热休克蛋白同源物Hsc70等5种基因在蜕皮激素信号途径中的功能及相互作用研究
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摘要
昆虫的蜕皮和变态受20-羟基蜕皮酮(20-hydroxyecdysone,20E)和倍半萜类保幼激素(juvenile hormone,JH)的双重作用。20E起始了蜕皮生理过程,而保幼激素则决定着蜕皮的性质。20E通过和蜕皮激素受体(ecdysteroid receptor,EcR)结合,再与超气门蛋白(ultraspiracle protein,USP)结合形成转录复合体,继而起始了蜕皮级联反应,一些早期的蜕皮受体超家族的转录因子开始转录,包括EcR、USP、E74、E75以及激素接受子3(HR3)。随后,激素信号途径中的若干晚期基因被诱导表达,从而介导了蜕皮进程。保幼激素是一种调控昆虫生活史的关键激素,在维持蜕皮期间的幼虫状态和引导昆虫的生殖成熟方面发挥了重要作用。保幼激素可能通过与其可能受体Met相结合来调控幼虫的生长发育。
EcR／USP转录复合体中还有伴侣蛋白参与,如热休克蛋白Hsp90和热休克蛋白同源物Hsc70是该异源复合物的组成部分,伴侣蛋白与EcR／USP形成的异源复合物对于EcR／USP的DNA结合活性的发挥是必不可少的。但对伴侣蛋白如何调控EcR／USP的DNA结合活性的分子机制并不清楚。
本文鉴定了一个新的棉铃虫表皮细胞系,建立了可被激素诱导和可进行RNA干扰的细胞系模型。在此基础上检测了一系列受20E调控的基因的表达模式。借助细胞系RNAi鉴定了蜕皮激素受体EcRB1和超气门蛋白USP1及热休克蛋白同源物Hsc70在20E信号途径中的功能及作用机理。此外,还鉴定了两个受20E上调的激酶(鸟苷酸激酶和腺苷酸激酶1),为研究20E信号途径提供了有效的分子靶标。
1.棉铃虫表皮细胞系鉴定及蜕皮激素诱导模型和RNA干扰模型的建立
通过标记基因鉴定了HaEpi为表皮细胞系。棉铃虫表皮细胞系HaEpi由本实验室邵红莲建立,该论文的工作是用各种标记基因鉴定细胞系,证明其没有其它组织污染。研究结果表明,两种表皮蛋白(Ha-cup1和Ha-cup4)与Ha-trypsin2在表皮组织和表皮细胞系中表达,但在血淋巴中无表达。相反,Ha-cathL在血淋巴中特异表达,但在表皮细胞和其它三种组织中无表达。在细胞系上未检测到在中肠组织特异表达的hmg176和在脂肪体中特异表达的hexamerine。这些结果表明该HaEpi系的基因表达模式与表皮组织一致,但不同于血细胞、中肠和脂肪体,说明HaEpi系确属表皮组织来源的表皮细胞系。
建立了在HaEpi系上进行20E诱导和进行RNA干扰的方法。Northern blot结果表明,非甾醇类的20E类似物RH-2485处理后的细胞中蜕皮激素信号途径中的标记分子HHR3的表达量上调。HHR3的主带在处理后3小时内就开始表达,12小时后达到最高峰,然后逐渐下降,而其他三条带的表达相对较弱。当我们用HHR3的双链RNA转染细胞沉默HHR3后,RT-PCR结果显示HHR3的表达量显著降低。这些结果表明HaEpi是一个可被20E诱导和可进行RNA干扰的细胞系。这为我们利用RNAi检测基因的功能提供了一个良好的实验模型。
利用该可诱导的细胞系模型检测了20E对一系列基因表达的调控。这些基因包括HaEcRB1, HaUSPl, E74A, E75B, HHR3, ecdysteroid-regulated gene (ecdy), carbA2, nuclear transfer factor 2 (NTF2), gamma subunit of guanine nucleotide binding protein(G-pro-γ)和Ha-cup1。结果显示,用1μM 20E处理细胞不同时间段后,几乎所有被检测的基因的表达量都上调。蜕皮激素受体EcRB1的表达量在处理后的3-12 h内最高,而USP1要在处理12h后才有明显表达。其它两个转录因子E74a和E75b的表达在处理3h后也明显上调。HHR3经20E处理后在3h内就开始转录表达并且在12小时后达到高峰,随后逐渐下降。蜕皮激素相关蛋白ecdy和羧肽酶A2(carbA2)的表达量也上调。核转移因子2(NTF2)和G蛋白γ亚基(G-proγ)也可以被20E诱导上调表达。当用20E先处理细胞12h而后再撤掉激素继续培养不同时间段后,EcRB1,USP1和HHR3的表达在撤掉激素6 h后就消失了,其它基因的表达量也相继下降,而表皮蛋白1(cup1)在撤除激素后仍持续表达。这些结果说明20E调控这一组基因的表达,并且EcRB1,USP1和HHR3等上游转录因子的表达依赖于20E的存在。
该部分研究得到如下结论：本实验室前期建立的HaEpi系确属表皮组织来源的表皮细胞系；HaEpi系是一个可诱导和可干扰的细胞系；20E可以诱导HaEcRB1等一组基因的上调表达。综上,该细胞系可以用来研究激素的调控效应,并且通过进行RNAi极大地方便了基因功能的研究。
2.棉铃虫蜕皮激素受体HaEcRB1、超气门蛋白HaUSPl和热休克蛋白同源物Hsc70在20E信号途径中的功能研究
为了研究蜕皮激素信号转导的分子机理,分别克隆得到了棉铃虫蜕皮激素受体HaEcRB1,超气门蛋白HaUSP1及热休克蛋白同系物HaHsc70的全长序列。HaEcRB1全长1871bp,编码545个氨基酸。HaUSPl全长2290bp,编码414个氨基酸。HaHsc70全长2145bp,编码654个氨基酸。DNAman多重序列比对结果表明,HaEcRB1，HaUSP1和HaHsc70分别与同源物种的相似性非常高。HaEcRB1与烟芽夜蛾Heliothis virescens(018473)的相似性达96%；HaUSP1与印度古螟P.interpunctella(AY619987)的相似性为91%；HaHsc70与烟草天蛾M. sexta(AY220911)的相似性高达98%。
进一步用半定量RT-PCR研究了HaEcRBl.HaUSP1和HaHsc70在棉铃虫的组织分布和发育阶段表达模式。结果显示,HaEcRB1和HaUSP1的转录子在表皮、中肠、脂肪体和血细胞中都有表达,而在5龄蜕皮和6龄变态时期的表达量明显高于5龄取食时期。HaHsc70在四种组织中也都有表达,但它的转录本水平在各个时期是
一致的。说明这三种基因在各种组织中广泛表达,但HaEcRB1和HaUSP1在蜕皮和变态时存在转录水平调控,而HaHsc70属于组成性表达。体外20E诱导细胞和体内注射20E的结果显示,20E可以诱导HaHsc70上调表达,而保幼激素类似物methoprene未影响HaHsc70的表达水平。
为了阐明这些基因在蜕皮激素信号转导途径中的功能,我们在表皮细胞系上将HaEcRB1、HaUSP1和HaHsc70分别沉默,然后用RT-PCR检测了HaEcRB1,HaUSP1, E74A,E75B,HHR3,ecdy,carbA2,NTF2,G-pro-γ, HaHsc70,Hsp90和apoptosis inhibitor(apoi)等12个基因的表达情况。结果显示,分别干扰HaUSP1、HaEcRB1和HaHsc70后,一系列基因的表达被抑制,包括转录因子E74A,E75B和HHR3,还有效应基因ecdy以及伴侣蛋白Hsc70,但NTF2、G-pro-γ、Hsp90和apoi的表达量未变化。有趣的是,在沉默HaHsc70后,HaEcRB1和HaUSP1表达明显被抑制了,而分别干扰HaEcRB1、HaUSP1并不互相抑制对方的表达。这些结果说明HaEcRB1、HaUSP1和HaHsc70可能位于20E信号途径的上游发挥作用。
为了阐明HaHsc70参与蜕皮激素信号转导的机理,进行了HaHsc70细胞定位研究。免疫细胞化学结果显示,在DMSO处理的对照组细胞中,HaHsc70定位于细胞质中,当用20E处理细胞后,HaHsc70部分迁入核内。然而,在methoprene处理细胞后,未观察到HaHsc70的质核迁移。相反,HaEcRB1和HaUSP1在未处理的细胞中主要位于细胞核中,用20E处理细胞后,HaEcRB1和HaUSP1在细胞核中的表达量明显增加。然而,将HaHsc70干扰后,HaEcRB1和HaUSP1在细胞核中的信号明显减弱。说明20E可使HaHsc70入核表达,并且沉默HaHsc70后影响HaEcRB1和]HaUSP1的表达。
体内和体外的pull down结果进一步表明,HaHsc70和HaUSP1之间存在相互作用。因此,HaHsc70可能在20E作用下入核,通过与HaUSP1相结合进而促进了EcR/USP的DNA结合活性,启动20E信号途径中的基因转录,从而起始了20E信号通路。
该部分研究得到如下结论：HaEcRB1、HaUSP1和HaHsc70位于20E信号通路的上游；HaEcRB1和HaUSP1主要位于细胞核中,HaHsc70在正常情况下位于细胞质中,20E可使HaHsc70入核；HaHsc70通过与HaUSP1结合来介导20E信号通路中的基因的转录表达。
3.鸟苷酸激酶HaGK和腺苷酸激酶1HaAK1在20E信号途径中的功能
a.鸟苷酸激酶
我们从棉铃虫的cDNA文库中得到了棉铃虫鸟苷酸激酶HaGK。全长1216 bp,编码202个氨基酸。HaGK含有一个鸟苷酸激酶功能域(Gln5-Met191)。HaGK除与家蚕的鸟苷酸激酶具有最高的相似性(84%)外,与埃及伊蚊鸟苷酸激酶有67%的相似性,与豌豆蚜鸟苷酸激酶有61%的相似性,与赤拟古盗鸟苷酸激酶有60%的相似性。
为了研究该基因在20E信号途径中的功能,用qRT-PCR的方法检测了5龄取食幼虫、5龄蜕皮幼虫及6龄变态幼虫的mRNA表达模式。结果发现HaGK的转录子在三个时期的幼虫的表皮、中肠和脂肪体中都有表达,而血细胞中的表达量极低。此外,HaGK在蜕皮期和变态期的表达水平明显高于取食期。HaGK的转录本在5龄24 h开始就有表达,在5龄36 h(头壳爆裂期)时达到峰值,然后在6龄0 h(刚蜕完皮)开始下降。有趣的是,HaGK的表达在6龄72 h又达到第二次高峰,随后逐渐降低,到6龄96 h表达水平极低。当幼虫进入预蛹期时,HaGK的mRNA又开始表达,直到新蛹形成一直维持在一定的水平。这样的表达模式说明HaGK与蜕皮和变态密切相关,极有可能受蜕皮激素调控。
为了验证该假设,用激素处理表皮细胞不同时间段后检测其表达模式。结果表明,HaGK可以被20E明显的上调。它的表达量在20E诱导后的1h内就开始上升，在3 h时达到最高峰,然后逐渐下降。相反,methoprene处理细胞后HaGK的表达量和对照组相比明显下降。当同时用两种激素处理细胞时,HaGK的表达量较对照组也增加了,但却是在激素处理3h后才开始上升,说明methoprene延迟了20E的作用。
为了确定HaGK在细胞内的分布及激素对其定位的影响,取棉铃虫的表皮细胞做免疫细胞化学,以不添加抗HaGK血清的兔前血清作为阴性对照,结果显示,在DMSO处理的细胞中,HaGK主要位于细胞质。当用20E处理细胞1 h或6 h时,HaGK仍然主要集中于胞质中,表明该激素未引起HaGK的亚细胞定位变化。当用methoprene处理细胞后,得到了与20E诱导类似的结果,HaGK主要定位于胞质中。
RNAi实验结果表明,在分别干扰掉20E受体复合体的EcRB1和USP1后,在20E作用下HaGK未能被上调,说明HaGK在20E信号途径中可能位于EcRB1和USP1的下游。
该部分研究得到如下结论：HaGK在蜕皮与变态时期高表达；20E可使HaGK的表达量上调,而methoprene则抑制其表达；HaGK可能在20E信号途径中位于EcRB1和USP1的下游来发挥作用。这些结果表明,鸟苷酸激酶除了在核苷酸代谢方面发挥作用外,可能还参与了昆虫蜕皮和变态的生理过程。
b.腺苷酸激酶1
我们从棉铃虫的cDNA文库中得到了棉铃虫腺苷酸激酶1HaAK1。全长1125 bp,编码299个氨基酸。HaAK1含有一个腺苷酸激酶功能域(Ile48-Tyr205)。HaAK1与埃及伊蚊腺苷酸激酶有52%的相似性,与果蝇腺苷酸激酶1有44%的相似性,与线虫腺苷酸激酶1有50%的相似性。
为了鉴定HaAK1存在于哪个组织,用qRT-PCR的方法检测了5龄取食幼虫、5龄蜕皮幼虫及6龄变态幼虫的mRNA。结果发现HaAK1的转录子在三个时期的幼虫的表皮,中肠和脂肪体中都有表达,而血细胞中基本检测不到。此外,HaAK1基因在蜕皮期的表达水平明显高于取食期。HaAK1的转录本在5龄24 h开始就有表达,在5龄36 h(头壳爆裂期)有一个表达高峰。说明HaAK1与蜕皮密切相关,极有可能受蜕皮激素调控。
我们用激素处理表皮细胞不同时间段后检测其表达模式。结果表明,HaAK1的表达量在被20E处理6 h后明显的上调。相反,在methoprene处理细胞后的6 h内HaAK1的表达量无明显变化,但在12 h后开始显著下调。值得注意的是,当同时用两种激素处理细胞时,HaAK1的表达量较对照组也增加了,但主要是在激素处理12 h后显著上升,说明methoprene延迟了20E的作用。
我们用免疫细胞化学检测了HaAK1在细胞内的分布及激素对其定位的影响。结果显示,在对照组中,HaAK1主要位于细胞质。当用20E处理细胞时,HaAK1的定位未发生变化。当用methoprene处理细胞后,得到了与20E诱导类似的结果,HaAK1主要定位于胞质中。
在表皮细胞系上干扰掉20E受体复合体组分之一的EcRB1后,检测发现20E作用后HaAK1的表达量未受影响,说明EcRB1并未直接调控HaAK1,可能存在EcR的其它异构体来调控HaAK1。
该部分研究得到如下结论：HaAK1在蜕皮时期高表达；体外20E可以促进HaAK1的表达；HaAK1主要位于细胞质。这些结果表明,腺苷酸激酶1可能参与了昆虫蜕皮的生理过程。
Insect molting and metamorphosis are regulated by two major hormones:the steroid 20-hydroxyecdysone (20E) and the sesquiterpenoid juvenile hormone (JH).20E initiates molting while JH governs the nature of the developmental transition. Upon the binding of 20E to the ecdysone (20E) receptor (EcR), which subsequently binds to its heterodimeric partner ultraspiracle protein (USP) and forms the receptor complex, a molting cascade is initiated by the transcription of a number of transcription factors in the nuclear receptor superfamily including EcR, USP, E74, E75, and hormone receptor 3 (HR3). Subsequently, several late genes in the hormone pathway are upregulated and help mediate the molting process.
It has been reported that some chaperone proteins are involved in the maturing of the EcR/USP transcription complex. Two proteins, Hsp90 and Hsc70, have been found as the components of the complex. A chaperone-EcR/USP heterocomplex is required for activation of EcR/USP DNA binding activity. However, little is know about the role of chaperone proteins in the up-or down-regulation of its activity.
In the current research, we identified an epidermal cell line from Helicoverpa armigera, which could be used for investigating hormonal regulation on the gene expression in the hormonal signal pathway and performing RNAi to investigate gene function. To probe the role of the Hsc70 in 20E signaling transduction pathway, we have analyzed the role of Hsc70 and its interaction with USP, utilizing molecular cloning, expression analysis, and functional determination of EcRB1, USP1, and Hsc70. The results reveal that Hsc70 plays important roles in regulating the expression of a set of genes involved in the 20E signaling transduction pathway by binding with USP1 and facilitating the expression of EcRB1 and USP1. We also characterized two 20E induced genes (guanylate kinase and adenylate kinase 1) from Helicoverpa armigera. These provided useful target molecules for understanding the signal cascades of 20E. It was also helpful to investigate the mechanisms during the molting and metamorphosis of insects.
1. Identification of an epidermal cell line from Helicoverpa armigera and establishment of a model for investigating hormonal regulation on the gene expression and gene function
An epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was identified by maker genes. This cell line was established by Shao Honglian in our lab。My own work was the identification of the cell line using several maker genes and conforming that it wasn't contaminated by other tissues. Results showed that two cuticle proteins(Ha-cup1 and Ha-cup4) and Ha-trypsin2 expressed in both HaEpi cell line and epidermis but did not express in haemocytes. In contrast, Ha-cathL expressed only in haemocytes and not in other tissues or the HaEpi cell line. Hmg176 expressed only in the midgut and hexamerin only expressed in the fat body but not in HaEpi cell line and epidermis. These facts indicated that HaEpi cell line was not derived from haemocytes, midgut or fat body but from integument.
