草鱼PKZ的功能分析
摘要
PKR-like(PKZ)是近年来在鲫鱼、斑马鱼(Danio rerio)、大西洋鲑(Salmo salar)、稀有鮈鲫(Gobiocypris rarus)和草鱼(Ctenopharyngodon idella)中报道的一个蛋白激酶,由于其N端为Z-DNA结合域(Za),因此,将它命名为PKZ(protein kinase containing Z-DNA binding domain)。有趣的是,PKZ的C-端也具有12个保守的eIF2a激酶催化域,与PKR(double-stranded RNA dependent protein kinase)非常相似。
初步研究表明PKZ与PKR非常相似,也与宿主细胞抗病毒有关。如鱼类PKZ基因的本底表达非常微弱,当干扰素等诱导后,表达上调。此外,与其eIF2a激酶家族其他成员一样,斑马鱼PKZ、大西洋鲑PKZ也能够抑制细胞蛋白质的合成。尽管如此,对Z-DNA如何调节及激活鱼类PKZ还不清楚。
基于此,我们根据本实验室已克隆的草鱼PKR-like(PKZ)(GU299765),构建了原核表达载体,并在体外进行表达、纯化。对所得草鱼PKZ全长蛋白进行功能分析表明：原核表达的草鱼PKZ本身就已经被磷酸化了。以磷酸酶使之去自磷酸化后,PKZ就失去了激酶活性。我们还发现Z-DNA可以使去自磷酸化的PKZ重新激活,然后磷酸化eIF2α。而Poly I:C却无法使之重新活化。体内实验表明：草鱼PKZ能够在翻译水平上极大地抑制荧光素酶的产生,说明它能够抑制蛋白质的翻译。其198位点突变后,几乎完全失去阻碍翻译的功能。此外,调节区Zα的缺失对其功能也影响极大。
另外,草鱼PKZ启动子的序列分析表明其上存在干扰素调节因子1(IRF-1)的结合位点。基于此,我们利用原核表达的IRF-1的多肽与PKZ启动子序列进行了琼脂糖凝胶阻滞实验,结果表明,IRF-1可与PKZ启动子结合。这个结果为我们构建真核表达载体,进一步在体内分析二者的结合情况,以及探索IRF-1调控草鱼PKZ的转录做好了准备。
依据本实验中所得结果,以及分析以往对PKZ的研究成果,我们在不排除前人提出的PKZ抗病毒通路的前提下,提出一条新路径以供参考。这条通路与以往PKZ激活的“被动”模式不同,我们认为PKZ有可能存在一条内源性激活通路,这种激活模式对于细胞应激占据主动地位具有重要意义。而此通路的提出在一定程度上弥补了旧通路的不足之处。
In recent years, the kinase PKR-like (PKZ) genes had been reported successively in some teleost fish, such as CaPKZ in Goldfish (Carassius auratus)(Hu et al.,2004), DrPKZ in Zebrafish (Danio rerio)(Rothenburg et al.,2005), SsPKZ in Atlantic salmon (Salmo salar)(Bergan et al.,2008) and GrPKZ in Rare minnow (Gobiocypris rarus)(Su et al.,2008). Because the proteins encoded by these genes contain two Za domains and can bind Z-DNA, so they were designated as "PKZ"(protein kinase containing Z-DNA binding domain)(Rothenburg et al.,2005). It is interested that the C-terminus of PKZ also contain12conserved subdomains which are more closely to fish PKR (dsRNA-activated protein kinase), so Rothenburg et al (2008) considered PKZ as replication of fish PKR and stop at tetrapod (Rothenburg et al.,2008). Hence, PKZ is the special protein kinase, not only is a kind of Z-DNA binding protein but also belongs to an eIF2a protein kinase family.
The research on the function of PKZ has a good beginning. The preliminary conclusion is that the PKZ is extremely similar to PKR, so it is likely related to the antivirus of cell (Hu et al.,2004; Rothenburg et al.,2005). For example, the constitutive expression of PKZ is lower, but have a highly up-regulation induced by IFN etc (Hu et al.,2004; Rothenburg et al.,2005; Bergan et al.,2008). Moreover, like the others member of eIF2a kinase, DrPKZ and SsPKZ could inhibit protein synthesis (Rothenburg et al.,2005; Bergan et al.,2008). Despite for all this, we also puzzle about how to regulate and activate the fish PKZ by Z-DNA.
Ctenopharyngodon idella Grass carp (Ctenopharyngodon idella) is one of the major fresh fish in china. Because it is very sensitive to many stresses, so the PKZ from grass carp may be beneficial for us to comprehend the function of fish PKZ.On account of it,we constructed the prokaryotic expression plasmids based on grass carp PKZ(CiPKZ, GU299765), expressed in vitro,and purified.We contrasted the yield of protein PKZ between BL21(DE3) and Rosseta, found that Rosseta were higher yield.then we analysed the function of PKZ,and showed that the fusion PKZ protein extracted from E.coli have been activated in the absence of activators. When we added phosphatase,found that PKZ was not able to phosphorylate eIF2a..Moreover, when Z-DNA or Poly I:C were incubated with dephosphorylated PKZ, we found that Z-DNA but not Poly I:C could revive PKZ.In vivo,we found that CiPKZ could restrain the expression of luciferase extremely. Zα and the K198amino acid residue play a key role in its function.
Moreover,the analysis of CiPKZ prompter showed us a site that might combine IFN regulatory factor1(I1).So we examined the binding activity of His-tag I1fusion protein and PKZ prompter by agarose gel mobility shift assay. The results demonstrated that I1could combine I1.Our results was a beginning for analysis of I1and CiPKZ prompter in vivo,and exploration the response of CiPKZ transcription to I1.
