Roles Of The Phosphorylation Of Silkworm Transcription Factor BR-C On Its Transcriptional Activities

Insect growth and development are cooperatively controlled by two endocrine hormones,namely 20-hydroxyecdysone(20E)and juvenile hormone(JH).Through the receptor complex Ec R/USP,20 E induces a cascade of primary response genes and secondary response genes,and the secondary response genes in turn regulates the expression of their targets involved in insect growth and development.Transcription factor Broad Complex(BR-C)is a 20 E primary response gene and contains a BTB domain that mediates protein-protein interaction and two zinc finger domains.In our previous study,we performed a yeast two-hybrid scanning by using the BTB domain of silkworm BR-C as bait and identified that the receptor for activated C-kinase 1(RACK1),which is involved in protein kinase C(PKC)-mediated phosphorylation of its substrate proteins,as the potential interacting partner of BR-C,suggesting that BR-C may be phosphorylated by PKC.Therefore,elucidating the phosphorylation of insect BR-C will contribute to improve 20 E signaling pathway.Given that BR-C is a member of the BTB domain-containing protein gene family,we firstly performed a genome-wide comparison of the BTB protein genes from the silkworm and other insects to analyze the evolution of the BR-C gene.In addition,by using bioinformatics analysis,LC-MS/MS method and biochemical and molecular biological experiments,we explored the roles of the BTB domain in PKC-mediated phosphorylation of silkworm BR-C,and further investigated the effects of protein kinase A(PKA)-mediated phosphorylation of silkworm BR-C on its transcriptional activity and how the BR-C phosphorylation is regulated by 20 E.The main results are listed as follows:1.Genome-wide comparison of silkworm BR-C and other BTB domain-contain ing protein genesThe transcription factor BR-C from the silkworm and other insects contains a BTB domain.To better understand the evolution of insect BR-C genes,we first performed a genome-wide identification of the BTB domain-containing protein gene family in the silkworm.Through a BLAST search against silkworm genome assembly by using the amino acid sequence for the conserved BTB domain of silkworm BR-C,a total of 56 BTB protein genes were identified from the silkworm genome,and 53 of which could be mapped on the different chromosomes.Based on their domain architecture,silkworm BTB proteins were classified into BTBD subfamily containing BTB only and other eight subfamilies containing additional domains in addition to BTB domain,including ABTB(ANK),BBTB(BACK),BBP(BACK and PHR),KBTB(Kelch),BBK(BACK and Kelch),ZBTB or BTB-ZF(zinc finger),HBTB(HTH),and MBTB(MATH).Comparatively,the BTBD and ZBTB subfamilies contain more members.Silkworm BR-C belongs to the ZBTB subfamily and is named by ZBTB6.Phylogenetic analysis revealed that BBK,BBP and three members of the BTB-ZF subfamily,namely ZBTB14,ZBTB15 and ZBTB16,were tightly grouped together,suggesting that these BTB protein genes have evolved through gene duplication and may play similar physiological functions.Moreover,comparative analysis showed that there are 44,46,55,and 53 BTB protein genes in the fruit fly,honey bee,red flour beetle,and monarch butterfly,respectively.13 BTB protein genes showed a rigorous 1:1:1:1:1 orthologous relationship among silkworm and other four insect species,such as the BR-C gene,indicating conserved functions of these genes during insect evolution.Furthermore,based on the microarray data of genome-wide gene expression and RT-PCR examination,we observed that the silkworm BR-C(ZBTB6)gene exhibted no sex-specific or tissue-specific expressions,but was highly expressed before entering into prepupa and adult.In addition,several members of silkworm BTB protein gene family exhibited sex-specific expression in the tissues of silkworm larvae on day 3 of the fifth instar or at different stages during metamorphosis.2.Silkworm BR-C interacts with RACK1 that functions as a key factor in PKC phosphorylation pathway to activate its transcriptional activity1)BR-C interacts with RACK1 as a key anchoring protein of the PKC pathwayIn our previous study,we performed a yeast two-hybrid screen using the BTB domain of the silkworm BR-C as bait