Futhermore, a model for investigating hormonal regulation on the gene expression and gene function using RNAi was established. Northern blot results showed that HHR3, the maker gene of the 20E signal transduction pathway, was upregulated after being induced by RH-2485. A Dig-labeled HHR3 probe was used to detect 4 isoforms of HHR3 transcripts. Band 1 was dominant and could be induced within 3 h, peaked at 12 h, and declined thereafter. The other bands were notably fainter. Additionally, HHR3 expression could be knocked down using the RNAi method. These characters present us with a model for investigating gene function by RNAi in the cell line.
During the 20E induction, nearly all of the examined genes were upregulated by 20E treatment. Ecdysone receptor (EcRb) expression peaked at 3 h、12 h, while ultraspiracle protein (USP1) appeared obviously after 12 h induction with 20E. Ecdysone induced protein E74 (E74a) and E75 (E75b) increased along with the culture over 24 h. Hormone receptor 3 (HHR3) was not detected in the absence of 20E, but rapidly elevated after culturing for 3 h with 20E, peaked at 12 h, and declined gradually thereafter. The ecdysteroid-regulated gene (ecdy)、carboxypeptidase (carbA2)、nuclear transfer factor 2 (NTF2) and G-protein y subunit (G-proy). Otherwise, withdrawing of 20E after 12 h culture in it, EcRb, USP1 and HHR3 stopped expression at 6 h. Other genes also showed decreased expression along with the incubation time from 6 to 24 h after withdrawal of 20E. The cuticle protein 1 (cup1) did not demonstrate a close relationship to the withdrawing of 20E. This result indicated that the cell line responded well to the 20E analogs, which suggested that a new epidermal cell line which could be induced by ecdysone was successfully developed.
In conclusion, HaEpi cell line was indeed derived from integument; it could be used to analyse the hormonal regulation on the gene expression and investigate gene function by RNAi.
2. Regulation of USP1, EcRB1 and Hsc70 on the gene expression in the 20E signal transduction pathway in Helicoverpa armigera
The complete sequences of HaEcRB1、HaUSP1 and HaHsc70 was respectively cloned from Helicoverpa armigera. HaEcRB1 encodes a 545-amino acid protein while HaUSPl and HaHsc70 encode a 414 amino acid protein、a 654-amino acid residue protein respectively. The results from multi-alignment revealed that HaEcRB1, HaUSP1, and HaHsc70 all exhibited a high amino acid sequence homology with corresponding counterparts from other species. Semi-quantitative RT-PCR showed that HaEcRBl and HaUSP1 were obviously upregulated in feeding 5th instar larvae and molting 5th instar larvae in all tested tissues. However, the transcript of HaHsc70 reflected a constitutive expression in all tested tissues and at all three developmental stages. Western blot results revealed that HaHsc70 was upregulated by 20E both in the 20E-injected larvae and in 20E-treated HaEpi cells compared to controls. However, methoprene administered to larvae or HaEpi cells exhibited no obvious effect on the HaHsc70 content.
To explore the function of these genes in 20E signal transduction pathway, HaEcRB1、HaUSPl and HaHsc70 were silenced in the HaEpi respectively. Then RT-PCR was used to analyse the expression of several genes, including HaEcRB1, HaUSP1, E74A, E75B, HHR3, ecdy, carbA2, NTF2, G-pro-γ,HaHsc70, Hsp90 and apoptosis inhibitor (apoi). Silencing of HaEcRBl、HaUSP1 and HaHsc70 respectively by RNAi resulted in suppression of a set of 20E induced genes, including the transcription factors E74A, E75B, and HHR3, effector genes ecdy and carbA, and chaperone protein Hsc70, when compared to the control cells treated with GFP dsRNA. However, silencing of them did not result in the suppression of the induction of NTF2, G-pro-γ,Hsp90, and apoi. Interestingly, 20E-induced expression of HaEcRBl and HaUSP1 was significantly suppressed after HaHsc70 was silenced. These results suggest that HaEcRB1、HaUSP1 and HaHsc70 were functioned upstream in the 20E signal transduction pathway.
To clarify the mechanism how HaHsc70 is involed in the 20E signal transduction pathway, the subcellular location of HaHsc70 was investigated. It was found to be located in the cytoplasm in control cells. After the cells were incubated with 20E, HaHsc70 was detected not only in the cytoplasm, but also in the nuclei. In contrast, HaUSP1 and HaEcRB1 were predominantly located in the control cells'nuclei. Following induction with 20E, they were notably upregulated in the nucleus. However, when HaHsc70 was knocked down by RNAi, the HaUSP1 and HaEcRB1 signal in the nucleus decreased compared with that in the dsGFP-control cells. These results suggest that HaHsc70 could translocate to nucleus after 20E induction and the expression of HaUSP1 and HaEcRB1 was regulated by HaHsc70.
HaHsc70 was capable of binding to HaUSPl in pull-down assays.
In sum, HaEcRB1、HaUSP1 and HaHsc70 function upstream in the 20E signal transduction pathway; HaEcRB1 and HaUSP1 mainly locate in nucleus while HaHsc70 locates in cytoplasm; HaHsc70 could translocate to nucleus after 20E induction; Hsc70 participates in the 20E signal transduction pathway via binding to USP1 and mediating the expression of EcRB1, USP1 and then a set of 20E responsive genes.
3. Function of guanylate kinase and adenylate kinase 1 in 20E signal transduction pathway
a. Guanylate kinase
A 1,216-bp full length cDNA of HaGK was obtained from the cDNA library of H. armigera. It contains a 606-bp ORF encoding a 202-amino acid residue protein. Analysis of HaGK using the SMART software showed that HaGK contained a GuKc domain (Gln5-Met191). HaGK had the highest similarity to guanylate kinase from Bombyx. mori (84%identity). In addition, the identities of HaGK to the guanylate kinase from Aedes aegypti, Acyrthosiphon pisum and Tribolium castaneum were 67%,61%, and 60%, respectively.
To explore its roles in 20E signal pathway, qRT-PCR results showed that HaGK was obviously upregulated in 5th molting larvae in epidermis, midgut and fat body compared to those at 5th feeding larvae. Furthermore, the gene was detected at all developmental stages and it reached the peaks at the 5th instar 36 h with head capsule slippage and 6th instar 72 h (6-72h, metamorphic molting). This expression pattern suggests that HaGK could be closely related to molting and metamorphosis and might be regulated by ecdysone.
To validate this hypothesis, cells were treated by hormones and the expression pattern was detected. HaGK was upregulated by 20E in 20E-treated HaEpi cells compared to controls. Its expression began to increase within 1 h after induction, peaked at 3 h, and then declined. In contrast, the expression of HaGK decreased after treatment with methoprene as compared to the control group. Notably, it was also upregulated by 20E and methoprene together, but the upregulation appeared only after treatment after 3 h.
To determine its subcellular location, immunocytochemistry was performed in the HaEpi. HaGK was mainly detected in the cytoplasm in the control group.20E and methoprene had no effect on the location of HaGK.
RNAi experiments revealed that the expression of HaGK was largely decreased after HaEcRB1 and HaUSP1 was silenced, showing that HaGK mightbe located downstream in 20E pathway.
In summary, HaGK was obviously upregulated in 5th molting and 6th metamorphic molting larvae; its expression could be upregulated by 20E and suppressed by methoprene; HaGK might act downstream EcRB1 and USP1 in 20E signal pathway. These results suggest that HaGK participate in insect molting and metamorphosis in addition to function in energy metabolism.
b. Adenylate kinase 1
A 1,125-bp full length cDNA for HaAKl was obtained from the cDNA library of H. armigera. It contains a 897-bp ORF encoding a 299-amino acid residue protein. Analysis of HaAK1 using the SMART software showed that HaAK1 contained an AK domain (Ile48-Tyr205). HaAK1 had the highest similarity to adenylate kinase from A.aegypti (78% identity). In addition, the identities of HaAK1 to the adenylate kinase from C. elegans and D. rerio were 63%,60%, respectively.
To determine its tissue distribution, qRT-PCR was used. Results showed that HaAK1 were obviously upregulated in 5th molting larvae in epidermis, midgut and fat body compared to those at 5th feeding larvae. However, a rather weak signal was observed in the haemocyte. To investigate the expression pattern of HaAKl in the epidermis during different developmental stages, the cDNAs of the epidermis from the 5th instar 24 h (5-24) to the 1st day pupae (p0) were determined by qRT-PCR. The gene was detected at all developmental stages and it reached the peaks at the 5th instar 36 h with head capsule slippage and 6th instar 72 h (6-72h, metamorphic molting). This expression pattern suggests that HaAK1 could be closely related to molting and metamorphosis and might be regulated by ecdysone.
To confirm this, HaEpi cells were treated by 20E and methoprene. The results revealed that HaAK1 was upregulated by 20E in 20E-treated HaEpi cells compared to controls. Its expression peaked at 3 h, and then declined, In contrast, the expression of HaAK1 decreased after treatment with methoprene as compared to the control group. Notably, it was also upregulated by 20E and methoprene together, but the upregulation appeared only after treatment after 12 h.
Immunocytochemistry was performed in the HaEpi to determine its subcellular location. HaAK1 was mainly detected in the cytoplasm in the control group.20E and methoprene had no effect on the location of HaGK.
RNAi experiments revealed that silence of HaEcRB1 had no effect on the expression of HaAK1, suggesting that HaAK1 could not be directly regulated by EcRB1 but maybe other isoforms of EcRB1.
In brief, HaAK1 was upregulated in 5th molting larvae; its expression could be upregulated by 20E in vitro. These results suggest that HaAK1 might participate in insect molting process.
EcR／USP转录复合体中还有伴侣蛋白参与,如热休克蛋白Hsp90和热休克蛋白同源物Hsc70是该异源复合物的组成部分,伴侣蛋白与EcR／USP形成的异源复合物对于EcR／USP的DNA结合活性的发挥是必不可少的。但对伴侣蛋白如何调控EcR／USP的DNA结合活性的分子机制并不清楚。
本文鉴定了一个新的棉铃虫表皮细胞系,建立了可被激素诱导和可进行RNA干扰的细胞系模型。在此基础上检测了一系列受20E调控的基因的表达模式。借助细胞系RNAi鉴定了蜕皮激素受体EcRB1和超气门蛋白USP1及热休克蛋白同源物Hsc70在20E信号途径中的功能及作用机理。此外,还鉴定了两个受20E上调的激酶(鸟苷酸激酶和腺苷酸激酶1),为研究20E信号途径提供了有效的分子靶标。
1.棉铃虫表皮细胞系鉴定及蜕皮激素诱导模型和RNA干扰模型的建立
通过标记基因鉴定了HaEpi为表皮细胞系。棉铃虫表皮细胞系HaEpi由本实验室邵红莲建立,该论文的工作是用各种标记基因鉴定细胞系,证明其没有其它组织污染。研究结果表明,两种表皮蛋白(Ha-cup1和Ha-cup4)与Ha-trypsin2在表皮组织和表皮细胞系中表达,但在血淋巴中无表达。相反,Ha-cathL在血淋巴中特异表达,但在表皮细胞和其它三种组织中无表达。在细胞系上未检测到在中肠组织特异表达的hmg176和在脂肪体中特异表达的hexamerine。这些结果表明该HaEpi系的基因表达模式与表皮组织一致,但不同于血细胞、中肠和脂肪体,说明HaEpi系确属表皮组织来源的表皮细胞系。
建立了在HaEpi系上进行20E诱导和进行RNA干扰的方法。Northern blot结果表明,非甾醇类的20E类似物RH-2485处理后的细胞中蜕皮激素信号途径中的标记分子HHR3的表达量上调。HHR3的主带在处理后3小时内就开始表达,12小时后达到最高峰,然后逐渐下降,而其他三条带的表达相对较弱。当我们用HHR3的双链RNA转染细胞沉默HHR3后,RT-PCR结果显示HHR3的表达量显著降低。这些结果表明HaEpi是一个可被20E诱导和可进行RNA干扰的细胞系。这为我们利用RNAi检测基因的功能提供了一个良好的实验模型。
利用该可诱导的细胞系模型检测了20E对一系列基因表达的调控。这些基因包括HaEcRB1, HaUSPl, E74A, E75B, HHR3, ecdysteroid-regulated gene (ecdy), carbA2, nuclear transfer factor 2 (NTF2), gamma subunit of guanine nucleotide binding protein(G-pro-γ)和Ha-cup1。结果显示,用1μM 20E处理细胞不同时间段后,几乎所有被检测的基因的表达量都上调。蜕皮激素受体EcRB1的表达量在处理后的3-12 h内最高,而USP1要在处理12h后才有明显表达。其它两个转录因子E74a和E75b的表达在处理3h后也明显上调。HHR3经20E处理后在3h内就开始转录表达并且在12小时后达到高峰,随后逐渐下降。蜕皮激素相关蛋白ecdy和羧肽酶A2(carbA2)的表达量也上调。核转移因子2(NTF2)和G蛋白γ亚基(G-proγ)也可以被20E诱导上调表达。当用20E先处理细胞12h而后再撤掉激素继续培养不同时间段后,EcRB1,USP1和HHR3的表达在撤掉激素6 h后就消失了,其它基因的表达量也相继下降,而表皮蛋白1(cup1)在撤除激素后仍持续表达。这些结果说明20E调控这一组基因的表达,并且EcRB1,USP1和HHR3等上游转录因子的表达依赖于20E的存在。
该部分研究得到如下结论：本实验室前期建立的HaEpi系确属表皮组织来源的表皮细胞系；HaEpi系是一个可诱导和可干扰的细胞系；20E可以诱导HaEcRB1等一组基因的上调表达。综上,该细胞系可以用来研究激素的调控效应,并且通过进行RNAi极大地方便了基因功能的研究。
2.棉铃虫蜕皮激素受体HaEcRB1、超气门蛋白HaUSPl和热休克蛋白同源物Hsc70在20E信号途径中的功能研究
为了研究蜕皮激素信号转导的分子机理,分别克隆得到了棉铃虫蜕皮激素受体HaEcRB1,超气门蛋白HaUSP1及热休克蛋白同系物HaHsc70的全长序列。HaEcRB1全长1871bp,编码545个氨基酸。HaUSPl全长2290bp,编码414个氨基酸。HaHsc70全长2145bp,编码654个氨基酸。DNAman多重序列比对结果表明,HaEcRB1，HaUSP1和HaHsc70分别与同源物种的相似性非常高。HaEcRB1与烟芽夜蛾Heliothis virescens(018473)的相似性达96%；HaUSP1与印度古螟P.interpunctella(AY619987)的相似性为91%；HaHsc70与烟草天蛾M. sexta(AY220911)的相似性高达98%。
进一步用半定量RT-PCR研究了HaEcRBl.HaUSP1和HaHsc70在棉铃虫的组织分布和发育阶段表达模式。结果显示,HaEcRB1和HaUSP1的转录子在表皮、中肠、脂肪体和血细胞中都有表达,而在5龄蜕皮和6龄变态时期的表达量明显高于5龄取食时期。HaHsc70在四种组织中也都有表达,但它的转录本水平在各个时期是
一致的。说明这三种基因在各种组织中广泛表达,但HaEcRB1和HaUSP1在蜕皮和变态时存在转录水平调控,而HaHsc70属于组成性表达。体外20E诱导细胞和体内注射20E的结果显示,20E可以诱导HaHsc70上调表达,而保幼激素类似物methoprene未影响HaHsc70的表达水平。
为了阐明这些基因在蜕皮激素信号转导途径中的功能,我们在表皮细胞系上将HaEcRB1、HaUSP1和HaHsc70分别沉默,然后用RT-PCR检测了HaEcRB1,HaUSP1, E74A,E75B,HHR3,ecdy,carbA2,NTF2,G-pro-γ, HaHsc70,Hsp90和apoptosis inhibitor(apoi)等12个基因的表达情况。结果显示,分别干扰HaUSP1、HaEcRB1和HaHsc70后,一系列基因的表达被抑制,包括转录因子E74A,E75B和HHR3,还有效应基因ecdy以及伴侣蛋白Hsc70,但NTF2、G-pro-γ、Hsp90和apoi的表达量未变化。有趣的是,在沉默HaHsc70后,HaEcRB1和HaUSP1表达明显被抑制了,而分别干扰HaEcRB1、HaUSP1并不互相抑制对方的表达。这些结果说明HaEcRB1、HaUSP1和HaHsc70可能位于20E信号途径的上游发挥作用。
为了阐明HaHsc70参与蜕皮激素信号转导的机理,进行了HaHsc70细胞定位研究。免疫细胞化学结果显示,在DMSO处理的对照组细胞中,HaHsc70定位于细胞质中,当用20E处理细胞后,HaHsc70部分迁入核内。然而,在methoprene处理细胞后,未观察到HaHsc70的质核迁移。相反,HaEcRB1和HaUSP1在未处理的细胞中主要位于细胞核中,用20E处理细胞后,HaEcRB1和HaUSP1在细胞核中的表达量明显增加。然而,将HaHsc70干扰后,HaEcRB1和HaUSP1在细胞核中的信号明显减弱。说明20E可使HaHsc70入核表达,并且沉默HaHsc70后影响HaEcRB1和]HaUSP1的表达。
体内和体外的pull down结果进一步表明,HaHsc70和HaUSP1之间存在相互作用。因此,HaHsc70可能在20E作用下入核,通过与HaUSP1相结合进而促进了EcR/USP的DNA结合活性,启动20E信号途径中的基因转录,从而起始了20E信号通路。
该部分研究得到如下结论：HaEcRB1、HaUSP1和HaHsc70位于20E信号通路的上游；HaEcRB1和HaUSP1主要位于细胞核中,HaHsc70在正常情况下位于细胞质中,20E可使HaHsc70入核；HaHsc70通过与HaUSP1结合来介导20E信号通路中的基因的转录表达。
3.鸟苷酸激酶HaGK和腺苷酸激酶1HaAK1在20E信号途径中的功能
a.鸟苷酸激酶
我们从棉铃虫的cDNA文库中得到了棉铃虫鸟苷酸激酶HaGK。全长1216 bp,编码202个氨基酸。HaGK含有一个鸟苷酸激酶功能域(Gln5-Met191)。HaGK除与家蚕的鸟苷酸激酶具有最高的相似性(84%)外,与埃及伊蚊鸟苷酸激酶有67%的相似性,与豌豆蚜鸟苷酸激酶有61%的相似性,与赤拟古盗鸟苷酸激酶有60%的相似性。
为了研究该基因在20E信号途径中的功能,用qRT-PCR的方法检测了5龄取食幼虫、5龄蜕皮幼虫及6龄变态幼虫的mRNA表达模式。结果发现HaGK的转录子在三个时期的幼虫的表皮、中肠和脂肪体中都有表达,而血细胞中的表达量极低。此外,HaGK在蜕皮期和变态期的表达水平明显高于取食期。HaGK的转录本在5龄24 h开始就有表达,在5龄36 h(头壳爆裂期)时达到峰值,然后在6龄0 h(刚蜕完皮)开始下降。有趣的是,HaGK的表达在6龄72 h又达到第二次高峰,随后逐渐降低,到6龄96 h表达水平极低。当幼虫进入预蛹期时,HaGK的mRNA又开始表达,直到新蛹形成一直维持在一定的水平。这样的表达模式说明HaGK与蜕皮和变态密切相关,极有可能受蜕皮激素调控。
为了验证该假设,用激素处理表皮细胞不同时间段后检测其表达模式。结果表明,HaGK可以被20E明显的上调。它的表达量在20E诱导后的1h内就开始上升，在3 h时达到最高峰,然后逐渐下降。相反,methoprene处理细胞后HaGK的表达量和对照组相比明显下降。当同时用两种激素处理细胞时,HaGK的表达量较对照组也增加了,但却是在激素处理3h后才开始上升,说明methoprene延迟了20E的作用。