初步研究表明PKZ与PKR非常相似,也与宿主细胞抗病毒有关。如鱼类PKZ基因的本底表达非常微弱,当干扰素等诱导后,表达上调。此外,与其eIF2a激酶家族其他成员一样,斑马鱼PKZ、大西洋鲑PKZ也能够抑制细胞蛋白质的合成。尽管如此,对Z-DNA如何调节及激活鱼类PKZ还不清楚。
基于此,我们根据本实验室已克隆的草鱼PKR-like(PKZ)(GU299765),构建了原核表达载体,并在体外进行表达、纯化。对所得草鱼PKZ全长蛋白进行功能分析表明：原核表达的草鱼PKZ本身就已经被磷酸化了。以磷酸酶使之去自磷酸化后,PKZ就失去了激酶活性。我们还发现Z-DNA可以使去自磷酸化的PKZ重新激活,然后磷酸化eIF2α。而Poly I:C却无法使之重新活化。体内实验表明：草鱼PKZ能够在翻译水平上极大地抑制荧光素酶的产生,说明它能够抑制蛋白质的翻译。其198位点突变后,几乎完全失去阻碍翻译的功能。此外,调节区Zα的缺失对其功能也影响极大。
另外,草鱼PKZ启动子的序列分析表明其上存在干扰素调节因子1(IRF-1)的结合位点。基于此,我们利用原核表达的IRF-1的多肽与PKZ启动子序列进行了琼脂糖凝胶阻滞实验,结果表明,IRF-1可与PKZ启动子结合。这个结果为我们构建真核表达载体,进一步在体内分析二者的结合情况,以及探索IRF-1调控草鱼PKZ的转录做好了准备。
依据本实验中所得结果,以及分析以往对PKZ的研究成果,我们在不排除前人提出的PKZ抗病毒通路的前提下,提出一条新路径以供参考。这条通路与以往PKZ激活的“被动”模式不同,我们认为PKZ有可能存在一条内源性激活通路,这种激活模式对于细胞应激占据主动地位具有重要意义。而此通路的提出在一定程度上弥补了旧通路的不足之处。
In recent years, the kinase PKR-like (PKZ) genes had been reported successively in some teleost fish, such as CaPKZ in Goldfish (Carassius auratus)(Hu et al.,2004), DrPKZ in Zebrafish (Danio rerio)(Rothenburg et al.,2005), SsPKZ in Atlantic salmon (Salmo salar)(Bergan et al.,2008) and GrPKZ in Rare minnow (Gobiocypris rarus)(Su et al.,2008). Because the proteins encoded by these genes contain two Za domains and can bind Z-DNA, so they were designated as "PKZ"(protein kinase containing Z-DNA binding domain)(Rothenburg et al.,2005). It is interested that the C-terminus of PKZ also contain12conserved subdomains which are more closely to fish PKR (dsRNA-activated protein kinase), so Rothenburg et al (2008) considered PKZ as replication of fish PKR and stop at tetrapod (Rothenburg et al.,2008). Hence, PKZ is the special protein kinase, not only is a kind of Z-DNA binding protein but also belongs to an eIF2a protein kinase family.
The research on the function of PKZ has a good beginning. The preliminary conclusion is that the PKZ is extremely similar to PKR, so it is likely related to the antivirus of cell (Hu et al.,2004; Rothenburg et al.,2005). For example, the constitutive expression of PKZ is lower, but have a highly up-regulation induced by IFN etc (Hu et al.,2004; Rothenburg et al.,2005; Bergan et al.,2008). Moreover, like the others member of eIF2a kinase, DrPKZ and SsPKZ could inhibit protein synthesis (Rothenburg et al.,2005; Bergan et al.,2008). Despite for all this, we also puzzle about how to regulate and activate the fish PKZ by Z-DNA.
Ctenopharyngodon idella Grass carp (Ctenopharyngodon idella) is one of the major fresh fish in china. Because it is very sensitive to many stresses, so the PKZ from grass carp may be beneficial for us to comprehend the function of fish PKZ.On account of it,we constructed the prokaryotic expression plasmids based on grass carp PKZ(CiPKZ, GU299765), expressed in vitro,and purified.We contrasted the yield of protein PKZ between BL21(DE3) and Rosseta, found that Rosseta were higher yield.then we analysed the function of PKZ,and showed that the fusion PKZ protein extracted from E.coli have been activated in the absence of activators. When we added phosphatase,found that PKZ was not able to phosphorylate eIF2a..Moreover, when Z-DNA or Poly I:C were incubated with dephosphorylated PKZ, we found that Z-DNA but not Poly I:C could revive PKZ.In vivo,we found that CiPKZ could restrain the expression of luciferase extremely. Zα and the K198amino acid residue play a key role in its function.
Moreover,the analysis of CiPKZ prompter showed us a site that might combine IFN regulatory factor1(I1).So we examined the binding activity of His-tag I1fusion protein and PKZ prompter by agarose gel mobility shift assay. The results demonstrated that I1could combine I1.Our results was a beginning for analysis of I1and CiPKZ prompter in vivo,and exploration the response of CiPKZ transcription to I1.
引文
[1]Ronald C. Wek.eIF-2 kinases:regulators of general and gene-specific translation initiation. Trends Biochem Sci.1994,19:491-6.
[2]Clemens MJ. Regulation of eukaryotic protein synthesis by protein kinases that phosphorylate initiation factor eIF-2. Molecular biology reports.1994,19:201-10.
[3]de Haro C, Mendez R, Santoyo J. The eIF-2alpha kinases and the control of protein synthesis.FASEB J.1996,10:1378-87.
[4]Dever TE. Gene-specific regulation by general translation factors. Cell.2002,108:545-56.
[5]Lu L, Han AP, Chen JJ. Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol Cell Biol. 2001,21:7971-80.
[6]Chen J J, Throop M S, Gehrke L, et al. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2a (eIF-2α) kinase of rabbit retilocytes:homology to yeast GCN2 protein kinase and human double-stranded RNA-depentent eIF-2α kinase.Proc Natl Acad Sci USA.1991,88:7729-7733.