and identified the receptor for activated C-kinase 1(RACK1),a anchoring protein involved in PKC-mediated phosphorylation pathway,as the potential partner capable of interacting with BR-C.Based on this finding,we further confirmed the protein-protein interaction between BR-C and RACK1.Yeast two-hybrid analysis showed that RACK1 could efficiently interact with both full-length BR-C and its BTB domain alone,but not with a mutated BR-C protein deleting its BTB domain.Far-western blot and pull-down assays revealed that BR-C fused with SUMO tag(SUMO-BR-C)could interact with RACK1 fused with GST(GST-RACK1).These results demonstrated that BR-C directly interacts with RACK1 via the BTB domain of BR-C.2)The interaction between silkworm BR-C and RACK1 regulates PKC-mediated phosphorylation and nuclear import of BR-CWe previously found that silkworm BR-C was localized in the nucleus while RACK1 involved in PKC-mediated phosphorylation of its interacting partners was mainly localized in the cytoplasm.To decipher potential roles of the interaction between silkworm BR-C and RACK1,we disrupted the RACK1-BR-C interaction by either deleting the BTB domain of BR-C or performing RNA interference(RNAi)against endogenous RACK1 in silkworm ovary-derived Bm N4 cells.The results demonstrated both the RNAi of endogenous RACK1 and BTB domain deletion caused a failure of nuclear import of BR-C,being retained in the cytoplasm.These results strongly suggested that the nuclear import of silkworm BR-C is determined by its interaction with RACK1.Given that RACK1 is mainly involved in PKC-mediated phosphorylation and subsequent subcellular localization of its interacting partners,we proposed that the interaction of silkworm BR-C with RACK1 may be required for PKC-mediated BR-C phosphorylation.To test this hypothesis,we first performed RNAi against endogenous PKC in Bm N4-SID1 cells.Consistent with the effect of RACK1 RNAi,PKC RNAi also changed subcellular localization of BR-C from the nucleus to the cytoplasm.In addition,we predicted 12 potential PKC phosphorylation sites in silkworm BR-C protein via online program.Site-directed mutagenesis analysis demonstrated that mutating Ser373 or Thr406 in the zinc finger motifs into Ala(S373A and T406A),which can block phosphorylation,clearly shifted the nuclear localization of BR-C to the cytoplasm.Notably,although the BTB domain of silkworm BR-C was deleted,mutating Ser373 or Thr406 into Glu(E)(S373E and T406E),which mimic phosphorylation,had no effect on nuclear import of BR-C.These results suggested that the interaction of the BTB domain in BR-C with RACK1 is involved in PKC-mediated phosphorylation at both Ser373 and Thr406,which in turn contributes to the nuclear import of BR-C.3)PKC-mediated phosphorylation of BR-C activates its transcriptional activityGenerally,the phosphorylation of transcription factor is required for the activation of its transcriptional activity.To examine whether PKC-mediated phosphorylation of silkworm BR-C at Ser373 and Thr406 affects its transcriptional activity,we selected the promoter of the wing cuticle protein 10 gene(WCP10),whose transcription is directly activated by the silkworm BR-C,to conduct a dual-luciferase reporter analysis.The results showed that the incomplete BR-C lacking the BTB domain completely abolished its transcriptional activity of BR-C toward the WCP10 promoter.Furthermore,the phosphorylation-deficient mutation at S373 and T406 of silkworm BR-C also clearly decreased its transcriptional activity toward the WCP10 promoter.Taken together,our findings in the silkworm demonstrated that the protein-protein interaction between BR-C and RACK1 modulates the phosphorylation of BR-C by PKC probably at Ser373 and Thr406,and the phosphorylated BR-C subsequently translocates to the nucleus to activate the transcription of BR-C target genes.3.PKA-mediated phosphorylation of silkworm BR-C inhibits its transcriptional activity1)PKA mediates BR-C phosphorylation at Ser186To identify the phosphorylation sites of silkworm BR-C,we further conducted a liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis.Firstly,through western blot analysis with an antibody against silkworm BR-C,we detected two prominent protein bands with a molecular weight of approximately 60 k Da in