为了确定HaGK在细胞内的分布及激素对其定位的影响,取棉铃虫的表皮细胞做免疫细胞化学,以不添加抗HaGK血清的兔前血清作为阴性对照,结果显示,在DMSO处理的细胞中,HaGK主要位于细胞质。当用20E处理细胞1 h或6 h时,HaGK仍然主要集中于胞质中,表明该激素未引起HaGK的亚细胞定位变化。当用methoprene处理细胞后,得到了与20E诱导类似的结果,HaGK主要定位于胞质中。
RNAi实验结果表明,在分别干扰掉20E受体复合体的EcRB1和USP1后,在20E作用下HaGK未能被上调,说明HaGK在20E信号途径中可能位于EcRB1和USP1的下游。
该部分研究得到如下结论：HaGK在蜕皮与变态时期高表达；20E可使HaGK的表达量上调,而methoprene则抑制其表达；HaGK可能在20E信号途径中位于EcRB1和USP1的下游来发挥作用。这些结果表明,鸟苷酸激酶除了在核苷酸代谢方面发挥作用外,可能还参与了昆虫蜕皮和变态的生理过程。
b.腺苷酸激酶1
我们从棉铃虫的cDNA文库中得到了棉铃虫腺苷酸激酶1HaAK1。全长1125 bp,编码299个氨基酸。HaAK1含有一个腺苷酸激酶功能域(Ile48-Tyr205)。HaAK1与埃及伊蚊腺苷酸激酶有52%的相似性,与果蝇腺苷酸激酶1有44%的相似性,与线虫腺苷酸激酶1有50%的相似性。
为了鉴定HaAK1存在于哪个组织,用qRT-PCR的方法检测了5龄取食幼虫、5龄蜕皮幼虫及6龄变态幼虫的mRNA。结果发现HaAK1的转录子在三个时期的幼虫的表皮,中肠和脂肪体中都有表达,而血细胞中基本检测不到。此外,HaAK1基因在蜕皮期的表达水平明显高于取食期。HaAK1的转录本在5龄24 h开始就有表达,在5龄36 h(头壳爆裂期)有一个表达高峰。说明HaAK1与蜕皮密切相关,极有可能受蜕皮激素调控。
我们用激素处理表皮细胞不同时间段后检测其表达模式。结果表明,HaAK1的表达量在被20E处理6 h后明显的上调。相反,在methoprene处理细胞后的6 h内HaAK1的表达量无明显变化,但在12 h后开始显著下调。值得注意的是,当同时用两种激素处理细胞时,HaAK1的表达量较对照组也增加了,但主要是在激素处理12 h后显著上升,说明methoprene延迟了20E的作用。
我们用免疫细胞化学检测了HaAK1在细胞内的分布及激素对其定位的影响。结果显示,在对照组中,HaAK1主要位于细胞质。当用20E处理细胞时,HaAK1的定位未发生变化。当用methoprene处理细胞后,得到了与20E诱导类似的结果,HaAK1主要定位于胞质中。
在表皮细胞系上干扰掉20E受体复合体组分之一的EcRB1后,检测发现20E作用后HaAK1的表达量未受影响,说明EcRB1并未直接调控HaAK1,可能存在EcR的其它异构体来调控HaAK1。
该部分研究得到如下结论：HaAK1在蜕皮时期高表达；体外20E可以促进HaAK1的表达；HaAK1主要位于细胞质。这些结果表明,腺苷酸激酶1可能参与了昆虫蜕皮的生理过程。
Insect molting and metamorphosis are regulated by two major hormones:the steroid 20-hydroxyecdysone (20E) and the sesquiterpenoid juvenile hormone (JH).20E initiates molting while JH governs the nature of the developmental transition. Upon the binding of 20E to the ecdysone (20E) receptor (EcR), which subsequently binds to its heterodimeric partner ultraspiracle protein (USP) and forms the receptor complex, a molting cascade is initiated by the transcription of a number of transcription factors in the nuclear receptor superfamily including EcR, USP, E74, E75, and hormone receptor 3 (HR3). Subsequently, several late genes in the hormone pathway are upregulated and help mediate the molting process.
It has been reported that some chaperone proteins are involved in the maturing of the EcR/USP transcription complex. Two proteins, Hsp90 and Hsc70, have been found as the components of the complex. A chaperone-EcR/USP heterocomplex is required for activation of EcR/USP DNA binding activity. However, little is know about the role of chaperone proteins in the up-or down-regulation of its activity.
In the current research, we identified an epidermal cell line from Helicoverpa armigera, which could be used for investigating hormonal regulation on the gene expression in the hormonal signal pathway and performing RNAi to investigate gene function. To probe the role of the Hsc70 in 20E signaling transduction pathway, we have analyzed the role of Hsc70 and its interaction with USP, utilizing molecular cloning, expression analysis, and functional determination of EcRB1, USP1, and Hsc70. The results reveal that Hsc70 plays important roles in regulating the expression of a set of genes involved in the 20E signaling transduction pathway by binding with USP1 and facilitating the expression of EcRB1 and USP1. We also characterized two 20E induced genes (guanylate kinase and adenylate kinase 1) from Helicoverpa armigera. These provided useful target molecules for understanding the signal cascades of 20E. It was also helpful to investigate the mechanisms during the molting and metamorphosis of insects.
1. Identification of an epidermal cell line from Helicoverpa armigera and establishment of a model for investigating hormonal regulation on the gene expression and gene function
An epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was identified by maker genes. This cell line was established by Shao Honglian in our lab。My own work was the identification of the cell line using several maker genes and conforming that it wasn't contaminated by other tissues. Results showed that two cuticle proteins(Ha-cup1 and Ha-cup4) and Ha-trypsin2 expressed in both HaEpi cell line and epidermis but did not express in haemocytes. In contrast, Ha-cathL expressed only in haemocytes and not in other tissues or the HaEpi cell line. Hmg176 expressed only in the midgut and hexamerin only expressed in the fat body but not in HaEpi cell line and epidermis. These facts indicated that HaEpi cell line was not derived from haemocytes, midgut or fat body but from integument.
Futhermore, a model for investigating hormonal regulation on the gene expression and gene function using RNAi was established. Northern blot results showed that HHR3, the maker gene of the 20E signal transduction pathway, was upregulated after being induced by RH-2485. A Dig-labeled HHR3 probe was used to detect 4 isoforms of HHR3 transcripts. Band 1 was dominant and could be induced within 3 h, peaked at 12 h, and declined thereafter. The other bands were notably fainter. Additionally, HHR3 expression could be knocked down using the RNAi method. These characters present us with a model for investigating gene function by RNAi in the cell line.
During the 20E induction, nearly all of the examined genes were upregulated by 20E treatment. Ecdysone receptor (EcRb) expression peaked at 3 h、12 h, while ultraspiracle protein (USP1) appeared obviously after 12 h induction with 20E. Ecdysone induced protein E74 (E74a) and E75 (E75b) increased along with the culture over 24 h. Hormone receptor 3 (HHR3) was not detected in the absence of 20E, but rapidly elevated after culturing for 3 h with 20E, peaked at 12 h, and declined gradually thereafter. The ecdysteroid-regulated gene (ecdy)、carboxypeptidase (carbA2)、nuclear transfer factor 2 (NTF2) and G-protein y subunit (G-proy). Otherwise, withdrawing of 20E after 12 h culture in it, EcRb, USP1 and HHR3 stopped expression at 6 h. Other genes also showed decreased expression along with the incubation time from 6 to 24 h after withdrawal of 20E. The cuticle protein 1 (cup1) did not demonstrate a close relationship to the withdrawing of 20E. This result indicated that the cell line responded well to the 20E analogs, which suggested that a new epidermal cell line which could be induced by ecdysone was successfully developed.
In conclusion, HaEpi cell line was indeed derived from integument; it could be used to analyse the hormonal regulation on the gene expression and investigate gene function by RNAi.
2. Regulation of USP1, EcRB1 and Hsc70 on the gene expression in the 20E signal transduction pathway in Helicoverpa armigera
The complete sequences of HaEcRB1、HaUSP1 and HaHsc70 was respectively cloned from Helicoverpa armigera. HaEcRB1 encodes a 545-amino acid protein while HaUSPl and HaHsc70 encode a 414 amino acid protein、a 654-amino acid residue protein respectively. The results from multi-alignment revealed that HaEcRB1, HaUSP1, and HaHsc70 all exhibited a high amino acid sequence homology with corresponding counterparts from other species. Semi-quantitative RT-PCR showed that HaEcRBl and HaUSP1 were obviously upregulated in feeding 5th instar larvae and molting 5th instar larvae in all tested tissues. However, the transcript of HaHsc70 reflected a constitutive expression in all tested tissues and at all three developmental stages. Western blot results revealed that HaHsc70 was upregulated by 20E both in the 20E-injected larvae and in 20E-treated HaEpi cells compared to controls. However, methoprene administered to larvae or HaEpi cells exhibited no obvious effect on the HaHsc70 content.
To explore the function of these genes in 20E signal transduction pathway, HaEcRB1、HaUSPl and HaHsc70 were silenced in the HaEpi respectively. Then RT-PCR was used to analyse the expression of several genes, including HaEcRB1, HaUSP1, E74A, E75B, HHR3, ecdy, carbA2, NTF2, G-pro-γ,HaHsc70, Hsp90 and apoptosis inhibitor (apoi). Silencing of HaEcRBl、HaUSP1 and HaHsc70 respectively by RNAi resulted in suppression of a set of 20E induced genes, including the transcription factors E74A, E75B, and HHR3, effector genes ecdy and carbA, and chaperone protein Hsc70, when compared to the control cells treated with GFP dsRNA. However, silencing of them did not result in the suppression of the induction of NTF2, G-pro-γ,Hsp90, and apoi. Interestingly, 20E-induced expression of HaEcRBl and HaUSP1 was significantly suppressed after HaHsc70 was silenced. These results suggest that HaEcRB1、HaUSP1 and HaHsc70 were functioned upstream in the 20E signal transduction pathway.
To clarify the mechanism how HaHsc70 is involed in the 20E signal transduction pathway, the subcellular location of HaHsc70 was investigated. It was found to be located in the cytoplasm in control cells. After the cells were incubated with 20E, HaHsc70 was detected not only in the cytoplasm, but also in the nuclei. In contrast, HaUSP1 and HaEcRB1 were predominantly located in the control cells'nuclei. Following induction with 20E, they were notably upregulated in the nucleus. However, when HaHsc70 was knocked down by RNAi, the HaUSP1 and HaEcRB1 signal in the nucleus decreased compared with that in the dsGFP-control cells. These results suggest that HaHsc70 could translocate to nucleus after 20E induction and the expression of HaUSP1 and HaEcRB1 was regulated by HaHsc70.
HaHsc70 was capable of binding to HaUSPl in pull-down assays.
In sum, HaEcRB1、HaUSP1 and HaHsc70 function upstream in the 20E signal transduction pathway; HaEcRB1 and HaUSP1 mainly locate in nucleus while HaHsc70 locates in cytoplasm; HaHsc70 could translocate to nucleus after 20E induction; Hsc70 participates in the 20E signal transduction pathway via binding to USP1 and mediating the expression of EcRB1, USP1 and then a set of 20E responsive genes.
3. Function of guanylate kinase and adenylate kinase 1 in 20E signal transduction pathway
a. Guanylate kinase
A 1,216-bp full length cDNA of HaGK was obtained from the cDNA library of H. armigera. It contains a 606-bp ORF encoding a 202-amino acid residue protein. Analysis of HaGK using the SMART software showed that HaGK contained a GuKc domain (Gln5-Met191). HaGK had the highest similarity to guanylate kinase from Bombyx. mori (84%identity). In addition, the identities of HaGK to the guanylate kinase from Aedes aegypti, Acyrthosiphon pisum and Tribolium castaneum were 67%,61%, and 60%, respectively.
To explore its roles in 20E signal pathway, qRT-PCR results showed that HaGK was obviously upregulated in 5th molting larvae in epidermis, midgut and fat body compared to those at 5th feeding larvae. Furthermore, the gene was detected at all developmental stages and it reached the peaks at the 5th instar 36 h with head capsule slippage and 6th instar 72 h (6-72h, metamorphic molting). This expression pattern suggests that HaGK could be closely related to molting and metamorphosis and might be regulated by ecdysone.
To validate this hypothesis, cells were treated by hormones and the expression pattern was detected. HaGK was upregulated by 20E in 20E-treated HaEpi cells compared to controls. Its expression began to increase within 1 h after induction, peaked at 3 h, and then declined. In contrast, the expression of HaGK decreased after treatment with methoprene as compared to the control group. Notably, it was also upregulated by 20E and methoprene together, but the upregulation appeared only after treatment after 3 h.
To determine its subcellular location, immunocytochemistry was performed in the HaEpi. HaGK was mainly detected in the cytoplasm in the control group.20E and methoprene had no effect on the location of HaGK.
RNAi experiments revealed that the expression of HaGK was largely decreased after HaEcRB1 and HaUSP1 was silenced, showing that HaGK mightbe located downstream in 20E pathway.
In summary, HaGK was obviously upregulated in 5th molting and 6th metamorphic molting larvae; its expression could be upregulated by 20E and suppressed by methoprene; HaGK might act downstream EcRB1 and USP1 in 20E signal pathway. These results suggest that HaGK participate in insect molting and metamorphosis in addition to function in energy metabolism.
b. Adenylate kinase 1
A 1,125-bp full length cDNA for HaAKl was obtained from the cDNA library of H. armigera. It contains a 897-bp ORF encoding a 299-amino acid residue protein. Analysis of HaAK1 using the SMART software showed that HaAK1 contained an AK domain (Ile48-Tyr205). HaAK1 had the highest similarity to adenylate kinase from A.aegypti (78% identity). In addition, the identities of HaAK1 to the adenylate kinase from C. elegans and D. rerio were 63%,60%, respectively.
To determine its tissue distribution, qRT-PCR was used. Results showed that HaAK1 were obviously upregulated in 5th molting larvae in epidermis, midgut and fat body compared to those at 5th feeding larvae. However, a rather weak signal was observed in the haemocyte. To investigate the expression pattern of HaAKl in the epidermis during different developmental stages, the cDNAs of the epidermis from the 5th instar 24 h (5-24) to the 1st day pupae (p0) were determined by qRT-PCR. The gene was detected at all developmental stages and it reached the peaks at the 5th instar 36 h with head capsule slippage and 6th instar 72 h (6-72h, metamorphic molting). This expression pattern suggests that HaAK1 could be closely related to molting and metamorphosis and might be regulated by ecdysone.
To confirm this, HaEpi cells were treated by 20E and methoprene. The results revealed that HaAK1 was upregulated by 20E in 20E-treated HaEpi cells compared to controls. Its expression peaked at 3 h, and then declined, In contrast, the expression of HaAK1 decreased after treatment with methoprene as compared to the control group. Notably, it was also upregulated by 20E and methoprene together, but the upregulation appeared only after treatment after 12 h.
Immunocytochemistry was performed in the HaEpi to determine its subcellular location. HaAK1 was mainly detected in the cytoplasm in the control group.20E and methoprene had no effect on the location of HaGK.