[7]Sudhakar A, Ramachandran A, Ghosh S, Hasnain SE, Kaufman RJ, Ramaiah KVA. Phosphorylation of serine 51 in initiation factor 2a (eIF2α) promotes complex formation between eIF2α (P) and eIF2B and causes inhibition in the guanine nucleotide exchange activity of zI2B.Biochemistry.2000,39:12929-12938.
[8]Chen JJ, London IM. Regulation of protein synthesis by heme-regulated eIF-2α kinase. Trends in Biochemical Sciences.1995,20:105-108.
[9]Chen JJ. Regulation of protein synthesis by the hemeregulated eIF2α kinase:relevance to anemias. Blood.2007,109:2693-2699.
[10]Crosby JS, Lee K, London IM, Chen JJ. Erythroid expression of the heme-regulated eIF-2akinase. Molecular and Cellular Biology.1994,14:3906-3914.
[11]Han AP, Yu C, Lu L, et al. Heme-regulated eIF2a kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency. EMBO.2001, 20:6909-6918.
[12]Chen JJ. Heme-regulated eIF2α kinase. in Translational Control of Gene Expression.2000,vol.39 of Cold Spring Harbor Monograph Series, pp.529-546, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
[13]Berlanga JJ, Santoyo J, de Haro C.Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2a kinase. Eur J Biochem.1999,265:754-762
[14]Hinnebusch AG.Mechanism and regulation of initiator methionyl-tRNA binding to ribosomes. In Translational Control of Gene Expression, Sonenberg N, Hershey JWB, Mathews MB (eds).2000,pp 185-243. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory Press.
[15]Deng J, Harding HP, Raught B, Gingras AC, Berlanga JJ, Scheuner D, Kaufman RJ, Ron D, Sonenberg N. Activation of GCN2 in UV-irradiated cells inhibits translation. Curr Biol. 2002,12:1279-1286.
[16]Wek RC, Jackson BM, Hinnebusch AG. Juxtaposition of domains homologous to protein kinases and histidyl-tRNA synthetases in GCN2 protein suggests a mechanism for coupling GCN4 expression to amino acid availability. Proc Natl Acad Sci USA.1989,86:4579-4583.
[17]Hinnebusch AG, Natarajan K.Gcn4p.a master regulator of gene expression, is controlled at multiple levels by diverse signals of starvation and stress. Eukaryot Cell,2002,1:22-32.
[18]Zhang F, Hinnebusch AG.An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA. Nucleic Acids Res. 2011,39:3128-40.
[19]Sood R, Porter AC, Olsen DA, Cavener DR, Wek RC.A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2a. Genetics.2000,154:787-801.
[20]Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D.Regulated translation initiation controls stress-inducedgene expression in mammalian cells. Mol Cell.2000,6: 1099-1108.
[21]Zhang P, McGrath BC, Reinert J, Olsen DS, Lei L, Gill S, Wek SA,Vattem KM, Wek RC, Kimball SR, Jefferson LS, Cavener DR.The GCN2 eIF2α kinase is required for adaptationto amino acid deprivation in mice. Mol Cell Biol.2002,22:6681-6688.
[22]Juan J Berlanga, Ivan Ventoso, Heather P Harding, Jing Deng, David Ron, Nahum Sonenberg, Luis Carrasco, Cesar de Haro.Antiviral effect of the mammalian translation initiation factor 2a kinase GCN2 against RNA viruses. EMBO J.2006,25:1730-1740.
[23]Hinnebusch AG. Mechanisms of gene regulation in the generalcontrol of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol.Rev.1988,52:248-273.
[24]Natarajan K,Meyer MR, Jackson BM,Slade D,Roberts C, Hinnebusch AG, Marton MJ.Transcriptional profiling shows thatGcn4p is a master regulator of gene expression during amino acid starvationin yeast. Mol Cell Biol.2001,21:4347-4368.
[25]Albrecht G., Mosch HU, Hoffman B, Reusser U, Braus G.H. Monitoring the Gcn4 protein-mediated response in the yeast Saccharomycescerevisiae. J Biol Chem. 1998,273:12696-12702.
[26]Shi Y, Vattem KM, Sood R, An J, Liang J, Stramm L, et al. Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol Cell Biol.1998 18:7499-509.
[27]Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Molecular cell.2000 5:897-904.
[28]Bi M, Naczki C, Koritzinsky M, et al. ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. EMBO J.2005,4:3470-81.
[29]Blais JD, Addison CL, Edge R, et al. Perk-dependent translational regulation promotes tumor celladaptation and angiogenesis in response to hypoxic stress. Mol Cell Biol.2006,26:9517-32.
[30]Sequeira SJ, Ranganathan AC, Adam AP, Iglesias BV, Farias EF, Aguirre-Ghiso JA. Inhibition ofproliferation by PERK regulates mammary acinar morphogenesis and tumor formation. PLoS ONE.2007,2:e615.
[31]Ranganathan AC, Ojhal S, Kourtidis A, Conklin DS, Aguirre-Ghiso J. Dual function of PERK in tumor cell growth arrest and survival. NIH. 2008,68:3260-3268.
[32]Williams BRG. PKR; a sentinel kinase for cellular stress. Oncogene.1999,18:6112-6120.
[33]Sudhakar A, Ramachandran A, Ghosh S, Hasnain SE, Kaufman RJ, Ramaiah KV. Phosphorylation of serine 51 in initiation factor 2a (eIF2a) promotes complex formation between eIF2a(P) and eIF2B and causes inhibition in the guanine nucleotide exchange activity of eIF2B. Biochemistry.2000,39:12929e38.
[34]Nanduri S, Carpick BW, Yang Y, Williams BRG and Qin J. Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation. Embo J.1998,17:5458-65.
