACK1 gene inhibits the nuclear import of exogenously expressed BR-C in BmN4-SID1 cells. Double-stranded RNA (dsRNA) targeted the RACK1 gene or the control EGFP gene was added to the culture medium separately for BmN4-SID1 cells, and five days later, the plasmid expressing full-length BR-C fused to DsRed was transfected into BmN4-SID1 cells. Confocal microscopy analysis was performed two days after transfection. Treatment with dsRNA against enhanced green fluorescent protein (EGFP) gene was used as a control. DAPI was used to stain the nuclear DNA of the cells.
Figure 5.
RNAi against the endogenous PKC gene alters the nuclear import of BR-C.
dsRNA against the PKC gene was used to treat BmN4-SID1 cells. Five days later, the construct expressing full-length BR-C fused to DsRed was transfected into BmN4-SID1 cells. The nuclear import of exogenously expressed BR-C was checked at two days after transfection using confocal microscopy. EGFP RNAi was used as a control.
Figure 6.
Mutating the predicted PKC phosphorylation sites in BR-C disrupts the nuclear import of BR-C.
Each construct expressing full-length BR-C with the mutation of a single predicted phosphorylation site to Ala (A; blocks phosphorylation) was transfected into BmN4 cells, and the nuclear import of the expressed BR-C protein was observed by confocal microscopy two days after transfection. The construct encoding wild-type BR-C without any phosphorylation site mutation was used as a control. Two predicted phosphorylation site mutations, S373A and T406A, led to the failure of BR-C nuclear import. However, mutating the predicted PKC phosphorylation site at Ser44 could not affect the nuclear localization of BR-C.
Figure 7.
Mutations of phosphorylation sites to Glu in BR-C lacking BTB domain have no impact on nuclear import of BR-C.
Construct expressing BR-C both lacking BTB domain and mutating the predicted phosphorylation sites of either Ser373 or Thr406 into Glu (E; mimics phosphorylation) was transfected into BmN4 cells. Confocal microscopy analysis revealed that compared to the control of BR-C only lacking BTB domain, the mutants of either S373E or T406E in BR-C without BTB domain could not change nuclear localization of BR-C.
Figure 8.
Both BTB domain deletion and PKC phosphorylation site mutants impaired the transcriptional activity of BR-C.
(A) Effects of the deletion of the BR-C BTB domain on the transcriptional activity of BR-C. DsRed fused to BR-C lacking the BTB domain was co-transfected into BmN4 cells with a plasmid containing a luciferase reporter gene under the control of the promoter of WCP10 gene, which is a direct target of BR-C. Luciferase activity was measured two days after transfection. Constructs containing either DsRed fused to full-length BR-C or DsRed alone were used as controls. (B) Effects of mutating the PKC phosphorylation sites in BR-C on the transcriptional activity of BR-C. The constructs containing BR-C with mutations at the phosphorylation sites S373 or T406 or non-mutated BR-C were each co-transfected into BmN4 cells with a plasmid containing a luciferase reporter gene under the control of the WCP10 gene promoter. Luciferase activity was measured two days after transfection.
Figure 9.
Proposed model for the nuclear import of BR-C upon interaction between BR-C and RACK1.
The scaffolding protein RACK1 recruits and activates PKC in the cytoplasm. After being translated in the cytoplasm, BR-C interacts with RACK1 and is phosphorylated by RACK1-anchored PKC at amino acid residues Ser373 and Thr406. Phosphorylated BR-C then translocates into the nucleus and subsequently activates the transcription of its target genes.