RNAi experiments revealed that silence of HaEcRB1 had no effect on the expression of HaAK1, suggesting that HaAK1 could not be directly regulated by EcRB1 but maybe other isoforms of EcRB1.
In brief, HaAK1 was upregulated in 5th molting larvae; its expression could be upregulated by 20E in vitro. These results suggest that HaAK1 might participate in insect molting process.
引文
Agui, N., Granger, N. A., Gilbert, L. I., Bollenbacher, W. E.,1979. Cellular localization of the insect prothoracicotropic hormone:In vitro assay of a single neurosecretory cell. Proc Natl Acad Sci U S A.76,5694-5698.
Ahsen, Pfanner,1997. Molecular chaperones:towards a characterization of the heat-shock protein 70 family. Trends Cell Biol.7,129-33.
Anderson, J. M.,1996. Cell signalling:MAGUK magic. Curr Biol.6,382-4.
Andres, A. J., Thummel, C. S.,1992. Hormones, puffs and flies:the molecular control of metamorphosis by ecdysone. Trends Genet.8,132-8.
Arbeitman, M. N., Hogness, D. S.,2000. Molecular chaperones activate the Drosophila ecdysone receptor, an RXR heterodimer. Cell.101,67-77.
Arner, E. S., Eriksson, S.,1995. Mammalian deoxyribonucleoside kinases. Pharmacol Ther. 67,155-86.
Bae, E., Phillips, G. N., Jr.,2006. Roles of static and dynamic domains in stability and catalysis of adenylate kinase. Proc Natl Acad Sci U S A.103,2132-7.
Bayer, C. A., von Kalm, L., Fristrom, J. W.,1997. Relationships between protein isoforms and genetic functions demonstrate functional redundancy at the Broad-Complex during Drosophila metamorphosis. Dev Biol.187,267-82.
Beckage, N. E., Riddiford, L. M.,1982. Effects of parasitism by Apanteles congregatus on the endocrine physiology of the tobacco hornworm Manduca sexta. Gen Comp Endocrinol.47,308-22.
Beckstead, R. B., Lam, G., Thummel, C. S.,2005. The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis. Genome Biol.6, R99.
Benayahu, D.,1997. Estrogen effects on protein expressed by marrow stromal osteoblasts. Biochem Biophys Res Commun.233,30-5.
Bender, M., Imam, F. B., Talbot, W. S., Ganetzky, B., Hogness, D. S.,1997. Drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms. Cell.91,777-88.
Bialecki, M., Shilton, A., Fichtenberg, C., Segraves, W. A., Thummel, C. S.,2002. Loss of the ecdysteroid-inducible E75A orphan nuclear receptor uncouples molting from metamorphosis in Drosophila. Dev Cell.3,209-20.
Billas, I. M., Iwema, T., Gamier, J. M., Mitschler, A., Rochel, N., Moras, D.,2003. Structural adaptability in the ligand-binding pocket of the ecdysone hormone receptor. Nature.426,91-6.
Billas, I. M., Moulinier, L., Rochel, N., Moras, D.,2001. Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid X receptors in insects. J Biol Chem.276, 7465-74.
Bonilha, V. L., Bhattacharya, S. K., West, K. A., Sun, J., Crabb, J. W., Rayborn, M. E., Hollyfield, J. G., 2004. Proteomic characterization of isolated retinal pigment epithelium microvilli. Mol Cell Proteomics.3,1119-27.
Brady, W. A., Kokoris, M. S., Fitzgibbon, M., Black, M. E.,1996. Cloning, characterization, and modeling of mouse and human guanylate kinases. J Biol Chem.271,16734-40.
Brennan, C. A., Li, T. R., Bender, M., Hsiung, F., Moses, K.,2001. Broad-complex, but not ecdysone receptor, is required for progression of the morphogenetic furrow in the Drosophila eye. Development.128,1-11.
Cao, W., Haig-Ladewig, L., Gerton, G. L., Moss, S. B.,2006. Adenylate kinases 1 and 2 are part of the accessory structures in the mouse sperm flagellum. Biol Reprod.75,492-500.
Chen, L., Reece, C., O'Keefe, S. L., Hawryluk, G. W., Engstrom, M. M., Hodgetts, R. B.,2002. Induction of the early-late Ddc gene during Drosophila metamorphosis by the ecdysone receptor. Mech Dev. 114,95-107.
Cho, K. O., Hunt, C. A., Kennedy, M. B.,1992. The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron.9,929-42.
Clayton, G. M., Peak-Chew, S. Y., Evans, R. M., Schwabe, J. W.,2001. The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation. Proc Natl Acad Sci U S A.98,1549-54.
Clemens, J. C., Worby, C. A., Simonson-Leff, N., Muda, M., Maehama, T., Hemmings, B. A., Dixon, J. E., 2000. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci U S A.97,6499-503.
Collavin, L., Lazarevic, D., Utrera, R., Marzinotto, S., Monte, M., Schneider, C.,1999. wt p53 dependent expression of a membrane-associated isoform of adenylate kinase. Oncogene.18,5879-88.
Cronan, J. E., Jr., Godson, G. N.,1972. Mutants of Escherichia coli with temperature-sensitive lesions in membrane phospholipid synthesis:genetic analysis of glycerol-3-phosphate acyltransferase mutants. Mol Gen Genet.116,199-210.
DeFranco, D. B., Ramakrishnan, C., Tang, Y.,1998. Molecular chaperones and subcellular trafficking of steroid receptors. J Steroid Biochem Mol Biol.65,51-8.
DePasquale, J. A., Samsonoff, W. A., Gierthy, J. F.,1994.17-beta-Estradiol induced alterations of cell-matrix and intercellular adhesions in a human mammary carcinoma cell line. J Cell Sci.107 (Pt 5),1241-54.
Devarakonda, S., Harp, J. M., Kim, Y., Ozyhar, A., Rastinejad, F.,2003. Structure of the heterodimeric ecdysone receptor DNA-binding complex. EMBO J.22,5827-40.
Dimitratos, S. D., Woods, D. F., Bryant, P. J.,1997. Camguk, Lin-2, and CASK:novel membrane-associated guanylate kinase homologs that also contain CaM kinase domains. Mech Dev.63,127-30.
Dimitratos, S. D., Woods, D. F., Stathakis, D. G., Bryant, P. J.,1999. Signaling pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family. Bioessays.21,912-21.
Ding, X. Z., Tsokos, G. C., Smallridge, R. C., Kiang, J. G.,1997. Heat shock gene-expression in HSP-70 and HSF1 gene-transfected human epidermoid A-431 cells. Mol Cell Biochem.167,145-52.
Dong, D. J., He, H. J., Chai, L. Q., Jiang, X. J., Wang, J. X., Zhao, X. F.,2007. Identification of genes differentially expressed during larval molting and metamorphosis of Helicoverpa armigera. BMC Dev Biol.7,73.
Duina, A. A., Kalton, H. M., Gaber, R. F.,1998. Requirement for Hsp90 and a CyP-40-type cyclophilin in negative regulation of the heat shock response. J Biol Chem.273,18974-8.
Dunne, J. C., Kondylis, V., Rabouille, C.,2002. Ecdysone triggers the expression of Golgi genes in Drosophila imaginal discs via broad-complex. Dev Biol.245,172-86.
Dzeja, P., Kalvenas, A., Toleikis, A., Praskevicius, A.,1985. The effect of adenylate kinase activity on the rate and efficiency of energy transport from mitochondria to hexokinase. Biochem Int.10,259-65.
Dzeja, P. P., Bortolon, R., Perez-Terzic, C., Holmuhamedov, E. L., Terzic, A.,2002. Energetic communication between mitochondria and nucleus directed by catalyzed phosphotransfer. Proc Natl Acad Sci U S A.99,10156-61.
Dzeja, P. P., Zeleznikar, R. J., Goldberg, N. D.,1998. Adenylate kinase:kinetic behavior in intact cells indicates it is integral to multiple cellular processes. Mol Cell Biochem.184,169-82.
Elbashir, S. M., Lendeckel, W., Tuschl, T.,2001. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev.15,188-200.
Eystathioy, T., Swevers, L., Iatrou, K.,2001. The orphan nuclear receptor BmHR3A of Bombyx mori: hormonal control, ovarian expression and functional properties. Mech Dev.103,107-15.
Fang, F., Xu, Y, Jones, D., Jones, G.,2005. Interactions of ultraspiracle with ecdysone receptor in the transduction of ecdysone-and juvenile hormone-signaling. FEBS J.272,1577-89.
Fernandez-Gonzalez, A., Kourembanas, S., Wyatt, T. A., Mitsialis, S. A.,2009. Mutation of murine adenylate kinase 7 underlies a primary ciliary dyskinesia phenotype. Am J Respir Cell Mol Biol. 40,305-13.
Fink, A. L.,1999. Chaperone-mediated protein folding. Physiol Rev.79,425-49.
Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., Ahringer, J.,2000. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature. 408,325-30.
Fujisawa, K., Murakami, R., Horiguchi, T., Noma, T.,2009. Adenylate kinase isozyme 2 is essential for growth and development of Drosophila melanogaster. Comp Biochem Physiol B Biochem Mol Biol.
Fujiwara, H., Jindra, M., Newitt, R., Palli, S. R., Hiruma, K., Riddiford, L. M.,1995. Cloning of an ecdysone receptor homolog from Manduca sexta and the developmental profile of its mRNA in wings. Insect Biochem Mol Biol.25,845-56.
Gaidarov, I. O., Suslov, O. N., Abdulaev, N. G,1993. Enzymes of the cyclic GMP metabolism in bovine retina. I. Cloning and expression of the gene for guanylate kinase. FEBS Lett.335,81-4.
Gentry, D., Bengra, C, Ikehara, K., Cashel, M.,1993. Guanylate kinase of Escherichia coli K-12. J Biol Chem.268,14316-21.
Gilbert, L. I., Granger, N. A., Roe, R. M.,2000. The juvenile hormones:historical facts and speculations on future research directions. Insect Biochem Mol Biol.30,617-44.
Ginger, M. L., Ngazoa, E. S., Pereira, C. A., Pullen, T.J., Kabiri, M., Becker, K., Gull, K., Steverding, D., 2005. Intracellular positioning of isoforms explains an unusually large adenylate kinase gene family in the parasite Trypanosoma brucei. J Biol Chem.280,11781-9.
Grace, T. D.,1962. Establishment of four strains of cells from insect tissues grown in vitro. Nature.195, 788-9.
Grebe, M., Przibilla, S., Henrich, V. C., Spindler-Barth, M.,2003. Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster. Biol Chem.384,105-16.
Grenert, J. P., Johnson, B. D., Toft, D. O.,1999. The importance of ATP binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes. J Biol Chem.274,17525-33.
Grenert, J. P., Sullivan, W. P., Fadden, P., Haystead, T. A., Clark, J., Mimnaugh, E., Krutzsch, H., Ochel, H. J., Schulte, T. W., Sausville, E., Neckers, L. M., Toft, D. O.,1997. The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation. J Biol Chem.272,23843-50.
Hall, B. L., Thummel, C. S.,1998. The RXR homolog ultraspiracle is an essential component of the Drosophila ecdysone receptor. Development.125,4709-17.
Hannon, G. J.,2002. RNA interference. Nature.418,244-51.
Hartl, F. U.,1996. Molecular chaperones in cellular protein folding. Nature.381,571-9.
Hayward, D. C., Dhadialla, T. S., Zhou, S., Kuiper, M. J., Ball, E. E., Wyatt, G. R., Walker, V. K.,2003. Ligand specificity and developmental expression of RXR and ecdysone receptor in the migratory locust. J Insect Physiol.49,1135-44.
He, H. J., Wang, Q., Zheng, W. W., Wang, J. X., Song, Q. S., Zhao, X. F., Function of nuclear transport factor 2 and Ran in the 20E signal transduction pathway in the cotton bollworm, Helicoverpa armigera. BMC Cell Biol.11,1.
Henrich, V. C.,2005. The ecdysteroid receptor. In "Comprehensive Molecular Insect Science". Eds L.I. Gilbert, K. Iatrou and S.S. Gill, vol.3, pp.243-285, Elsevier, Oxford.
Henrich, V. C., Rybczynski, R., Gilbert, L. I.,1999. Peptide hormones, steroid hormones, and puffs: mechanisms and models in insect development. Vitam Horm.55,73-125.
Henrich, V. C., Sliter, T. J., Lubahn, D. B., Maclntyre, A., Gilbert, L. I.,1990. A steroid/thyroid hormone receptor superfamily member in Drosophila melanogaster that shares extensive sequence similarity with a mammalian homologue. Nucleic Acids Res.18,4143-8.
Henrich, V. C., Szekely, A. A., Kim, S. J., Brown, N. E., Antoniewski, C., Hayden, M. A., Lepesant, J. A., Gilbert, L. I.,1994. Expression and function of the ultraspiracle (usp) gene during development of Drosophila melanogaster. Dev Biol.165,38-52.
Hiruma, K., Bocking, D., Lafont, R., Riddiford, L. M.,1997. Action of different ecdysteroids on the regulation of mRNAs for the ecdysone receptor, MHR3, dopa decarboxylase, and a larval cuticle protein in the larval epidermis of the tobacco hornworm, Manduca sexta. Gen Comp Endocrinol. 107,84-97.
Hiruma, K., Carter, M. S., Riddiford, L. M.,1995. Characterization of the dopa decarboxylase gene of Manduca sexta and its suppression by 20-hydroxyecdysone. Dev Biol.169,195-209.
Hiruma, K., Riddiford, L. M.,1984. Regulation of melanization of tobacco hornworm larval cuticle in vitro. J Exp Zool.230,393-403.
Hiruma, K., Riddiford, L. M.,1990. Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone. Dev Biol.138, 214-24.
Hiruma, K., Riddiford, L. M.,2001. Regulation of transcription factors MHR4 and betaFTZ-F1 by 20-hydroxyecdysone during a larval molt in the tobacco hornworm, Manduca sexta. Dev Biol.232, 265-74.
Hiruma, K., Riddiford, L. M.,2004. Differential control of MHR3 promoter activity by isoforms of the ecdysone receptor and inhibitory effects of E75A and MHR3. Dev Biol.272,510-21.
Hiruma, K., Riddiford, L. M., Hopkins, T. L., Morgan, T. D.,1985. Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco homworm and their control by 20-hydroxyecdysone. J Comp Physiol B.155,659-69.
Hiruma, K., Shinoda, T., Malone, F., Riddiford, L. M.,1999. Juvenile hormone modulates 20-hydroxyecdysone-inducible ecdysone receptor and ultraspiracle gene expression in the tobacco hornworm, Manduca sexta. Dev Genes Evol.209,18-30.
Horodyski, F. M., Ewer, J., Riddiford, L. M., Truman, J. W.,1993. Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster. Eur J Biochem.215,221-8.
Hoskins, R., Hajnal, A. F., Harp, S. A., Kim, S. K.,1996. The C. elegans vulval induction gene lin-2 encodes a member of the MAGUK family of cell junction proteins. Development.122,97-111.
Janssen, E., de Groof, A., Wijers, M., Fransen, J., Dzeja, P. P., Terzic, A., Wieringa, B.,2003. Adenylate kinase 1 deficiency induces molecular and structural adaptations to support muscle energy metabolism. J Biol Chem.278,12937-45.
Janssen, E., Dzeja, P. P., Oerlemans, F., Simonetti, A. W., Heerschap, A., de Haan, A., Rush, P. S., Terjung, R. R., Wieringa, B., Terzic, A.,2000. Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement. EMBO J.19,6371-81.
Janssen, E., Kuiper, J., Hodgson, D., Zingman, L. V., Alekseev, A. E., Terzic, A., Wieringa, B.,2004. Two structurally distinct and spatially compartmentalized adenylate kinases are expressed from the AK1 gene in mouse brain. Mol Cell Biochem.256-257,59-72.
Jibard, N., Meng, X., Leclerc, P., Rajkowski, K., Fortin, D., Schweizer-Groyer, G., Catelli, M. G., Baulieu, E. E., Cadepond, F.,1999. Delimitation of two regions in the 90-kDa heat shock protein (Hsp90) able to interact with the glucocorticosteroid receptor (GR). Exp Cell Res.247,461-74.
Jindra, M., Huang, J. Y., Malone, F., Asahina, M., Riddiford, L. M.,1997. Identification and mRNA developmental profiles of two ultraspiracle isoforms in the epidermis and wings of Manduca sexta. Insect Mol Biol.6,41-53.
Jindra, M., Malone, F., Hiruma, K., Riddiford, L. M.,1996. Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm, Manduca sexta. Dev Biol.180,258-72.
Johnson, D. E., Brookhart, G. L., Kramer, K. J., Barnett, B. D., McGaughey, W. H.,1990. Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella:comparison of midgut proteinases from susceptible and resistant larvae. J Invertebr Pathol.55,235-44.
Kamimura, M., Tomita, S., Kiuchi, M., Fujiwara, H.,1997. Tissue-specific and stage-specific expression of two silkworm ecdysone receptor isoforms - ecdysteroid-dependent transcription in cultured anterior silk glands. Eur J Biochem.248,786-93.
Kataoka, H., Nagasawa, H., Isogai, A., Ishizaki, H., Suzuki, A.,1991. Prothoracicotropic hormone of the silkworm, Bombyx mori:amino acid sequence and dimeric structure. Agric Biol Chem.55,73-86.