[35]Manche L, Green S, Schmedt C and Mathews M.Interactions between double-stranded RNAregulators and the protein kinase DAI. Mol Cell Biol.1992,12:5238-48.
[36]Carpick BW, Graziano V, Schneider D, Maitra RK, Lee X, Williams BRG. Characterization of the solution complex between the interferon-induced, doublestranded RNA-activated protein kinase PKR and HIV-1 TAR RNA. JBiol Chem.1997,272:9510-9516.
[37]Rice AP, Kostura M, Mathews MB. Identification of a 90-kDa polypeptide which associates with adenovirus VA RNAI and is phosphorylated by the double-stranded RNA-dependent protein kinase. JBiol Chem.1989,264:20632-20637.
[38]Langland JO, Jacobs BL. Cytosolic double-stranded RNA-dependent protein kinase is likely a dimer of partially phosphorylated Mr= 66,000 subunits. J Biol Chem.1992,267:10729-36.
[39]Thomis DC, Samuel CE. Mechanism of interferon action:evidence for intermolecular autophosphorylation and autoactivation ofthe interferon-induced, RNA-dependent protein kinase PKR. J Virol.1993,67:7695-7700.
[40]Romano PR, Green SR, Barber G.N, Mathews MB, Hinnebusch AG. Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF2a kinase DAI in Saccharomyces cerevisiae. Mol Cell Biol.1995,15:365-378.
[41]Patel RC, Stanton P, Mcmillan NMJ, Williams BRG, Sen G.C. The interferon-inducibledouble-stranded RNA-activated protein kinase self-associates invitro and in vivo. Proc Nati Acad Sci USA.1995,92:8283-8287.
[42]Ortega LG, Mccotter MD, Henry GL, Mccormack SJ, Thomis DC, Samuel CE. Mechanism of interferon action-biochemical and genetic evidence forthe intcrmolccular association of the RNA-dependent protein ki-nase PKR from human cells. Virology.1996,215:31-39.
[43]Wu S, Rehemtulla A, Gupta NK, Kaufman RJ. A eukaryotic translation initiation factor 2-associated 67 kDa glycoprolein partially reverses protein synthesis inhibi-tion by activated double-stranded RNA-dependent protein kinasein intact cells. Biochemistry.1996,35:S275-8280.
[44]Tan SL, Gale MJ Jr, Katze MG. Double-stranded RNA-independent dimerization of interferon-induced protein kinase PKR and inhibition of dimerization by the cellular P58IPK inhibitor. Mol. Cell. Biol.1998,18:2431-2443.
[45]Patel RC, STANTON, P., and SEN, G.C. Specificmutations near the amino terminus of double-stranded RNA-depcndcnlprolein kinase (PKR) differentially affect its doublestrandedRNA binding and dimerization properties. J. Biol. Chem.1996,271: 25657-25663.
[46]Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells respond to interferons.Ann Biochem.1998,67:227-264.
[47]Tan SL, Katze MG. The emerging role of the interferon-induced PKR protein kinase as an apoptotic effector:a new face of death? J Interferon Cytokine Res.1999,19:543-54.
[48]Balachandran S, Roberts PC, Brown LE, Truong H, Pattnaik AK, Archer DR, Barber GN.Essential role for the dsRN A-dependent protein kinase PKR in innate immunity to viral infection. Immunity.2000,13:129-41.
[49]Stojdl DF, Abraham N, Knowles S, Marius R, Brasey A, Lichty BD, Brown EG, Sonenberg N, Bell JC. The murine double-stranded RNA-dependent protein kinase PKR is required for resistance to vesicular stomatitis virus.J Virol.2000,74:9580-5.
[50]Cuddihy AR, Li S, Tam NW, Wong AH, Taya Y, Abraham N, Bell JC, Koromilas AE.Double-stranded-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53. Mol Cell Biol.1999,19:2475-84.
[51]Yang YL, Reis LFL, Pavlovic J, Aguzzi,.,SCHAFER, R., RUMAR,., WILLIAMS, B.R.G., AGUET, M.,and WEISSMANN, C.Deficient signaling in mice devoidof douhle-stranded RNA-dependent protein kinase, PRR. EMBOJ.1995,14:6095-6106.
[52]Berlanga JJ, Ventoso I, Harding HP, Deng J, Ron D, Sonenberg N, Carrasco L, de Haro C. Antiviral effect of the mammalian translation initiation factor 2alpha kinase GCN2 against RNA viruses. EMBO J.2006,25:1730-40.
[53]del Castillo CS, Hikima J, Ohtani M, Jung TS, Aoki T. Characterization and functional analysis of two PKR genes in fugu (Takifugu rubripes). Fish Shellfish Immunol. 2012,32:79-88.
[54]Clemens MJ, Elia A.The double-stranded RNA-dependent protein kinase PKR:structure and function. J Interferon Cytokine Res.1997,17:503-24.
[55]Kumar AJ, Haque J, Lacoste J, Hiscott, BRG Williams. Double-stranded RNA-dependent protein kinase activates transcription factorNF-kB by phosphorylating IkB. Proc. Natl. Acad. Sci. USA.1994,91:6288-6292.
[56]Offermann MK, Zimring J, Mellits KH, Hagan MK, Shaw R, Medford RM,Mathews MB,Goodbourn S,Jagus R. Activation ofthe double-stranded-RNA-activated protein kinase and induction of vascularcell adhesion molecule-1 by poly(I)×poly(C) in endothelial cells. Eur.J. Biochem.1995,232:28-36.
[57]Galabru J,Hovanessian A. Two interferon-induced proteins areinvolved in the protein kinase complex dependent on double-stranded RNA.Cell.1985,43:685-694.
[58]Rice AR, Kostura M,Mathews MB. Identification of a 90 KD polypeptide which associates with adenovirus VA RNA1 and is phosphorylatedby the double-stranded RNA dependent protein kinase. J. Biol. Chem.1989,264:20632-20637.