the total proteins prepared from both silkworm embryo-derived Bm E cells transiently overexpressing exogenous silkworm BR-C gene and fat bodies of silkworm larvae on day 3 of the 5th instar.The lower and upper bands were likely the non-phosphorylated and phosphorylated forms of silkworm BR-C proteins,respectively.Subsequent LC-MS/MS analysis identified the Ser186 residue of silkworm BR-C was phosphorylated.Accordingly,we designed a phospho-BR-C antibody against Ser186 site.Further examination in Bm E cells confirmed that either site-specific mutagenesis at Ser186 or protein phosphatase treatment toward Bm E cells overexpressing silkworm BR-C decreased and even eliminated the BR-C phosphorylation level at Ser186,confirming that silkworm BR-C was phosphorylated at Ser186.Online computational prediction revealed that the Ser186 of silkworm BR-C is likely a PKA phosphorylation site.In vivo analysis in Bm E cells overexpressing silkworm BR-C revealed that the treatment with PKA activators of c AMP or forskolin could increase the phosphorylation levels of BR-C at Ser186.By contrast,following the treatment with PKA inhibitors of H89 or KT5720,the phosphorylation level of BR-C at at Ser186 was significantly decreased.Similar changes in BR-C phosphorylation levels in response to PKA activators or inhibitors were observed in the cultured fat bodies of silkworm larvae.Taken together,our data strongly demonstrated that silkworm BR-C is phosphorylated by PKA at Ser186.2)PKA-mediated phosphorylation of BR-C does not affect its nuclear import but inhibits its transcriptional activityTo investigate the potential roles of PKA-mediated phosphorylation of silkworm BR-C,we mutated Ser186 to Ala(S186A)to block BR-C phosphorylation or to Glu(S186E)to mimic forced BR-C phosphorylation.Subsequent immunostaining analyses in silkworm Bm N4-SID1 cells revealed that both the S186 A mutation and the S186 E mutation had no effect on nuclear localization of BR-C.The same is as the observation in PKA inhibitors treatment.Because the WCP10 and sericin-1(Ser1)genes are two direct downstream targets that are positively and negatively regulated by silkworm BR-C,respectively,we therefore selected the promoters of these two genes to elucidate the effect of PKA-mediated phosphorylation at S186 A on transcriptional activity of silkworm BR-C.Dual luciferase reporter assay showed that compared with the empty control,the S186 A mutation mimicking dephosphorylation elevated the activity of the WCP10 promoter and inhibited the activity of the Ser1 promoter.Conversely,the S186 E mutation mimicking phosphorylation resulted in a loss of its transcriptional activity.Either c AMP or forskolin,two PKA activators,could obviously inhibit the transcriptional activation of BR-C toward WCP10 promoter.Moreover,electrophoretic mobility shift assay (EMSA)and chromatin immunoprecipitation(Ch IP)experiments confirmed that the phosphorylation of BR-C at Ser186 decreased its DNA-binding ability.These data together demonstrated that PKA-mediated BR-C phosphorylation at Ser186 was not involved in the regulation of nuclear import of BR-C itself,but suppressed its transcriptional activities on direct targets by abolishing its DNA-binding activity.3)20E inhibits BR-C phosphorylation by repressing the c AMP/PKA pathwayGiven that insect BR-C is a transcriptionally activated by 20 E,we further checked whether 20 E is also involved in PKA-mediated BR-C phosphorylation at Ser186.The results demonstrated that continuous 20 E treatment on the ex vivo cultured fat bodies of silkworm larvae at the beginning of the wandering stage or Bm E cells overexpressing silkworm BR-C could reduce the PKA-mediated phosphorylation level of BR-C at Ser186.Moreover,continuous 20 E signal also inhibited the production of c AMP as PKA activator,and reduced the phosphorylation levels of both PKA catalytic subunit PKA-C as the activated form of PKA and cyclic AMP response element-binding(CREB)protein as a marker of PKA activation.Taken together,our data revealed that PKA-mediated BR-C phosphorylation at Ser186 inhibits its transcriptional activity,and continuous 20 E signal suppresses PKA-mediated BR-C phosphorylation by repressing c AMP/PKA pathway,thereby maintaining transcriptional activity of silkworm BR-C.