Keshan, B., Hiruma, K., Riddiford, L. M.,2006. Developmental expression and hormonal regulation of different isoforms of the transcription factor E75 in the tobacco hornworm Manduca sexta. Dev Biol.295,623-32.
Khoo, J. C., Russell, P. J.,1972. Isoenzymes of adenylate kinase in human tissue. Biochim Biophys Acta. 268,98-101.
Kiang, J. G., Tsokos, G. C.,1998. Heat shock protein 70 kDa:molecular biology, biochemistry, and physiology. Pharmacol Then. 80,183-201.
Kim, S. K.,1995. Tight junctions, membrane-associated guanylate kinases and cell signaling. Curr Opin Cell Biol.7,641-9.
Koelle, M. R., Segraves, W. A., Hogness, D. S.,1992. DHR3:a Drosophila steroid receptor homolog. Proc Natl Acad Sci U S A.89,6167-71.
Koelle, M. R., Talbot, W. S., Segraves, W. A., Bender, M. T., Cherbas, P., Hogness, D. S.,1991. The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell.67,59-77.
Konrad, M.,1988. Analysis and in vivo disruption of the gene coding for adenylate kinase (ADK1) in the yeast Saccharomyces cerevisiae. J Biol Chem.263,19468-74.
Konrad, M.,1992. Cloning and expression of the essential gene for guanylate kinase from yeast. J Biol Chem.267,25652-5.
Konrad, M.,1993. Molecular analysis of the essential gene for adenylate kinase from the fission yeast Schizosaccharomyces pombe. J Biol Chem.268,11326-34.
Kosano, H., Stensgard, B., Charlesworth, M. C., McMahon, N., Toft, D.,1998. The assembly of progesterone receptor-hsp90 complexes using purified proteins. J Biol Chem.273,32973-9.
Kuhlendahl, S., Spangenberg, O., Konrad, M., Kim, E., Garner, C. C.,1998. Functional analysis of the guanylate kinase-like domain in the synapse-associated protein SAP97. Eur J Biochem.252, 305-13.
Kumar, V., Spangenberg, O., Konrad, M.,2000. Cloning of the guanylate kinase homologues AGK-1 and AGK-2 from Arabidopsis thaliana and characterization of AGK-1. Eur J Biochem.267,606-15.
Lagunas, L., Bradbury, C. M., Laszlo, A., Hunt, C. R., Gius, D.,2004. Indomethacin and ibuprofen induce Hsc70 nuclear localization and activation of the heat shock response in HeLa cells. Biochem Biophys Res Commun.313,863-70.
Lan, Q., Hiruma, K., Hu, X., Jindra, M., Riddiford, L. M.,1999. Activation of a delayed-early gene encoding MHR3 by the ecdysone receptor heterodimer EcR-B1-USP-1 but not by EcR-B1-USP-2. Mol Cell Biol.19,4897-906.
Lan, Q., Riddiford, L. M.,1997. DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta. In Vitro Cell Dev Biol Anim.33,615-21.
Lange, S., Auerbach, D., McLoughlin, P., Perriard, E., Schafer, B. W., Perriard, J. C., Ehler, E.,2002. Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2. J Cell Sci.115,4925-36.
Langelan, R. E., Fisher, J. E., Hiruma, K., Palli, S. R., Riddiford, L. M.,2000. Patterns of MHR3 expression in the epidermis during a larval molt of the tobacco hornworm Manduca sexta. Dev Biol.227,481-94.
Lavie, A., Konrad, M., Brundiers, R., Goody, R. S., Schlichting, I., Reinstein, J.,1998. Crystal structure of yeast thymidylate kinase complexed with the bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-thymidyl) pentaphosphate (TP5A) at 2.0 A resolution:implications for catalysis and AZT activation. Biochemistry.37,3677-86.
Lezzi, M., Bergman, T., Henrich, V. C., Vogtli, M., Fromel, C., Grebe, M., Przibilla, S., Spindler-Barth, M., 2002. Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle. Eur J Biochem.269,3237-45.
Liu, E., Restifo, L. L.,1998. Identification of a broad complex-regulated enhancer in the developing visual system of Drosophila. J Neurobiol.34,253-70.
Liu, J., Shi, G. P., Zhang, W. Q., Zhang, G. R., Xu, W. H.,2006. Cathepsin L function in insect moulting: molecular cloning and functional analysis in cotton bollworm, Helicoverpa armigera. Insect Mol Biol.15,823-34.
Liu, R., Strom, A. L., Zhai, J., Gal, J., Bao, S., Gong, W., Zhu, H.,2009. Enzymatically inactive adenylate kinase 4 interacts with mitochondrial ADP/ATP translocase. Int J Biochem Cell Biol.41,1371-80.
Lonergan, K. M., Chari, R., Deleeuw, R. J., Shadeo, A., Chi, B., Tsao, M. S., Jones, S., Marra, M., Ling, V, Ng, R., Macaulay, C., Lam, S., Lam, W. L.,2006. Identification of novel lung genes in bronchial epithelium by serial analysis of gene expression. Am J Respir Cell Mol Biol.35,651-61.
Lynn, D. E., Miller, S. G., Oberlander, H.,1982. Establishment of a cell line from lepidopteran wing imaginal discs:Induction of newly synthesized proteins by 20-hydroxyecdysone. Proc Natl Acad Sci U S A.79,2589-2593.
Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., Evans, R. M.,1995. The nuclear receptor superfamily:the second decade. Cell.83,835-9.
Miech, R. P., Parks, R. E., Jr.,1965. Adenosine Triphosphate:Guanosine Monophosphate Phosphotransferase. Partial Purification and Substrate Specificity. J Biol Chem.240,351-7.
Minakuchi, C., Nakagawa, Y., Kiuchi, M., Tomita, S., Kamimura, M.,2002. Molecular cloning, expression analysis and functional confirmation of two ecdysone receptor isoforms from the rice stem borer Chilo suppressalis. Insect Biochem Mol Biol.32,999-1008.
Montpied, P., Sobrier, M. L., Chapel, S., Couderc, J. L., Dastugue, B.,1988.20-Hydroxyecdysone induces the expression of one beta-tubulin gene in Drosophila Kc cells. Biochim Biophys Acta.949, 79-86.
Mottier, V., Siaussat, D., Bozzolan, F., Auzoux-Bordenave, S., Porcheron, P., Dcbernard, S.,2004. The 20-hydroxyecdysone-induced cellular arrest in G2 phase is preceded by an inhibition of cyclin expression. Insect Biochem Mol Biol.34,51-60.
Mugat, B., Brodu, V., Kejzlarova-Lepesant, J., Antoniewski, C., Bayer, C. A., Fristrom, J. W., Lepesant, J. A.,2000. Dynamic expression of broad-complex isoforms mediates temporal control of an ecdysteroid target gene at the onset of Drosophila metamorphosis. Dev Biol.227,104-17.
Nijhout, H. F.,1984. Colour pattern modification by coldshock in Lepidoptera. J Embryol Exp Morphol.81, 287-305.
Nijhout, H. F.,2003. The control of body size in insects. Dev Biol.261,1-9.
Nijhout, H. F., Sheffield, H. G.,1979. Antennal hair erection in male mosquitoes:a new mechanical effector in insects. Science.206,595-6.
Nishita, Y., Takiya, S.,2004. Structure and expression of the gene encoding a Broad-Complex homolog in the silkworm, Bombyx mori. Gene.339,161-72.
Noma, T.,2005. Dynamics of nucleotide metabolism as a supporter of life phenomena. J Med Invest.52, 127-36.
Oberlander, H., Leach, C. E., Shaaya, E.,2000. Juvenile hormone and juvenile hormone mimics inhibit proliferation in a lepidopteran imaginal disc cell line. J Insect Physiol.46,259-265.
Obermann, W. M., Sondermann, H., Russo, A. A., Pavletich, N. P., Hartl, F. U.,1998. In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis. J Cell Biol.143,901-10.
Okajima, T., Fukamizo, T., Goto, S., Fukui, T., Tanizawa, K.,1998. Exchange of nucleoside monophosphate-binding domains in adenylate kinase and UMP/CMP kinase. J Biochem.124, 359-67.
Oro, A. E., McKeown, M., Evans, R. M.,1992. The Drosophila retinoid X receptor homolog ultraspiracle functions in both female reproduction and eye morphogenesis. Development.115,449-62.
Ote, M., Mita, K., Kawasaki, H., Daimon, T., Kobayashi, M., Shimada, T.,2005. Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori. Comp Biochem Physiol B Biochem Mol Biol.141,314-22.
Palli, S. R., Caputo, G. F., Sohi, S. S., Brownwright, A. J., Ladd, T. R., Cook, B. J., Primavera, M., Arif, B. M., Retnakaran, A.,1996. CfMNPV blocks AcMNPV-induced apoptosis in a continuous midgut cell line. Virology.222,201-13.
Palli, S. R., Hiruma, K., Riddiford, L. M.,1992. An ecdysteroid-inducible Manduca gene similar to the Drosophila DHR3 gene, a member of the steroid hormone receptor superfamily. Dev Biol.150, 306-18.
Palli, S. R., Ladd, T. R., Ricci, A. R., Sohi, S. S., Retnakaran, A.,1997. Cloning and development expression of Choristoneura hormone receptor 75:a homologue of the Drosophila E75A gene. Dev Genet.20,36-46.
Panaretou, B., Prodromou, C., Roe, S. M., O'Brien, R., Ladbury, J. E., Piper, P. W., Pearl, L. H.,1998. ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo. EMBO J.17,4829-36.
Perrimon, N., Engstrom, L., Mahowald, A. P.,1985. Developmental genetics of the 2C-D region of the Drosophila X chromosome. Genetics. 111,23-41.
Prapapanich, V., Chen, S., Smith, D. F.,1998. Mutation of Hip's carboxy-terminal region inhibits a transitional stage of progesterone receptor assembly. Mol Cell Biol.18,944-52.
Prapapanich, V., Chen, S., Toran, E. J., Rimerman, R. A., Smith, D. F.,1996. Mutational analysis of the hsp70-interacting protein Hip. Mol Cell Biol.16,6200-7.
Pucar, D., Janssen, E., Dzeja, P. P., Juranic, N., Macura, S., Wieringa, B., Terzic, A.,2000. Compromised energetics in the adenylate kinase AK1 gene knockout heart under metabolic stress. J Biol Chem. 275,41424-9.
Qualtieri, A., Pedace, V., Bisconte, M. G., Bria, M., Gulino, B., Andreoli, V., Brancati, C.,1997. Severe erythrocyte adenylate kinase deficiency due to homozygous A-->G substitution at codon 164 of human AK1 gene associated with chronic haemolytic anaemia. Br J Haematol.99,770-6.
Ren, H., Wang, L., Bennett, M., Liang, Y., Zheng, X., Lu, F., Li, L., Nan, J., Luo, M., Eriksson, S., Zhang, C., Su, X. D.,2005. The crystal structure of human adenylate kinase 6:An adenylate kinase localized to the cell nucleus. Proc Natl Acad Sci U S A.102,303-8.
Rewitz, K. F., Yamanaka, N., Gilbert, L. I., O'Connor, M. B.,2009. The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis. Science.326,1403-5.
Riddiford, L. M.,1993. Hormone receptors and the regulation of insect metamorphosis. Receptor.3,203-9.
Riddiford, L. M.,2008. Juvenile hormone action:a 2007 perspective. J Insect Physiol.54,895-901.
Riddiford, L. M., Hiruma, K., Zhou, X., Nelson, C. A.,2003. Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster. Insect Biochem Mol Biol.33,1327-38.
Rutherford, S. L., Lindquist, S.,1998. Hsp90 as a capacitor for morphological evolution. Nature.396, 336-42.
Rybczynski, R., Bell, S. C., Gilbert, L. I.,2001. Activation of an extracellular signal-regulated kinase (ERK) by the insect prothoracicotropic hormone. Mol Cell Endocrinol.184,1-11.
Rybczynski, R., Gilbert, L. I.,2000. cDNA cloning and expression of a hormone-regulated heat shock protein (hsc 70) from the prothoracic gland of Manduca sexta. Insect Biochem Mol Biol.30, 579-89.
Samuels, R. I., Charnley, A. K., Reynolds, S. E.,1993. A cuticle-degrading proteinase from the moulting fluid of the tobacco hornworm, Manduca sexta. Insect Biochem Mol Biol.23,607-14.
Scherrer, L. C., Hutchison, K. A., Sanchez, E. R., Randall, S. K., Pratt, W. B.,1992. A heat shock protein complex isolated from rabbit reticulocyte lysate can reconstitute a functional glucocorticoid receptor-Hsp90 complex. Biochemistry.31,7325-9.
Schett, G., Steiner, C. W., Groger, M., Winkler, S., Graninger, W., Smolen, J., Xu, Q., Steiner, G.,1999. Activation of Fas inhibits heat-induced activation of HSF1 and up-regulation of hsp70. FASEB J. 13,833-42.
Schneider, I.,1972. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol.27,353-65.
Schubiger, M., Truman, J. W.,2000. The RXR ortholog USP suppresses early metamorphic processes in Drosophila in the absence of ecdysteroids. Development.127,1151-9.
Schubiger, M., Wade, A. A., Carney, G. E., Truman, J. W., Bender, M.,1998. Drosophila EcR-B ecdysone receptor isoforms are required for larval molting and for neuron remodeling during metamorphosis. Development.125,2053-62.
Schulz, G. E., Schiltz, E., Tomasselli, A. G., Frank, R., Brune, M., Wittinghofer, A., Schirmer, R. H.,1986. Structural relationships in the adenylate kinase family. Eur J Biochem.161,127-32.
Segraves, W. A., Hogness, D. S.,1990. The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily. Genes Dev.4, 204-19.
Segraves, W. A., Woldin, C.,1993. The E75 gene of Manduca sexta and comparison with its Drosophila homolog. Insect Biochem Mol Biol.23,91-7.
Shaknovich, R., Shue, G., Kohtz, D. S.,1992. Conformational activation of a basic helix-loop-helix protein (MyoD1) by the C-terminal region of murine HSP90 (HSP84). Mol Cell Biol.12,5059-68.
Shao, H. L., Zheng, W. W., Liu, P. C., Wang, Q., Wang, J. X., Zhao, X. F.,2008. Establishment of a new cell line from lepidopteran epidermis and hormonal regulation on the genes. PLoS One.3, e3127.
Sharp, P. A.,1999. RNAi and double-strand RNA. Genes Dev.13,139-41.
Siaussat, D., Bozzolan, F., Porcheron, P., Debernard, S.,2007. Identification of steroid hormone signaling pathway in insect cell differentiation. Cell Mol Life Sci.64,365-76.
Siaussat, D., Bozzolan, F., Queguiner, I., Porcheron, P., Debernard, S.,2004a. Effects of juvenile hormone on 20-hydroxyecdysone-inducible EcR, HR3, E75 gene expression in imaginal wing cells of Plodia interpunctella lepidoptera. Eur J Biochem.271,3017-27.
Siaussat, D., Mottier, V., Bozzolan, F., Porcheron, P., Debernard, S.,2004b. Synchronization of Plodia interpunctella lepidopteran cells and effects of 20-hydroxyecdysone. Insect Mol Biol.13,179-87.
Smith, D. F., Toft, D. O.,1993. Steroid receptors and their associated proteins. Mol Endocrinol.7,4-11.
Smith, W. A., Gilbert, L. I.,1989. Early events in peptide-stimulated ecdysteroid secretion by the prothoracic glands of Manduca sexta. J Exp Zool.252,264-70.
Sobrier, M. L., Couderc, J. L., Chapel, S., Dastugue, B.,1986. Expression of a new beta tubulin subunit is induced by 20-hydroxyecdysone in Drosophila cultured cells. Biochem Biophys Res Commun. 134,191-200.
Somma, M. P., Fasulo, B., Cenci, G., Cundari, E., Gatti, M.,2002. Molecular dissection of cytokincsis by RNA interference in Drosophila cultured cells. Mol Biol Cell.13,2448-60.
Spindler, K.-D., Betanska, K, Nieva, C., Gwozdz, T. Dutko-Gwozdz, Ozyhar, A. and Spindler-Barth, M., 2009. Intracellular localization of the ecdysteroid receptor. In "Ecdysone:Structures and Functions", Ed. G. Smagghe, pp.389-409. Springer, Berlin
Stanojevic, V., Habener, J. F., Holz, G. G., Leech, C. A.,2008. Cytosolic adenylate kinases regulate K-ATP channel activity in human beta-cells. Biochem Biophys Res Commun.368,614-9.
Stehle, T., Schulz, G. E.,1992. Refined structure of the complex between guanylate kinase and its substrate GMP at 2.0 A resolution. J Mol Biol.224,1127-41.
Stephanou, A., Isenberg, D. A., Nakajima, K., Latchman, D. S.,1999. Signal transducer and activator of transcription-1 and heat shock factor-1 interact and activate the transcription of the Hsp-70 and Hsp-90beta gene promoters. J Biol Chem.274,1723-8.
Stilwell, G. E., Nelson, C. A., Weller, J., Cui, H., Hiruma, K., Truman, J. W., Riddiford, L. M.,2003. E74 exhibits stage-specific hormonal regulation in the epidermis of the tobacco hornworm, manduca sexta. Dev Biol.258,76-90.