[59]Brand SR,Kobayashi R,Mathews MB. The Tat protein of humanimmunodeficiency virus type 1 is a substrate and inhibitor of the interferoninduced,virus-activated protein kinase, PKR. Submitted for publication.
[60]McMillan NA, Chun RF, Siderovski DP, Galabru J, Toone WM, Samuel CE, Mak TW, Hovanessian AG, Jeang KT, Williams BR..HIV-1 Tat directly interacts with the interferon-induced,double-stranded RNA-dependent kinase, PKR. Virology 1995,213:413-424.
[61]彭悟,汤雅男,胡成钰CaPKR-like在鲫鱼与草鱼组织中的表达特性分析.动物学研究.2007 28：465-9.
[62]Hu CY, Zhang YB, Huang GP, Zhang QY, Gui JF. Molecular cloning and characterisation of a fish PKR-like gene from cultured CAB cells induced by UV-inactivated virus. Fish & shellfish immunology.2004 17:353-66.
[63]Wu CX, Wang SJ, Lin G, Hu CY. The Za domain of PKZ from Carassius auratus can bind to d(GC)n in negative supercoils. Fish Shellfish Immunol,2010,28:783-788.
[64]卢普忠.鲫鱼PKR-like(PKZ)Za能将B-DNA翻转为Z-DNA:南昌大学硕士学位论文；2011.
[65]Rothenburg S, Deigendesch N, Dittmar K, Koch-Nolte F, Haag F, Lowenhaupt K... A PKR-like eukaryotic initiation factor2a kinase from zebrafish contains Z-DNA binding domains instead of dsRNA binding domains. Proc Nati Acad Sci USA 2005; 102:1602-1607.
[66]Rothenburg S, Deigendesch N, Dey M, Dever TE, Tazi L. Double-stranded RNA-activated protein kinase PKR of fishes and amphibians:varying the number of double-stranded RNA binding domains and lineage-specific duplications. BMC Biol.2008,6:12.
[67]Bergan V, Jagus R, Lauksund S, Kileng (?), Robertsen B. The Atlantic salmon Z-DNA binding protein kinase phosphorylates translation initiation factor 2 alpha and constitutes a unique orthologue to the mammalian dsRNA activated protein kinase R. FEBS J 2008;275:184-197.
[68]Su JG, Zhu ZY, Wang YP. Molecular cloning, characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus. Fish Shellfish Immunol 2008;25:106-113.
[69]Liu TK, Zhang YB, Liu Y, Sun F, Gui JF. Cooperative roles offish protein kinase containing Z-DNA binding domains and double-stranded RNA-dependent protein kinase in interferon-mediated antiviral response.. J Virol.2011,85:12769-80.
[70]Romano PR, Garcia-Barrio MT, Zhang X, Wang Q,Taylor DR, Zhang F, Herring C, Mathews MB, Qin J& Hinnebusch AG.Autophosphorylation in the activation loop is required for full kinase activity invivo of human and yeast eukaryotic initiation factor2alpha kinases PKR and GCN2.Mol Cell Biol.1998,18:2282-2297.
[71]Barber GN, Tomita J, Hovanessian AG, Meurs E &Katze MG.Functional expression and characterizationof the interferon-induced double-stranded RNAactivated P68 protein kinase from Escherichia coli.Biochemistry.1991,30:10356-10361.
[72]Jammi NV & Beal PA.Phosphorylation of theRNA-dependent protein kinase regulates its RNA-bindingactivity. Nucleic Acids Res.2001,29:3020-3029.
[73]Taylor DR, Lee SB, Romano PR, Marshak DR,Hinnebusch AG, Esteban M & Mathews MB.Autophosphorylation sites participate in the activationof the double-stranded-RNA-activated protein kinasePKR.Mol Cell Biol.1996,16:6295-6302.
[74]Lemaire PA, Lary J, Cole JL. Mechanism of PKR activation:dimerization and kinase activation in the absence of double-stranded RNA. Journal of molecular biology.2005 345:81-90.
[75]Tian B, Mathews MB. Functional characterization of and cooperation between the double-stranded RNA-binding motifs of the protein kinase PKR. The Journal of biological chemistry.2001 276:9936-44.
[76]胡成钰.鲫鱼PKR-like基因的鉴定及其特征分析：中国科学院理学博十学位论文：2004.
[77]汤雅男.PKZ在草鱼组织中表达特性及原子力显微镜观察PKZ PZa结合d(GC)13重组质粒：南昌大学硕十学位论文；2008.
[78]Kim D, Lee YH, Hwang HY, Kim KK, Park HJ. Z-DNA binding proteins as targets for structure-based virtual screening.Current Drug Targets.2010,11:335-344.
[79]Rich A, Zhang S. Z-DNA:the long road to biological function. Nature Reviews Genetics.2003,4:566-572.
[80]Bae S, Kim D, Kim KK, Kim YG, Hohng S. Intrinsic Z-DNA is stabilized by the conformational selection mechanism of Z-DNA-binding proteins. J Am Chem Soc,2011, 13:668-671.
[81]Wang G, Vasquez KM. Z-DNA, an active element in the genome. Frontiers in Bioscience, 2007,12:4424-4438.
[82]de Rosa M, de Sanctis D, Rosario AL, Archer M, Rich A, Athanasiadis A, Carrondo MA. Crystal structure of a junction between two Z-DNA helices. Proc Nati Acad Sci USA, 2010,107:9088-9092.
[2]Clemens MJ. Regulation of eukaryotic protein synthesis by protein kinases that phosphorylate initiation factor eIF-2. Molecular biology reports.1994,19:201-10.
[3]de Haro C, Mendez R, Santoyo J. The eIF-2alpha kinases and the control of protein synthesis.FASEB J.1996,10:1378-87.