Sudeep, A. B., Shouche, Y. S., Mourya, D. T., Pant, U.,2002. New Helicoverpa armigera Hbn cell line from larval hemocyte for baculovirus studies. Indian J Exp Biol.40,69-73.
Sui, Y. P., Liu, X. B., Chai, L. Q., Wang, J. X., Zhao, X. F.,2009. Characterization and influences of classical insect hormones on the expression profiles of a molting carboxypeptidase A from the cotton bollworm (Helicoverpa armigera). Insect Mol Biol.18,353-63.
Sun, G. C., Hirose, S., Ueda, H.,1994. Intermittent expression of BmFTZ-F1, a member of the nuclear hormone receptor superfamily during development of the silkworm Bombyx mori. Dev Biol.162, 426-37.
Sutherland, J. D., Kozlova, T., Tzertzinis, G., Kafatos, F. C.,1995. Drosophila hormone receptor 38:a second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type. Proc Natl Acad Sci U S A.92,7966-70.
Swevers, L., Cherbas, L., Cherbas, P., Iatrou, K.,1996. Bombyx EcR (BmEcR) and Bombyx USP (BmCF1) combine to form a functional ecdysone receptor. Insect Biochem Mol Biol.26,217-21.
Swevers, L., Eystathioy, T., Iatrou, K.,2002. The orphan nuclear receptors BmE75A and BmE75C of the silkmoth Bombyx mori:hornmonal control and ovarian expression. Insect Biochem Mol Biol.32, 1643-52.
Talbot, W. S., Swyryd, E. A., Hogness, D. S.,1993. Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell.73,1323-37.
Tan, A., Palli, S. R.,2008. Ecdysone [corrected] receptor isoforms play distinct roles in controlling molting and metamorphosis in the red flour beetle, Tribolium castaneum. Mol Cell Endocrinol.291,42-9.
Tanabe, T., Yamada, M., Noma, T., Kajii, T., Nakazawa, A.,1993. Tissue-specific and developmentally regulated expression of the genes encoding adenylate kinase isozymes. J Biochem.113,200-7.
Terashima, J., Bownes, M.,2006. E75A and E75B have opposite effects on the apoptosis/development choice of the Drosophila egg chamber. Cell Death Differ.13,454-64.
Thummel, C. S.,1995. From embryogenesis to metamorphosis:the regulation and function of Drosophila nuclear receptor superfamily members. Cell.83,871-7.
Thummel, C. S.,2001. Steroid-triggered death by autophagy. Bioessays.23,677-82.
Tomasselli, A. G., Noda, L. H.,1979. Mitochondrial GTP-AMP phosphotransferase.2. Kinetic and equilibrium dialysis studies. Eur J Biochem.93,263-7.
Tomasselli, A. G, Schirmer, R. H., Noda, L. H.,1979. Mitochondrial GTP-AMP phosphotransferase.1. Purification and properties. Eur J Biochem.93,257-62.
Truman, J. W., Riddiford, L. M.,1974. Physiology of insect rhythms.3. The temporal organization of the endocrine events underlying pupation of the tobacco hornworm. J Exp Biol.60,371-82.
Truman, J. W., Sokolove, P. G.,1972. Silk Moth Eclosion:Hormonal Triggering of a Centrally Programmed Pattern of Behavior. Science.175,1491-1493.
Truss, M., Beato, M.,1993. Steroid hormone receptors:interaction with deoxyribonucleic acid and transcription factors. Endocr Rev.14,459-79.
Uhlirova, M., Foy, B. D., Beaty, B. J., Olson, K. E., Riddiford, L. M., Jindra, M.,2003. Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis. Proc Natl Acad Sci U S A.100,15607-12.
van Horssen, R., Janssen, E., Peters, W., van de Pasch, L., Lindert, M. M., van Dommelen, M. M., Linssen, P. C., Hagen, T. L., Fransen, J. A., Wieringa, B.,2009. Modulation of cell motility by spatial repositioning of enzymatic ATP/ADP exchange capacity. J Biol Chem.284,1620-7.
Vasseur, S., Malicet, C., Calvo, E. L., Dagorn, J. C., Iovanna, J. L.,2005. Gene expression profiling of tumours derived from rasV12/E1A-transformed mouse embryonic fibroblasts to identify genes required for tumour development. Mol Cancer.4,4.
Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., McCawley, P.,1977. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro.13,213-7.
Velazquez, J. M., Lindquist, S.,1984. hsp70:nuclear concentration during environmental stress and cytoplasmic storage during recovery. Cell.36,655-62.
Wagner, G. P., Chiu, C. H., Hansen, T. F.,1999. Is Hsp90 a regulator of evolvability? J Exp Zool.285, 116-8.
Wang, J. L., Jiang, X. J., Wang, Q., Hou, L. J., Xu, D. W., Wang, J. X., Zhao, X. F.,2007. Identification and expression profile of a putative basement membrane protein gene in the midgut of Helicoverpa armigera. BMC Dev Biol.7,76.
Wang, J. L., Zhang, Y. P., Gu, Y. Y., Wang, J. X., Zhao, X. F.,2009. Function of a TGF-beta inducible nuclear protein in the silk gland in Bombyx mori. Insect Mol Biol.18,243-51.
Wei, Z. J., Zhang, Q. R., Kang, L., Xu, W. H., Denlinger, D. L.,2005. Molecular characterization and expression of prothoracicotropic hormone during development and pupal diapause in the cotton bollworm, Helicoverpa armigera. J Insect Physiol.51,691-700.
Weller, J., Sun, G. C., Zhou, B., Lan, Q., Hiruma, K., Riddiford, L. M.,2001. Isolation and developmental expression of two nuclear receptors, MHR4 and betaFTZ-F1, in the tobacco hornworm, Manduca sexta. Insect Biochem Mol Biol.31,827-37.
Wild, K., Grafmuller, R., Wagner, E., Schulz, G. E.,1997. Structure, catalysis and supramolecular assembly of adenylate kinase from maize. Eur J Biochem.250,326-31.
Wilson, R., Ainscough, R., Anderson, K., Baynes, C., Berks, M., Bonfield, J., Burton, J., Connell, M., Copsey, T., Cooper, J., et al.,1994.2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature.368,32-8.
Woodard, C. T., Baehrecke, E. H., Thummel, C. S.,1994. A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone. Cell.79,607-15.
Woods, D. F., Bryant, P. J.,1991. The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell.66,451-64.
Xu, L., Glass, C. K., Rosenfeld, M. G.,1999. Coactivator and corepressor complexes in nuclear receptor function. Curr Opin Genet Dev.9,140-7.
Yao, T. P., Forman, B. M., Jiang, Z., Cherbas, L., Chen, J. D., McKeown, M., Cherbas, P., Evans, R. M., 1993. Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature.366, 476-9.
Yao, T. P., Segraves, W. A., Oro, A. E., McKeown, M., Evans, R. M.,1992. Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. Cell.71,63-72.
Yussa, M., Lohr, U., Su, K., Pick. L.,2001. The nuclear receptor Ftz-F1 and homeodomain protein Ftz interact through evolutionarily conserved protein domains. Mech Dev.107,39-53.
Zelhof, A. C., Yao, T. P., Evans, R. M., McKeown, M.,1995. Identification and characterization of a Drosophila nuclear receptor with the ability to inhibit the ecdysone response. Proc Natl Acad Sci U S A.92,10477-81.
Zhai, R., Meng, G, Zhao, Y, Liu, B., Zhang, G., Zheng, X.,2006. A novel nuclear-localized protein with special adenylate kinase properties from Caenorhabditis elegans. FEBS Lett.580,3811-7.
Zhao, X. F., Wang, J. X., Xu, X. L., Li, Z. M., Kang, C. J.,2004. Molecular cloning and expression patterns of the molt-regulating transcription factor HHR3 from Helicoverpa armigera. Insect Mol Biol.13, 407-12.
Zhao, X. F., Wang, J. X., Xu, X. L., Schmid, R., Wieczorek, H.,2002. Molecular cloning and characterization of the cathepsin B-like proteinase from the cotton boll worm, Helicoverpa armigera. Insect Mol Biol.11,567-75.
Zheng, W. W., Yang, D. T., Wang, J. X., Song, Q. S., Gilbert, L. I., Zhao, X. F., Hsc70 binds to ultraspiracle resulting in the upregulation of 20-hydroxyecdsone-responsive genes in Helicoverpa armigera. Mol Cell Endocrinol.315,282-91.
Zhou, B., Hiruma, K., Jindra, M., Shinoda, T., Segraves, W. A., Malone, F., Riddiford, L. M.,1998a. Regulation of the transcription factor E75 by 20-hydroxyecdysone and juvenile hormone in the epidermis of the tobacco hornworm, Manduca sexta, during larval molting and metamorphosis. Dev Biol.193,127-38.
Zhou, B., Hiruma, K., Shinoda, T., Riddiford, L. M.,1998b. Juvenile hormone prevents ecdysteroid-induced expression of broad complex RNAs in the epidermis of the tobacco hornworm, Manduca sexta. Dev Biol.203,233-44.
Zou, J., Guo, Y, Guettouche, T., Smith, D. F., Voellmy, R.,1998. Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell.94,471-80.
Ahsen, Pfanner,1997. Molecular chaperones:towards a characterization of the heat-shock protein 70 family. Trends Cell Biol.7,129-33.
Anderson, J. M.,1996. Cell signalling:MAGUK magic. Curr Biol.6,382-4.
Andres, A. J., Thummel, C. S.,1992. Hormones, puffs and flies:the molecular control of metamorphosis by ecdysone. Trends Genet.8,132-8.
Arbeitman, M. N., Hogness, D. S.,2000. Molecular chaperones activate the Drosophila ecdysone receptor, an RXR heterodimer. Cell.101,67-77.
Arner, E. S., Eriksson, S.,1995. Mammalian deoxyribonucleoside kinases. Pharmacol Ther. 67,155-86.
Bae, E., Phillips, G. N., Jr.,2006. Roles of static and dynamic domains in stability and catalysis of adenylate kinase. Proc Natl Acad Sci U S A.103,2132-7.
Bayer, C. A., von Kalm, L., Fristrom, J. W.,1997. Relationships between protein isoforms and genetic functions demonstrate functional redundancy at the Broad-Complex during Drosophila metamorphosis. Dev Biol.187,267-82.
Beckage, N. E., Riddiford, L. M.,1982. Effects of parasitism by Apanteles congregatus on the endocrine physiology of the tobacco hornworm Manduca sexta. Gen Comp Endocrinol.47,308-22.
Beckstead, R. B., Lam, G., Thummel, C. S.,2005. The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis. Genome Biol.6, R99.
Benayahu, D.,1997. Estrogen effects on protein expressed by marrow stromal osteoblasts. Biochem Biophys Res Commun.233,30-5.
Bender, M., Imam, F. B., Talbot, W. S., Ganetzky, B., Hogness, D. S.,1997. Drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms. Cell.91,777-88.
Bialecki, M., Shilton, A., Fichtenberg, C., Segraves, W. A., Thummel, C. S.,2002. Loss of the ecdysteroid-inducible E75A orphan nuclear receptor uncouples molting from metamorphosis in Drosophila. Dev Cell.3,209-20.
Billas, I. M., Iwema, T., Gamier, J. M., Mitschler, A., Rochel, N., Moras, D.,2003. Structural adaptability in the ligand-binding pocket of the ecdysone hormone receptor. Nature.426,91-6.
Billas, I. M., Moulinier, L., Rochel, N., Moras, D.,2001. Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid X receptors in insects. J Biol Chem.276, 7465-74.
Bonilha, V. L., Bhattacharya, S. K., West, K. A., Sun, J., Crabb, J. W., Rayborn, M. E., Hollyfield, J. G., 2004. Proteomic characterization of isolated retinal pigment epithelium microvilli. Mol Cell Proteomics.3,1119-27.
Brady, W. A., Kokoris, M. S., Fitzgibbon, M., Black, M. E.,1996. Cloning, characterization, and modeling of mouse and human guanylate kinases. J Biol Chem.271,16734-40.
Brennan, C. A., Li, T. R., Bender, M., Hsiung, F., Moses, K.,2001. Broad-complex, but not ecdysone receptor, is required for progression of the morphogenetic furrow in the Drosophila eye. Development.128,1-11.
Cao, W., Haig-Ladewig, L., Gerton, G. L., Moss, S. B.,2006. Adenylate kinases 1 and 2 are part of the accessory structures in the mouse sperm flagellum. Biol Reprod.75,492-500.
Chen, L., Reece, C., O'Keefe, S. L., Hawryluk, G. W., Engstrom, M. M., Hodgetts, R. B.,2002. Induction of the early-late Ddc gene during Drosophila metamorphosis by the ecdysone receptor. Mech Dev. 114,95-107.
Cho, K. O., Hunt, C. A., Kennedy, M. B.,1992. The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron.9,929-42.
Clayton, G. M., Peak-Chew, S. Y., Evans, R. M., Schwabe, J. W.,2001. The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation. Proc Natl Acad Sci U S A.98,1549-54.
Clemens, J. C., Worby, C. A., Simonson-Leff, N., Muda, M., Maehama, T., Hemmings, B. A., Dixon, J. E., 2000. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci U S A.97,6499-503.
Collavin, L., Lazarevic, D., Utrera, R., Marzinotto, S., Monte, M., Schneider, C.,1999. wt p53 dependent expression of a membrane-associated isoform of adenylate kinase. Oncogene.18,5879-88.
Cronan, J. E., Jr., Godson, G. N.,1972. Mutants of Escherichia coli with temperature-sensitive lesions in membrane phospholipid synthesis:genetic analysis of glycerol-3-phosphate acyltransferase mutants. Mol Gen Genet.116,199-210.
DeFranco, D. B., Ramakrishnan, C., Tang, Y.,1998. Molecular chaperones and subcellular trafficking of steroid receptors. J Steroid Biochem Mol Biol.65,51-8.
DePasquale, J. A., Samsonoff, W. A., Gierthy, J. F.,1994.17-beta-Estradiol induced alterations of cell-matrix and intercellular adhesions in a human mammary carcinoma cell line. J Cell Sci.107 (Pt 5),1241-54.
Devarakonda, S., Harp, J. M., Kim, Y., Ozyhar, A., Rastinejad, F.,2003. Structure of the heterodimeric ecdysone receptor DNA-binding complex. EMBO J.22,5827-40.
Dimitratos, S. D., Woods, D. F., Bryant, P. J.,1997. Camguk, Lin-2, and CASK:novel membrane-associated guanylate kinase homologs that also contain CaM kinase domains. Mech Dev.63,127-30.
Dimitratos, S. D., Woods, D. F., Stathakis, D. G., Bryant, P. J.,1999. Signaling pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family. Bioessays.21,912-21.
Ding, X. Z., Tsokos, G. C., Smallridge, R. C., Kiang, J. G.,1997. Heat shock gene-expression in HSP-70 and HSF1 gene-transfected human epidermoid A-431 cells. Mol Cell Biochem.167,145-52.
Dong, D. J., He, H. J., Chai, L. Q., Jiang, X. J., Wang, J. X., Zhao, X. F.,2007. Identification of genes differentially expressed during larval molting and metamorphosis of Helicoverpa armigera. BMC Dev Biol.7,73.
Duina, A. A., Kalton, H. M., Gaber, R. F.,1998. Requirement for Hsp90 and a CyP-40-type cyclophilin in negative regulation of the heat shock response. J Biol Chem.273,18974-8.
Dunne, J. C., Kondylis, V., Rabouille, C.,2002. Ecdysone triggers the expression of Golgi genes in Drosophila imaginal discs via broad-complex. Dev Biol.245,172-86.
Dzeja, P., Kalvenas, A., Toleikis, A., Praskevicius, A.,1985. The effect of adenylate kinase activity on the rate and efficiency of energy transport from mitochondria to hexokinase. Biochem Int.10,259-65.
Dzeja, P. P., Bortolon, R., Perez-Terzic, C., Holmuhamedov, E. L., Terzic, A.,2002. Energetic communication between mitochondria and nucleus directed by catalyzed phosphotransfer. Proc Natl Acad Sci U S A.99,10156-61.
Dzeja, P. P., Zeleznikar, R. J., Goldberg, N. D.,1998. Adenylate kinase:kinetic behavior in intact cells indicates it is integral to multiple cellular processes. Mol Cell Biochem.184,169-82.
Elbashir, S. M., Lendeckel, W., Tuschl, T.,2001. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev.15,188-200.
Eystathioy, T., Swevers, L., Iatrou, K.,2001. The orphan nuclear receptor BmHR3A of Bombyx mori: hormonal control, ovarian expression and functional properties. Mech Dev.103,107-15.
Fang, F., Xu, Y, Jones, D., Jones, G.,2005. Interactions of ultraspiracle with ecdysone receptor in the transduction of ecdysone-and juvenile hormone-signaling. FEBS J.272,1577-89.
Fernandez-Gonzalez, A., Kourembanas, S., Wyatt, T. A., Mitsialis, S. A.,2009. Mutation of murine adenylate kinase 7 underlies a primary ciliary dyskinesia phenotype. Am J Respir Cell Mol Biol. 40,305-13.
Fink, A. L.,1999. Chaperone-mediated protein folding. Physiol Rev.79,425-49.
Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., Ahringer, J.,2000. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature. 408,325-30.
Fujisawa, K., Murakami, R., Horiguchi, T., Noma, T.,2009. Adenylate kinase isozyme 2 is essential for growth and development of Drosophila melanogaster. Comp Biochem Physiol B Biochem Mol Biol.
Fujiwara, H., Jindra, M., Newitt, R., Palli, S. R., Hiruma, K., Riddiford, L. M.,1995. Cloning of an ecdysone receptor homolog from Manduca sexta and the developmental profile of its mRNA in wings. Insect Biochem Mol Biol.25,845-56.
Gaidarov, I. O., Suslov, O. N., Abdulaev, N. G,1993. Enzymes of the cyclic GMP metabolism in bovine retina. I. Cloning and expression of the gene for guanylate kinase. FEBS Lett.335,81-4.
Gentry, D., Bengra, C, Ikehara, K., Cashel, M.,1993. Guanylate kinase of Escherichia coli K-12. J Biol Chem.268,14316-21.
Gilbert, L. I., Granger, N. A., Roe, R. M.,2000. The juvenile hormones:historical facts and speculations on future research directions. Insect Biochem Mol Biol.30,617-44.
Ginger, M. L., Ngazoa, E. S., Pereira, C. A., Pullen, T.J., Kabiri, M., Becker, K., Gull, K., Steverding, D., 2005. Intracellular positioning of isoforms explains an unusually large adenylate kinase gene family in the parasite Trypanosoma brucei. J Biol Chem.280,11781-9.
Grace, T. D.,1962. Establishment of four strains of cells from insect tissues grown in vitro. Nature.195, 788-9.
Grebe, M., Przibilla, S., Henrich, V. C., Spindler-Barth, M.,2003. Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster. Biol Chem.384,105-16.
Grenert, J. P., Johnson, B. D., Toft, D. O.,1999. The importance of ATP binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes. J Biol Chem.274,17525-33.
Grenert, J. P., Sullivan, W. P., Fadden, P., Haystead, T. A., Clark, J., Mimnaugh, E., Krutzsch, H., Ochel, H. J., Schulte, T. W., Sausville, E., Neckers, L. M., Toft, D. O.,1997. The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation. J Biol Chem.272,23843-50.
Hall, B. L., Thummel, C. S.,1998. The RXR homolog ultraspiracle is an essential component of the Drosophila ecdysone receptor. Development.125,4709-17.
Hannon, G. J.,2002. RNA interference. Nature.418,244-51.
Hartl, F. U.,1996. Molecular chaperones in cellular protein folding. Nature.381,571-9.
Hayward, D. C., Dhadialla, T. S., Zhou, S., Kuiper, M. J., Ball, E. E., Wyatt, G. R., Walker, V. K.,2003. Ligand specificity and developmental expression of RXR and ecdysone receptor in the migratory locust. J Insect Physiol.49,1135-44.
He, H. J., Wang, Q., Zheng, W. W., Wang, J. X., Song, Q. S., Zhao, X. F., Function of nuclear transport factor 2 and Ran in the 20E signal transduction pathway in the cotton bollworm, Helicoverpa armigera. BMC Cell Biol.11,1.
Henrich, V. C.,2005. The ecdysteroid receptor. In "Comprehensive Molecular Insect Science". Eds L.I. Gilbert, K. Iatrou and S.S. Gill, vol.3, pp.243-285, Elsevier, Oxford.
Henrich, V. C., Rybczynski, R., Gilbert, L. I.,1999. Peptide hormones, steroid hormones, and puffs: mechanisms and models in insect development. Vitam Horm.55,73-125.
Henrich, V. C., Sliter, T. J., Lubahn, D. B., Maclntyre, A., Gilbert, L. I.,1990. A steroid/thyroid hormone receptor superfamily member in Drosophila melanogaster that shares extensive sequence similarity with a mammalian homologue. Nucleic Acids Res.18,4143-8.
Henrich, V. C., Szekely, A. A., Kim, S. J., Brown, N. E., Antoniewski, C., Hayden, M. A., Lepesant, J. A., Gilbert, L. I.,1994. Expression and function of the ultraspiracle (usp) gene during development of Drosophila melanogaster. Dev Biol.165,38-52.
Hiruma, K., Bocking, D., Lafont, R., Riddiford, L. M.,1997. Action of different ecdysteroids on the regulation of mRNAs for the ecdysone receptor, MHR3, dopa decarboxylase, and a larval cuticle protein in the larval epidermis of the tobacco hornworm, Manduca sexta. Gen Comp Endocrinol. 107,84-97.
Hiruma, K., Carter, M. S., Riddiford, L. M.,1995. Characterization of the dopa decarboxylase gene of Manduca sexta and its suppression by 20-hydroxyecdysone. Dev Biol.169,195-209.
Hiruma, K., Riddiford, L. M.,1984. Regulation of melanization of tobacco hornworm larval cuticle in vitro. J Exp Zool.230,393-403.
Hiruma, K., Riddiford, L. M.,1990. Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone. Dev Biol.138, 214-24.
Hiruma, K., Riddiford, L. M.,2001. Regulation of transcription factors MHR4 and betaFTZ-F1 by 20-hydroxyecdysone during a larval molt in the tobacco hornworm, Manduca sexta. Dev Biol.232, 265-74.
Hiruma, K., Riddiford, L. M.,2004. Differential control of MHR3 promoter activity by isoforms of the ecdysone receptor and inhibitory effects of E75A and MHR3. Dev Biol.272,510-21.
Hiruma, K., Riddiford, L. M., Hopkins, T. L., Morgan, T. D.,1985. Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco homworm and their control by 20-hydroxyecdysone. J Comp Physiol B.155,659-69.
Hiruma, K., Shinoda, T., Malone, F., Riddiford, L. M.,1999. Juvenile hormone modulates 20-hydroxyecdysone-inducible ecdysone receptor and ultraspiracle gene expression in the tobacco hornworm, Manduca sexta. Dev Genes Evol.209,18-30.
Horodyski, F. M., Ewer, J., Riddiford, L. M., Truman, J. W.,1993. Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster. Eur J Biochem.215,221-8.
Hoskins, R., Hajnal, A. F., Harp, S. A., Kim, S. K.,1996. The C. elegans vulval induction gene lin-2 encodes a member of the MAGUK family of cell junction proteins. Development.122,97-111.
Janssen, E., de Groof, A., Wijers, M., Fransen, J., Dzeja, P. P., Terzic, A., Wieringa, B.,2003. Adenylate kinase 1 deficiency induces molecular and structural adaptations to support muscle energy metabolism. J Biol Chem.278,12937-45.
Janssen, E., Dzeja, P. P., Oerlemans, F., Simonetti, A. W., Heerschap, A., de Haan, A., Rush, P. S., Terjung, R. R., Wieringa, B., Terzic, A.,2000. Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement. EMBO J.19,6371-81.
Janssen, E., Kuiper, J., Hodgson, D., Zingman, L. V., Alekseev, A. E., Terzic, A., Wieringa, B.,2004. Two structurally distinct and spatially compartmentalized adenylate kinases are expressed from the AK1 gene in mouse brain. Mol Cell Biochem.256-257,59-72.
Jibard, N., Meng, X., Leclerc, P., Rajkowski, K., Fortin, D., Schweizer-Groyer, G., Catelli, M. G., Baulieu, E. E., Cadepond, F.,1999. Delimitation of two regions in the 90-kDa heat shock protein (Hsp90) able to interact with the glucocorticosteroid receptor (GR). Exp Cell Res.247,461-74.
Jindra, M., Huang, J. Y., Malone, F., Asahina, M., Riddiford, L. M.,1997. Identification and mRNA developmental profiles of two ultraspiracle isoforms in the epidermis and wings of Manduca sexta. Insect Mol Biol.6,41-53.
Jindra, M., Malone, F., Hiruma, K., Riddiford, L. M.,1996. Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm, Manduca sexta. Dev Biol.180,258-72.
Johnson, D. E., Brookhart, G. L., Kramer, K. J., Barnett, B. D., McGaughey, W. H.,1990. Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella:comparison of midgut proteinases from susceptible and resistant larvae. J Invertebr Pathol.55,235-44.
Kamimura, M., Tomita, S., Kiuchi, M., Fujiwara, H.,1997. Tissue-specific and stage-specific expression of two silkworm ecdysone receptor isoforms - ecdysteroid-dependent transcription in cultured anterior silk glands. Eur J Biochem.248,786-93.
Kataoka, H., Nagasawa, H., Isogai, A., Ishizaki, H., Suzuki, A.,1991. Prothoracicotropic hormone of the silkworm, Bombyx mori:amino acid sequence and dimeric structure. Agric Biol Chem.55,73-86.
Keshan, B., Hiruma, K., Riddiford, L. M.,2006. Developmental expression and hormonal regulation of different isoforms of the transcription factor E75 in the tobacco hornworm Manduca sexta. Dev Biol.295,623-32.
Khoo, J. C., Russell, P. J.,1972. Isoenzymes of adenylate kinase in human tissue. Biochim Biophys Acta. 268,98-101.
Kiang, J. G., Tsokos, G. C.,1998. Heat shock protein 70 kDa:molecular biology, biochemistry, and physiology. Pharmacol Then. 80,183-201.
Kim, S. K.,1995. Tight junctions, membrane-associated guanylate kinases and cell signaling. Curr Opin Cell Biol.7,641-9.
Koelle, M. R., Segraves, W. A., Hogness, D. S.,1992. DHR3:a Drosophila steroid receptor homolog. Proc Natl Acad Sci U S A.89,6167-71.
Koelle, M. R., Talbot, W. S., Segraves, W. A., Bender, M. T., Cherbas, P., Hogness, D. S.,1991. The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell.67,59-77.
Konrad, M.,1988. Analysis and in vivo disruption of the gene coding for adenylate kinase (ADK1) in the yeast Saccharomyces cerevisiae. J Biol Chem.263,19468-74.
Konrad, M.,1992. Cloning and expression of the essential gene for guanylate kinase from yeast. J Biol Chem.267,25652-5.
Konrad, M.,1993. Molecular analysis of the essential gene for adenylate kinase from the fission yeast Schizosaccharomyces pombe. J Biol Chem.268,11326-34.
Kosano, H., Stensgard, B., Charlesworth, M. C., McMahon, N., Toft, D.,1998. The assembly of progesterone receptor-hsp90 complexes using purified proteins. J Biol Chem.273,32973-9.
Kuhlendahl, S., Spangenberg, O., Konrad, M., Kim, E., Garner, C. C.,1998. Functional analysis of the guanylate kinase-like domain in the synapse-associated protein SAP97. Eur J Biochem.252, 305-13.
Kumar, V., Spangenberg, O., Konrad, M.,2000. Cloning of the guanylate kinase homologues AGK-1 and AGK-2 from Arabidopsis thaliana and characterization of AGK-1. Eur J Biochem.267,606-15.
Lagunas, L., Bradbury, C. M., Laszlo, A., Hunt, C. R., Gius, D.,2004. Indomethacin and ibuprofen induce Hsc70 nuclear localization and activation of the heat shock response in HeLa cells. Biochem Biophys Res Commun.313,863-70.
Lan, Q., Hiruma, K., Hu, X., Jindra, M., Riddiford, L. M.,1999. Activation of a delayed-early gene encoding MHR3 by the ecdysone receptor heterodimer EcR-B1-USP-1 but not by EcR-B1-USP-2. Mol Cell Biol.19,4897-906.
Lan, Q., Riddiford, L. M.,1997. DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta. In Vitro Cell Dev Biol Anim.33,615-21.
Lange, S., Auerbach, D., McLoughlin, P., Perriard, E., Schafer, B. W., Perriard, J. C., Ehler, E.,2002. Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2. J Cell Sci.115,4925-36.
Langelan, R. E., Fisher, J. E., Hiruma, K., Palli, S. R., Riddiford, L. M.,2000. Patterns of MHR3 expression in the epidermis during a larval molt of the tobacco hornworm Manduca sexta. Dev Biol.227,481-94.
Lavie, A., Konrad, M., Brundiers, R., Goody, R. S., Schlichting, I., Reinstein, J.,1998. Crystal structure of yeast thymidylate kinase complexed with the bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-thymidyl) pentaphosphate (TP5A) at 2.0 A resolution:implications for catalysis and AZT activation. Biochemistry.37,3677-86.
Lezzi, M., Bergman, T., Henrich, V. C., Vogtli, M., Fromel, C., Grebe, M., Przibilla, S., Spindler-Barth, M., 2002. Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle. Eur J Biochem.269,3237-45.
Liu, E., Restifo, L. L.,1998. Identification of a broad complex-regulated enhancer in the developing visual system of Drosophila. J Neurobiol.34,253-70.
Liu, J., Shi, G. P., Zhang, W. Q., Zhang, G. R., Xu, W. H.,2006. Cathepsin L function in insect moulting: molecular cloning and functional analysis in cotton bollworm, Helicoverpa armigera. Insect Mol Biol.15,823-34.
Liu, R., Strom, A. L., Zhai, J., Gal, J., Bao, S., Gong, W., Zhu, H.,2009. Enzymatically inactive adenylate kinase 4 interacts with mitochondrial ADP/ATP translocase. Int J Biochem Cell Biol.41,1371-80.
Lonergan, K. M., Chari, R., Deleeuw, R. J., Shadeo, A., Chi, B., Tsao, M. S., Jones, S., Marra, M., Ling, V, Ng, R., Macaulay, C., Lam, S., Lam, W. L.,2006. Identification of novel lung genes in bronchial epithelium by serial analysis of gene expression. Am J Respir Cell Mol Biol.35,651-61.
Lynn, D. E., Miller, S. G., Oberlander, H.,1982. Establishment of a cell line from lepidopteran wing imaginal discs:Induction of newly synthesized proteins by 20-hydroxyecdysone. Proc Natl Acad Sci U S A.79,2589-2593.
Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., Evans, R. M.,1995. The nuclear receptor superfamily:the second decade. Cell.83,835-9.
Miech, R. P., Parks, R. E., Jr.,1965. Adenosine Triphosphate:Guanosine Monophosphate Phosphotransferase. Partial Purification and Substrate Specificity. J Biol Chem.240,351-7.
Minakuchi, C., Nakagawa, Y., Kiuchi, M., Tomita, S., Kamimura, M.,2002. Molecular cloning, expression analysis and functional confirmation of two ecdysone receptor isoforms from the rice stem borer Chilo suppressalis. Insect Biochem Mol Biol.32,999-1008.
Montpied, P., Sobrier, M. L., Chapel, S., Couderc, J. L., Dastugue, B.,1988.20-Hydroxyecdysone induces the expression of one beta-tubulin gene in Drosophila Kc cells. Biochim Biophys Acta.949, 79-86.
Mottier, V., Siaussat, D., Bozzolan, F., Auzoux-Bordenave, S., Porcheron, P., Dcbernard, S.,2004. The 20-hydroxyecdysone-induced cellular arrest in G2 phase is preceded by an inhibition of cyclin expression. Insect Biochem Mol Biol.34,51-60.
Mugat, B., Brodu, V., Kejzlarova-Lepesant, J., Antoniewski, C., Bayer, C. A., Fristrom, J. W., Lepesant, J. A.,2000. Dynamic expression of broad-complex isoforms mediates temporal control of an ecdysteroid target gene at the onset of Drosophila metamorphosis. Dev Biol.227,104-17.
Nijhout, H. F.,1984. Colour pattern modification by coldshock in Lepidoptera. J Embryol Exp Morphol.81, 287-305.
Nijhout, H. F.,2003. The control of body size in insects. Dev Biol.261,1-9.
Nijhout, H. F., Sheffield, H. G.,1979. Antennal hair erection in male mosquitoes:a new mechanical effector in insects. Science.206,595-6.
Nishita, Y., Takiya, S.,2004. Structure and expression of the gene encoding a Broad-Complex homolog in the silkworm, Bombyx mori. Gene.339,161-72.
Noma, T.,2005. Dynamics of nucleotide metabolism as a supporter of life phenomena. J Med Invest.52, 127-36.
Oberlander, H., Leach, C. E., Shaaya, E.,2000. Juvenile hormone and juvenile hormone mimics inhibit proliferation in a lepidopteran imaginal disc cell line. J Insect Physiol.46,259-265.
Obermann, W. M., Sondermann, H., Russo, A. A., Pavletich, N. P., Hartl, F. U.,1998. In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis. J Cell Biol.143,901-10.
Okajima, T., Fukamizo, T., Goto, S., Fukui, T., Tanizawa, K.,1998. Exchange of nucleoside monophosphate-binding domains in adenylate kinase and UMP/CMP kinase. J Biochem.124, 359-67.
Oro, A. E., McKeown, M., Evans, R. M.,1992. The Drosophila retinoid X receptor homolog ultraspiracle functions in both female reproduction and eye morphogenesis. Development.115,449-62.
Ote, M., Mita, K., Kawasaki, H., Daimon, T., Kobayashi, M., Shimada, T.,2005. Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori. Comp Biochem Physiol B Biochem Mol Biol.141,314-22.
Palli, S. R., Caputo, G. F., Sohi, S. S., Brownwright, A. J., Ladd, T. R., Cook, B. J., Primavera, M., Arif, B. M., Retnakaran, A.,1996. CfMNPV blocks AcMNPV-induced apoptosis in a continuous midgut cell line. Virology.222,201-13.
Palli, S. R., Hiruma, K., Riddiford, L. M.,1992. An ecdysteroid-inducible Manduca gene similar to the Drosophila DHR3 gene, a member of the steroid hormone receptor superfamily. Dev Biol.150, 306-18.