[4]Dever TE. Gene-specific regulation by general translation factors. Cell.2002,108:545-56.
[5]Lu L, Han AP, Chen JJ. Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol Cell Biol. 2001,21:7971-80.
[6]Chen J J, Throop M S, Gehrke L, et al. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2a (eIF-2α) kinase of rabbit retilocytes:homology to yeast GCN2 protein kinase and human double-stranded RNA-depentent eIF-2α kinase.Proc Natl Acad Sci USA.1991,88:7729-7733.
[7]Sudhakar A, Ramachandran A, Ghosh S, Hasnain SE, Kaufman RJ, Ramaiah KVA. Phosphorylation of serine 51 in initiation factor 2a (eIF2α) promotes complex formation between eIF2α (P) and eIF2B and causes inhibition in the guanine nucleotide exchange activity of zI2B.Biochemistry.2000,39:12929-12938.
[8]Chen JJ, London IM. Regulation of protein synthesis by heme-regulated eIF-2α kinase. Trends in Biochemical Sciences.1995,20:105-108.
[9]Chen JJ. Regulation of protein synthesis by the hemeregulated eIF2α kinase:relevance to anemias. Blood.2007,109:2693-2699.
[10]Crosby JS, Lee K, London IM, Chen JJ. Erythroid expression of the heme-regulated eIF-2akinase. Molecular and Cellular Biology.1994,14:3906-3914.
[11]Han AP, Yu C, Lu L, et al. Heme-regulated eIF2a kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency. EMBO.2001, 20:6909-6918.
[12]Chen JJ. Heme-regulated eIF2α kinase. in Translational Control of Gene Expression.2000,vol.39 of Cold Spring Harbor Monograph Series, pp.529-546, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
[13]Berlanga JJ, Santoyo J, de Haro C.Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2a kinase. Eur J Biochem.1999,265:754-762
[14]Hinnebusch AG.Mechanism and regulation of initiator methionyl-tRNA binding to ribosomes. In Translational Control of Gene Expression, Sonenberg N, Hershey JWB, Mathews MB (eds).2000,pp 185-243. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory Press.
[15]Deng J, Harding HP, Raught B, Gingras AC, Berlanga JJ, Scheuner D, Kaufman RJ, Ron D, Sonenberg N. Activation of GCN2 in UV-irradiated cells inhibits translation. Curr Biol. 2002,12:1279-1286.
[16]Wek RC, Jackson BM, Hinnebusch AG. Juxtaposition of domains homologous to protein kinases and histidyl-tRNA synthetases in GCN2 protein suggests a mechanism for coupling GCN4 expression to amino acid availability. Proc Natl Acad Sci USA.1989,86:4579-4583.
[17]Hinnebusch AG, Natarajan K.Gcn4p.a master regulator of gene expression, is controlled at multiple levels by diverse signals of starvation and stress. Eukaryot Cell,2002,1:22-32.
[18]Zhang F, Hinnebusch AG.An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA. Nucleic Acids Res. 2011,39:3128-40.
[19]Sood R, Porter AC, Olsen DA, Cavener DR, Wek RC.A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2a. Genetics.2000,154:787-801.
[20]Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D.Regulated translation initiation controls stress-inducedgene expression in mammalian cells. Mol Cell.2000,6: 1099-1108.
[21]Zhang P, McGrath BC, Reinert J, Olsen DS, Lei L, Gill S, Wek SA,Vattem KM, Wek RC, Kimball SR, Jefferson LS, Cavener DR.The GCN2 eIF2α kinase is required for adaptationto amino acid deprivation in mice. Mol Cell Biol.2002,22:6681-6688.
[22]Juan J Berlanga, Ivan Ventoso, Heather P Harding, Jing Deng, David Ron, Nahum Sonenberg, Luis Carrasco, Cesar de Haro.Antiviral effect of the mammalian translation initiation factor 2a kinase GCN2 against RNA viruses. EMBO J.2006,25:1730-1740.
[23]Hinnebusch AG. Mechanisms of gene regulation in the generalcontrol of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol.Rev.1988,52:248-273.
[24]Natarajan K,Meyer MR, Jackson BM,Slade D,Roberts C, Hinnebusch AG, Marton MJ.Transcriptional profiling shows thatGcn4p is a master regulator of gene expression during amino acid starvationin yeast. Mol Cell Biol.2001,21:4347-4368.
[25]Albrecht G., Mosch HU, Hoffman B, Reusser U, Braus G.H. Monitoring the Gcn4 protein-mediated response in the yeast Saccharomycescerevisiae. J Biol Chem. 1998,273:12696-12702.
[26]Shi Y, Vattem KM, Sood R, An J, Liang J, Stramm L, et al. Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol Cell Biol.1998 18:7499-509.
[27]Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Molecular cell.2000 5:897-904.
[28]Bi M, Naczki C, Koritzinsky M, et al. ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. EMBO J.2005,4:3470-81.
[29]Blais JD, Addison CL, Edge R, et al. Perk-dependent translational regulation promotes tumor celladaptation and angiogenesis in response to hypoxic stress. Mol Cell Biol.2006,26:9517-32.
[30]Sequeira SJ, Ranganathan AC, Adam AP, Iglesias BV, Farias EF, Aguirre-Ghiso JA. Inhibition ofproliferation by PERK regulates mammary acinar morphogenesis and tumor formation. PLoS ONE.2007,2:e615.
[31]Ranganathan AC, Ojhal S, Kourtidis A, Conklin DS, Aguirre-Ghiso J. Dual function of PERK in tumor cell growth arrest and survival. NIH. 2008,68:3260-3268.
[32]Williams BRG. PKR; a sentinel kinase for cellular stress. Oncogene.1999,18:6112-6120.