Palli, S. R., Ladd, T. R., Ricci, A. R., Sohi, S. S., Retnakaran, A.,1997. Cloning and development expression of Choristoneura hormone receptor 75:a homologue of the Drosophila E75A gene. Dev Genet.20,36-46.
Panaretou, B., Prodromou, C., Roe, S. M., O'Brien, R., Ladbury, J. E., Piper, P. W., Pearl, L. H.,1998. ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo. EMBO J.17,4829-36.
Perrimon, N., Engstrom, L., Mahowald, A. P.,1985. Developmental genetics of the 2C-D region of the Drosophila X chromosome. Genetics. 111,23-41.
Prapapanich, V., Chen, S., Smith, D. F.,1998. Mutation of Hip's carboxy-terminal region inhibits a transitional stage of progesterone receptor assembly. Mol Cell Biol.18,944-52.
Prapapanich, V., Chen, S., Toran, E. J., Rimerman, R. A., Smith, D. F.,1996. Mutational analysis of the hsp70-interacting protein Hip. Mol Cell Biol.16,6200-7.
Pucar, D., Janssen, E., Dzeja, P. P., Juranic, N., Macura, S., Wieringa, B., Terzic, A.,2000. Compromised energetics in the adenylate kinase AK1 gene knockout heart under metabolic stress. J Biol Chem. 275,41424-9.
Qualtieri, A., Pedace, V., Bisconte, M. G., Bria, M., Gulino, B., Andreoli, V., Brancati, C.,1997. Severe erythrocyte adenylate kinase deficiency due to homozygous A-->G substitution at codon 164 of human AK1 gene associated with chronic haemolytic anaemia. Br J Haematol.99,770-6.
Ren, H., Wang, L., Bennett, M., Liang, Y., Zheng, X., Lu, F., Li, L., Nan, J., Luo, M., Eriksson, S., Zhang, C., Su, X. D.,2005. The crystal structure of human adenylate kinase 6:An adenylate kinase localized to the cell nucleus. Proc Natl Acad Sci U S A.102,303-8.
Rewitz, K. F., Yamanaka, N., Gilbert, L. I., O'Connor, M. B.,2009. The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis. Science.326,1403-5.
Riddiford, L. M.,1993. Hormone receptors and the regulation of insect metamorphosis. Receptor.3,203-9.
Riddiford, L. M.,2008. Juvenile hormone action:a 2007 perspective. J Insect Physiol.54,895-901.
Riddiford, L. M., Hiruma, K., Zhou, X., Nelson, C. A.,2003. Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster. Insect Biochem Mol Biol.33,1327-38.
Rutherford, S. L., Lindquist, S.,1998. Hsp90 as a capacitor for morphological evolution. Nature.396, 336-42.
Rybczynski, R., Bell, S. C., Gilbert, L. I.,2001. Activation of an extracellular signal-regulated kinase (ERK) by the insect prothoracicotropic hormone. Mol Cell Endocrinol.184,1-11.
Rybczynski, R., Gilbert, L. I.,2000. cDNA cloning and expression of a hormone-regulated heat shock protein (hsc 70) from the prothoracic gland of Manduca sexta. Insect Biochem Mol Biol.30, 579-89.
Samuels, R. I., Charnley, A. K., Reynolds, S. E.,1993. A cuticle-degrading proteinase from the moulting fluid of the tobacco hornworm, Manduca sexta. Insect Biochem Mol Biol.23,607-14.
Scherrer, L. C., Hutchison, K. A., Sanchez, E. R., Randall, S. K., Pratt, W. B.,1992. A heat shock protein complex isolated from rabbit reticulocyte lysate can reconstitute a functional glucocorticoid receptor-Hsp90 complex. Biochemistry.31,7325-9.
Schett, G., Steiner, C. W., Groger, M., Winkler, S., Graninger, W., Smolen, J., Xu, Q., Steiner, G.,1999. Activation of Fas inhibits heat-induced activation of HSF1 and up-regulation of hsp70. FASEB J. 13,833-42.
Schneider, I.,1972. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol.27,353-65.
Schubiger, M., Truman, J. W.,2000. The RXR ortholog USP suppresses early metamorphic processes in Drosophila in the absence of ecdysteroids. Development.127,1151-9.
Schubiger, M., Wade, A. A., Carney, G. E., Truman, J. W., Bender, M.,1998. Drosophila EcR-B ecdysone receptor isoforms are required for larval molting and for neuron remodeling during metamorphosis. Development.125,2053-62.
Schulz, G. E., Schiltz, E., Tomasselli, A. G., Frank, R., Brune, M., Wittinghofer, A., Schirmer, R. H.,1986. Structural relationships in the adenylate kinase family. Eur J Biochem.161,127-32.
Segraves, W. A., Hogness, D. S.,1990. The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily. Genes Dev.4, 204-19.
Segraves, W. A., Woldin, C.,1993. The E75 gene of Manduca sexta and comparison with its Drosophila homolog. Insect Biochem Mol Biol.23,91-7.
Shaknovich, R., Shue, G., Kohtz, D. S.,1992. Conformational activation of a basic helix-loop-helix protein (MyoD1) by the C-terminal region of murine HSP90 (HSP84). Mol Cell Biol.12,5059-68.
Shao, H. L., Zheng, W. W., Liu, P. C., Wang, Q., Wang, J. X., Zhao, X. F.,2008. Establishment of a new cell line from lepidopteran epidermis and hormonal regulation on the genes. PLoS One.3, e3127.
Sharp, P. A.,1999. RNAi and double-strand RNA. Genes Dev.13,139-41.
Siaussat, D., Bozzolan, F., Porcheron, P., Debernard, S.,2007. Identification of steroid hormone signaling pathway in insect cell differentiation. Cell Mol Life Sci.64,365-76.
Siaussat, D., Bozzolan, F., Queguiner, I., Porcheron, P., Debernard, S.,2004a. Effects of juvenile hormone on 20-hydroxyecdysone-inducible EcR, HR3, E75 gene expression in imaginal wing cells of Plodia interpunctella lepidoptera. Eur J Biochem.271,3017-27.
Siaussat, D., Mottier, V., Bozzolan, F., Porcheron, P., Debernard, S.,2004b. Synchronization of Plodia interpunctella lepidopteran cells and effects of 20-hydroxyecdysone. Insect Mol Biol.13,179-87.
Smith, D. F., Toft, D. O.,1993. Steroid receptors and their associated proteins. Mol Endocrinol.7,4-11.
Smith, W. A., Gilbert, L. I.,1989. Early events in peptide-stimulated ecdysteroid secretion by the prothoracic glands of Manduca sexta. J Exp Zool.252,264-70.
Sobrier, M. L., Couderc, J. L., Chapel, S., Dastugue, B.,1986. Expression of a new beta tubulin subunit is induced by 20-hydroxyecdysone in Drosophila cultured cells. Biochem Biophys Res Commun. 134,191-200.
Somma, M. P., Fasulo, B., Cenci, G., Cundari, E., Gatti, M.,2002. Molecular dissection of cytokincsis by RNA interference in Drosophila cultured cells. Mol Biol Cell.13,2448-60.
Spindler, K.-D., Betanska, K, Nieva, C., Gwozdz, T. Dutko-Gwozdz, Ozyhar, A. and Spindler-Barth, M., 2009. Intracellular localization of the ecdysteroid receptor. In "Ecdysone:Structures and Functions", Ed. G. Smagghe, pp.389-409. Springer, Berlin
Stanojevic, V., Habener, J. F., Holz, G. G., Leech, C. A.,2008. Cytosolic adenylate kinases regulate K-ATP channel activity in human beta-cells. Biochem Biophys Res Commun.368,614-9.
Stehle, T., Schulz, G. E.,1992. Refined structure of the complex between guanylate kinase and its substrate GMP at 2.0 A resolution. J Mol Biol.224,1127-41.
Stephanou, A., Isenberg, D. A., Nakajima, K., Latchman, D. S.,1999. Signal transducer and activator of transcription-1 and heat shock factor-1 interact and activate the transcription of the Hsp-70 and Hsp-90beta gene promoters. J Biol Chem.274,1723-8.
Stilwell, G. E., Nelson, C. A., Weller, J., Cui, H., Hiruma, K., Truman, J. W., Riddiford, L. M.,2003. E74 exhibits stage-specific hormonal regulation in the epidermis of the tobacco hornworm, manduca sexta. Dev Biol.258,76-90.
Sudeep, A. B., Shouche, Y. S., Mourya, D. T., Pant, U.,2002. New Helicoverpa armigera Hbn cell line from larval hemocyte for baculovirus studies. Indian J Exp Biol.40,69-73.
Sui, Y. P., Liu, X. B., Chai, L. Q., Wang, J. X., Zhao, X. F.,2009. Characterization and influences of classical insect hormones on the expression profiles of a molting carboxypeptidase A from the cotton bollworm (Helicoverpa armigera). Insect Mol Biol.18,353-63.
Sun, G. C., Hirose, S., Ueda, H.,1994. Intermittent expression of BmFTZ-F1, a member of the nuclear hormone receptor superfamily during development of the silkworm Bombyx mori. Dev Biol.162, 426-37.
Sutherland, J. D., Kozlova, T., Tzertzinis, G., Kafatos, F. C.,1995. Drosophila hormone receptor 38:a second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type. Proc Natl Acad Sci U S A.92,7966-70.
Swevers, L., Cherbas, L., Cherbas, P., Iatrou, K.,1996. Bombyx EcR (BmEcR) and Bombyx USP (BmCF1) combine to form a functional ecdysone receptor. Insect Biochem Mol Biol.26,217-21.
Swevers, L., Eystathioy, T., Iatrou, K.,2002. The orphan nuclear receptors BmE75A and BmE75C of the silkmoth Bombyx mori:hornmonal control and ovarian expression. Insect Biochem Mol Biol.32, 1643-52.
Talbot, W. S., Swyryd, E. A., Hogness, D. S.,1993. Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell.73,1323-37.
Tan, A., Palli, S. R.,2008. Ecdysone [corrected] receptor isoforms play distinct roles in controlling molting and metamorphosis in the red flour beetle, Tribolium castaneum. Mol Cell Endocrinol.291,42-9.
Tanabe, T., Yamada, M., Noma, T., Kajii, T., Nakazawa, A.,1993. Tissue-specific and developmentally regulated expression of the genes encoding adenylate kinase isozymes. J Biochem.113,200-7.
Terashima, J., Bownes, M.,2006. E75A and E75B have opposite effects on the apoptosis/development choice of the Drosophila egg chamber. Cell Death Differ.13,454-64.
Thummel, C. S.,1995. From embryogenesis to metamorphosis:the regulation and function of Drosophila nuclear receptor superfamily members. Cell.83,871-7.
Thummel, C. S.,2001. Steroid-triggered death by autophagy. Bioessays.23,677-82.
Tomasselli, A. G., Noda, L. H.,1979. Mitochondrial GTP-AMP phosphotransferase.2. Kinetic and equilibrium dialysis studies. Eur J Biochem.93,263-7.
Tomasselli, A. G, Schirmer, R. H., Noda, L. H.,1979. Mitochondrial GTP-AMP phosphotransferase.1. Purification and properties. Eur J Biochem.93,257-62.
Truman, J. W., Riddiford, L. M.,1974. Physiology of insect rhythms.3. The temporal organization of the endocrine events underlying pupation of the tobacco hornworm. J Exp Biol.60,371-82.
Truman, J. W., Sokolove, P. G.,1972. Silk Moth Eclosion:Hormonal Triggering of a Centrally Programmed Pattern of Behavior. Science.175,1491-1493.
Truss, M., Beato, M.,1993. Steroid hormone receptors:interaction with deoxyribonucleic acid and transcription factors. Endocr Rev.14,459-79.
Uhlirova, M., Foy, B. D., Beaty, B. J., Olson, K. E., Riddiford, L. M., Jindra, M.,2003. Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis. Proc Natl Acad Sci U S A.100,15607-12.
van Horssen, R., Janssen, E., Peters, W., van de Pasch, L., Lindert, M. M., van Dommelen, M. M., Linssen, P. C., Hagen, T. L., Fransen, J. A., Wieringa, B.,2009. Modulation of cell motility by spatial repositioning of enzymatic ATP/ADP exchange capacity. J Biol Chem.284,1620-7.
Vasseur, S., Malicet, C., Calvo, E. L., Dagorn, J. C., Iovanna, J. L.,2005. Gene expression profiling of tumours derived from rasV12/E1A-transformed mouse embryonic fibroblasts to identify genes required for tumour development. Mol Cancer.4,4.
Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., McCawley, P.,1977. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro.13,213-7.
Velazquez, J. M., Lindquist, S.,1984. hsp70:nuclear concentration during environmental stress and cytoplasmic storage during recovery. Cell.36,655-62.
Wagner, G. P., Chiu, C. H., Hansen, T. F.,1999. Is Hsp90 a regulator of evolvability? J Exp Zool.285, 116-8.
Wang, J. L., Jiang, X. J., Wang, Q., Hou, L. J., Xu, D. W., Wang, J. X., Zhao, X. F.,2007. Identification and expression profile of a putative basement membrane protein gene in the midgut of Helicoverpa armigera. BMC Dev Biol.7,76.
Wang, J. L., Zhang, Y. P., Gu, Y. Y., Wang, J. X., Zhao, X. F.,2009. Function of a TGF-beta inducible nuclear protein in the silk gland in Bombyx mori. Insect Mol Biol.18,243-51.
Wei, Z. J., Zhang, Q. R., Kang, L., Xu, W. H., Denlinger, D. L.,2005. Molecular characterization and expression of prothoracicotropic hormone during development and pupal diapause in the cotton bollworm, Helicoverpa armigera. J Insect Physiol.51,691-700.
Weller, J., Sun, G. C., Zhou, B., Lan, Q., Hiruma, K., Riddiford, L. M.,2001. Isolation and developmental expression of two nuclear receptors, MHR4 and betaFTZ-F1, in the tobacco hornworm, Manduca sexta. Insect Biochem Mol Biol.31,827-37.
Wild, K., Grafmuller, R., Wagner, E., Schulz, G. E.,1997. Structure, catalysis and supramolecular assembly of adenylate kinase from maize. Eur J Biochem.250,326-31.
Wilson, R., Ainscough, R., Anderson, K., Baynes, C., Berks, M., Bonfield, J., Burton, J., Connell, M., Copsey, T., Cooper, J., et al.,1994.2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature.368,32-8.
Woodard, C. T., Baehrecke, E. H., Thummel, C. S.,1994. A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone. Cell.79,607-15.
Woods, D. F., Bryant, P. J.,1991. The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell.66,451-64.
Xu, L., Glass, C. K., Rosenfeld, M. G.,1999. Coactivator and corepressor complexes in nuclear receptor function. Curr Opin Genet Dev.9,140-7.
Yao, T. P., Forman, B. M., Jiang, Z., Cherbas, L., Chen, J. D., McKeown, M., Cherbas, P., Evans, R. M., 1993. Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature.366, 476-9.
Yao, T. P., Segraves, W. A., Oro, A. E., McKeown, M., Evans, R. M.,1992. Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. Cell.71,63-72.
Yussa, M., Lohr, U., Su, K., Pick. L.,2001. The nuclear receptor Ftz-F1 and homeodomain protein Ftz interact through evolutionarily conserved protein domains. Mech Dev.107,39-53.
Zelhof, A. C., Yao, T. P., Evans, R. M., McKeown, M.,1995. Identification and characterization of a Drosophila nuclear receptor with the ability to inhibit the ecdysone response. Proc Natl Acad Sci U S A.92,10477-81.
Zhai, R., Meng, G, Zhao, Y, Liu, B., Zhang, G., Zheng, X.,2006. A novel nuclear-localized protein with special adenylate kinase properties from Caenorhabditis elegans. FEBS Lett.580,3811-7.
Zhao, X. F., Wang, J. X., Xu, X. L., Li, Z. M., Kang, C. J.,2004. Molecular cloning and expression patterns of the molt-regulating transcription factor HHR3 from Helicoverpa armigera. Insect Mol Biol.13, 407-12.
Zhao, X. F., Wang, J. X., Xu, X. L., Schmid, R., Wieczorek, H.,2002. Molecular cloning and characterization of the cathepsin B-like proteinase from the cotton boll worm, Helicoverpa armigera. Insect Mol Biol.11,567-75.
Zheng, W. W., Yang, D. T., Wang, J. X., Song, Q. S., Gilbert, L. I., Zhao, X. F., Hsc70 binds to ultraspiracle resulting in the upregulation of 20-hydroxyecdsone-responsive genes in Helicoverpa armigera. Mol Cell Endocrinol.315,282-91.
Zhou, B., Hiruma, K., Jindra, M., Shinoda, T., Segraves, W. A., Malone, F., Riddiford, L. M.,1998a. Regulation of the transcription factor E75 by 20-hydroxyecdysone and juvenile hormone in the epidermis of the tobacco hornworm, Manduca sexta, during larval molting and metamorphosis. Dev Biol.193,127-38.
Zhou, B., Hiruma, K., Shinoda, T., Riddiford, L. M.,1998b. Juvenile hormone prevents ecdysteroid-induced expression of broad complex RNAs in the epidermis of the tobacco hornworm, Manduca sexta. Dev Biol.203,233-44.
Zou, J., Guo, Y, Guettouche, T., Smith, D. F., Voellmy, R.,1998. Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell.94,471-80.