[33]Sudhakar A, Ramachandran A, Ghosh S, Hasnain SE, Kaufman RJ, Ramaiah KV. Phosphorylation of serine 51 in initiation factor 2a (eIF2a) promotes complex formation between eIF2a(P) and eIF2B and causes inhibition in the guanine nucleotide exchange activity of eIF2B. Biochemistry.2000,39:12929e38.
[34]Nanduri S, Carpick BW, Yang Y, Williams BRG and Qin J. Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation. Embo J.1998,17:5458-65.
[35]Manche L, Green S, Schmedt C and Mathews M.Interactions between double-stranded RNAregulators and the protein kinase DAI. Mol Cell Biol.1992,12:5238-48.
[36]Carpick BW, Graziano V, Schneider D, Maitra RK, Lee X, Williams BRG. Characterization of the solution complex between the interferon-induced, doublestranded RNA-activated protein kinase PKR and HIV-1 TAR RNA. JBiol Chem.1997,272:9510-9516.
[37]Rice AP, Kostura M, Mathews MB. Identification of a 90-kDa polypeptide which associates with adenovirus VA RNAI and is phosphorylated by the double-stranded RNA-dependent protein kinase. JBiol Chem.1989,264:20632-20637.
[38]Langland JO, Jacobs BL. Cytosolic double-stranded RNA-dependent protein kinase is likely a dimer of partially phosphorylated Mr= 66,000 subunits. J Biol Chem.1992,267:10729-36.
[39]Thomis DC, Samuel CE. Mechanism of interferon action:evidence for intermolecular autophosphorylation and autoactivation ofthe interferon-induced, RNA-dependent protein kinase PKR. J Virol.1993,67:7695-7700.
[40]Romano PR, Green SR, Barber G.N, Mathews MB, Hinnebusch AG. Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF2a kinase DAI in Saccharomyces cerevisiae. Mol Cell Biol.1995,15:365-378.
[41]Patel RC, Stanton P, Mcmillan NMJ, Williams BRG, Sen G.C. The interferon-inducibledouble-stranded RNA-activated protein kinase self-associates invitro and in vivo. Proc Nati Acad Sci USA.1995,92:8283-8287.
[42]Ortega LG, Mccotter MD, Henry GL, Mccormack SJ, Thomis DC, Samuel CE. Mechanism of interferon action-biochemical and genetic evidence forthe intcrmolccular association of the RNA-dependent protein ki-nase PKR from human cells. Virology.1996,215:31-39.
[43]Wu S, Rehemtulla A, Gupta NK, Kaufman RJ. A eukaryotic translation initiation factor 2-associated 67 kDa glycoprolein partially reverses protein synthesis inhibi-tion by activated double-stranded RNA-dependent protein kinasein intact cells. Biochemistry.1996,35:S275-8280.
[44]Tan SL, Gale MJ Jr, Katze MG. Double-stranded RNA-independent dimerization of interferon-induced protein kinase PKR and inhibition of dimerization by the cellular P58IPK inhibitor. Mol. Cell. Biol.1998,18:2431-2443.
[45]Patel RC, STANTON, P., and SEN, G.C. Specificmutations near the amino terminus of double-stranded RNA-depcndcnlprolein kinase (PKR) differentially affect its doublestrandedRNA binding and dimerization properties. J. Biol. Chem.1996,271: 25657-25663.
[46]Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells respond to interferons.Ann Biochem.1998,67:227-264.
[47]Tan SL, Katze MG. The emerging role of the interferon-induced PKR protein kinase as an apoptotic effector:a new face of death? J Interferon Cytokine Res.1999,19:543-54.
[48]Balachandran S, Roberts PC, Brown LE, Truong H, Pattnaik AK, Archer DR, Barber GN.Essential role for the dsRN A-dependent protein kinase PKR in innate immunity to viral infection. Immunity.2000,13:129-41.
[49]Stojdl DF, Abraham N, Knowles S, Marius R, Brasey A, Lichty BD, Brown EG, Sonenberg N, Bell JC. The murine double-stranded RNA-dependent protein kinase PKR is required for resistance to vesicular stomatitis virus.J Virol.2000,74:9580-5.
[50]Cuddihy AR, Li S, Tam NW, Wong AH, Taya Y, Abraham N, Bell JC, Koromilas AE.Double-stranded-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53. Mol Cell Biol.1999,19:2475-84.
[51]Yang YL, Reis LFL, Pavlovic J, Aguzzi,.,SCHAFER, R., RUMAR,., WILLIAMS, B.R.G., AGUET, M.,and WEISSMANN, C.Deficient signaling in mice devoidof douhle-stranded RNA-dependent protein kinase, PRR. EMBOJ.1995,14:6095-6106.
[52]Berlanga JJ, Ventoso I, Harding HP, Deng J, Ron D, Sonenberg N, Carrasco L, de Haro C. Antiviral effect of the mammalian translation initiation factor 2alpha kinase GCN2 against RNA viruses. EMBO J.2006,25:1730-40.
[53]del Castillo CS, Hikima J, Ohtani M, Jung TS, Aoki T. Characterization and functional analysis of two PKR genes in fugu (Takifugu rubripes). Fish Shellfish Immunol. 2012,32:79-88.
[54]Clemens MJ, Elia A.The double-stranded RNA-dependent protein kinase PKR:structure and function. J Interferon Cytokine Res.1997,17:503-24.
[55]Kumar AJ, Haque J, Lacoste J, Hiscott, BRG Williams. Double-stranded RNA-dependent protein kinase activates transcription factorNF-kB by phosphorylating IkB. Proc. Natl. Acad. Sci. USA.1994,91:6288-6292.
[56]Offermann MK, Zimring J, Mellits KH, Hagan MK, Shaw R, Medford RM,Mathews MB,Goodbourn S,Jagus R. Activation ofthe double-stranded-RNA-activated protein kinase and induction of vascularcell adhesion molecule-1 by poly(I)×poly(C) in endothelial cells. Eur.J. Biochem.1995,232:28-36.
[57]Galabru J,Hovanessian A. Two interferon-induced proteins areinvolved in the protein kinase complex dependent on double-stranded RNA.Cell.1985,43:685-694.
[58]Rice AR, Kostura M,Mathews MB. Identification of a 90 KD polypeptide which associates with adenovirus VA RNA1 and is phosphorylatedby the double-stranded RNA dependent protein kinase. J. Biol. Chem.1989,264:20632-20637.
[59]Brand SR,Kobayashi R,Mathews MB. The Tat protein of humanimmunodeficiency virus type 1 is a substrate and inhibitor of the interferoninduced,virus-activated protein kinase, PKR. Submitted for publication.
[60]McMillan NA, Chun RF, Siderovski DP, Galabru J, Toone WM, Samuel CE, Mak TW, Hovanessian AG, Jeang KT, Williams BR..HIV-1 Tat directly interacts with the interferon-induced,double-stranded RNA-dependent kinase, PKR. Virology 1995,213:413-424.
[61]彭悟,汤雅男,胡成钰CaPKR-like在鲫鱼与草鱼组织中的表达特性分析.动物学研究.2007 28：465-9.
[62]Hu CY, Zhang YB, Huang GP, Zhang QY, Gui JF. Molecular cloning and characterisation of a fish PKR-like gene from cultured CAB cells induced by UV-inactivated virus. Fish & shellfish immunology.2004 17:353-66.
[63]Wu CX, Wang SJ, Lin G, Hu CY. The Za domain of PKZ from Carassius auratus can bind to d(GC)n in negative supercoils. Fish Shellfish Immunol,2010,28:783-788.
[64]卢普忠.鲫鱼PKR-like(PKZ)Za能将B-DNA翻转为Z-DNA:南昌大学硕士学位论文；2011.
[65]Rothenburg S, Deigendesch N, Dittmar K, Koch-Nolte F, Haag F, Lowenhaupt K... A PKR-like eukaryotic initiation factor2a kinase from zebrafish contains Z-DNA binding domains instead of dsRNA binding domains. Proc Nati Acad Sci USA 2005; 102:1602-1607.
[66]Rothenburg S, Deigendesch N, Dey M, Dever TE, Tazi L. Double-stranded RNA-activated protein kinase PKR of fishes and amphibians:varying the number of double-stranded RNA binding domains and lineage-specific duplications. BMC Biol.2008,6:12.
[67]Bergan V, Jagus R, Lauksund S, Kileng (?), Robertsen B. The Atlantic salmon Z-DNA binding protein kinase phosphorylates translation initiation factor 2 alpha and constitutes a unique orthologue to the mammalian dsRNA activated protein kinase R. FEBS J 2008;275:184-197.
[68]Su JG, Zhu ZY, Wang YP. Molecular cloning, characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus. Fish Shellfish Immunol 2008;25:106-113.
[69]Liu TK, Zhang YB, Liu Y, Sun F, Gui JF. Cooperative roles offish protein kinase containing Z-DNA binding domains and double-stranded RNA-dependent protein kinase in interferon-mediated antiviral response.. J Virol.2011,85:12769-80.
[70]Romano PR, Garcia-Barrio MT, Zhang X, Wang Q,Taylor DR, Zhang F, Herring C, Mathews MB, Qin J& Hinnebusch AG.Autophosphorylation in the activation loop is required for full kinase activity invivo of human and yeast eukaryotic initiation factor2alpha kinases PKR and GCN2.Mol Cell Biol.1998,18:2282-2297.
[71]Barber GN, Tomita J, Hovanessian AG, Meurs E &Katze MG.Functional expression and characterizationof the interferon-induced double-stranded RNAactivated P68 protein kinase from Escherichia coli.Biochemistry.1991,30:10356-10361.
[72]Jammi NV & Beal PA.Phosphorylation of theRNA-dependent protein kinase regulates its RNA-bindingactivity. Nucleic Acids Res.2001,29:3020-3029.
[73]Taylor DR, Lee SB, Romano PR, Marshak DR,Hinnebusch AG, Esteban M & Mathews MB.Autophosphorylation sites participate in the activationof the double-stranded-RNA-activated protein kinasePKR.Mol Cell Biol.1996,16:6295-6302.
[74]Lemaire PA, Lary J, Cole JL. Mechanism of PKR activation:dimerization and kinase activation in the absence of double-stranded RNA. Journal of molecular biology.2005 345:81-90.
[75]Tian B, Mathews MB. Functional characterization of and cooperation between the double-stranded RNA-binding motifs of the protein kinase PKR. The Journal of biological chemistry.2001 276:9936-44.
[76]胡成钰.鲫鱼PKR-like基因的鉴定及其特征分析：中国科学院理学博十学位论文：2004.
[77]汤雅男.PKZ在草鱼组织中表达特性及原子力显微镜观察PKZ PZa结合d(GC)13重组质粒：南昌大学硕十学位论文；2008.
[78]Kim D, Lee YH, Hwang HY, Kim KK, Park HJ. Z-DNA binding proteins as targets for structure-based virtual screening.Current Drug Targets.2010,11:335-344.
[79]Rich A, Zhang S. Z-DNA:the long road to biological function. Nature Reviews Genetics.2003,4:566-572.
[80]Bae S, Kim D, Kim KK, Kim YG, Hohng S. Intrinsic Z-DNA is stabilized by the conformational selection mechanism of Z-DNA-binding proteins. J Am Chem Soc,2011, 13:668-671.
[81]Wang G, Vasquez KM. Z-DNA, an active element in the genome. Frontiers in Bioscience, 2007,12:4424-4438.
[82]de Rosa M, de Sanctis D, Rosario AL, Archer M, Rich A, Athanasiadis A, Carrondo MA. Crystal structure of a junction between two Z-DNA helices. Proc Nati Acad Sci USA, 2010,107:9088-9092.