Expression profiling of the 14-3-3 gene family in response to salt stress and potassium and iron

DOI：10.19904/14-1160/s.2022.09.004水稻耐盐研究进展及展望蒋子凡（扬州大学，江苏扬州225000）摘要：土地盐碱化是世界范围内农业面临的重大问题之一。

全面了解盐胁迫对植物的危害性以及植物盐胁迫响应机制，将为增强作物耐盐能力提供研究基础。

水稻作为全球最重要的粮食作物之一，日益严重的土地盐碱化制约了其产量与品质。

综述盐胁迫条件对水稻生长发育、生理生化产生的影响以及目前对于水稻耐盐相关基因的研究，以期通过分子生物技术培育耐盐水稻新品种，实现水稻种植面积和总产量提高，保障粮食安全。

关键词：水稻；耐盐性；数量性状基因座文章编号：1005-2690（2022）09-0010-03中国图书分类号：S511文献标志码：B作者简介：蒋子凡（1997—），女，汉族，江苏扬州人，在读硕士，研究方向为玉米遗传育种。

在世界范围内，盐渍土面积约8.33亿hm 2，占总耕地面积的1/5。

而且随着人类活动范围不断扩大、极端气候增多、淡水资源不断减少等问题日益严重，盐渍土面积还在不断扩大[1]。

水稻作为世界第二大粮食作物，全世界大约有1/3的人口以稻米为主食。

深入了解耐盐机理、提高水稻的耐盐能力，能够提高对于盐渍土地的利用率，提升经济效益，对缓解世界粮食危机具有重大意义。

造成土壤盐分过高的原因有很多，目前已知高盐地下水灌溉、沿海地区海水释放等因素导致土地盐分积累[2]。

盐胁迫对于作物的伤害主要是脱水、渗透性应激反应、积累离子毒害和离子不平衡，最终导致作物缺乏营养。

这些伤害会抑制作物生长，造成减产甚至死亡。

土壤中盐分过多会导致土壤板结，植物难以建立根系。

土壤含水量减少，水势降低，引起渗透胁迫，造成植物水分亏欠，影响作物吸收营养物质，导致植株营养缺乏。

已有研究表明，许多基因在盐胁迫下可发挥调节作用，提升作物耐盐性。

虽然不同作物的抗逆能力不同，但在盐胁迫下作物的产量和品质都会受不同程度的影响。

水稻耐盐性是指在盐害环境下水稻对抗外界盐胁迫的能力。

REVIEW ARTICLERegulation of gene expression in the nervous systemLezanne OOI and Ian C.WOOD 1Institute of Membrane and Systems Biology,Faculty of Biological Sciences,Garstang Building,University of Leeds,Leeds LS29JT,U.K.The nervous system contains a multitude of cell types which are specified during development by cascades of transcription factors acting combinatorially.Some of these transcription factors are only active during development,whereas others continue to function in the mature nervous system to maintain appropriate gene-expression patterns in differentiated cells.Underpinning the function of the nervous system is its plasticity in response to external stimuli,and many transcription factors are involved in regulating gene expression in response to neuronal activity,allowing us to learn,remember and make complex decisions.Here we review some of the recent findings that have uncovered the molecular mechanisms that underpin the control of gene regulatory networks within the nervous system.We highlight some recent insights into the gene-regulatory circuits in the development and differentiation of cells within the nervous system and discuss some of the mechanisms by which synaptictransmission influences transcription-factor activity in the mature nervous system.Mutations in genes that are important in epigenetic regulation (by influencing DNA methylation and post-translational histone modifications)have long been associated with neuronal disorders in humans such as Rett syndrome,Huntington’s disease and some forms of mental retardation,and recent work has focused on unravelling their mechanisms of action.Finally,the discovery of microRNAs has produced a paradigm shift in gene expression,and we provide some examples and discuss the contribution of microRNAs to maintaining dynamic gene regulatory networks in the brain.Key words:chromatin,gene expression,microRNA,nervous system,synaptic plasticity,transcription.INTRODUCTIONThe brain is the most complex organ in the human body,con-taining the largest diversity of cell types of any organ.Collect-ively,cells that form the nervous system express 80%of genes in the genome [1].However,each individual cell type expresses a distinct subset of those genes.Preservation of appropriate ex-pression of these genes is a highly regulated process during devel-opment to ensure production of correct numbers of the different cell types and to maintain essential neuronal signalling plexity within the brain continues into adulthood,and cells undergo phenotypic changes in response to environmental cues and neuronal signalling.Such plasticity is vital and underlies our higher cognitive functions,such as learning and memory.Development of the nervous system is brought about by waves of transcription factors,which act combinatorially to specify neural gene networks and determine cell fate.Many of these transcription factors are not expressed in the adult brain;rather,they wield their power during development,bringing about lasting gene-expression changes that extend into adulthood.Nevertheless a substantial number of transcription factors are expressed in the brain and are vital for regulating phenotypic plasticity by controlling expression of a multitude of genes.It is now well established that alterations in gene expression are important in learning and memory,and also that inappropriate regulation of gene expression is a cause of a multitude of neuronal diseases.Though many mechanisms that control gene expression in neurons have been uncovered,there is still much work to be done before we fully understand how these individual mechanisms are integrated and feed into neuronal gene networks to create a complex organ that maintains homoeostatic control of our bodies,allows us to interpret our environment and to make complex decisions.Here we review some of the recent advances that have been made in elucidating the mechanisms that regulate neuronal gene expression and highlight the insights that have contributed to our understanding of the progression of neuronal disease.Abbreviations used:AA-NAT,acylalkalamine N -acetyltransferase;AMPA,α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;ATRX,α-thalassaemia/mental retardation,X-linked;BMP ,bone morphogenetic protein;Bdnf ,BDNF (brain-derived neurotrophic factor)gene;bHLH,basic helix–loop–helix;BRAF35,BRCA2-associated factor 35;Calb1,calbindin gene;CaMKIV ,calmodulin kinase IV;Cdk1,cyclin-dependent kinase 1;CK1,casein kinase-1;CREB,cAMP-response-element-binding protein;CBP,CREB-binding protein;CCAT,calcium-channel-associated transcription regulator;CCR,C–C chemokine receptor;ChIP ,chromatin immunoprecipitation,CREM,cAMP-responsive-element modulator;DNMT,DNA methyltransferase;DRE,downstream regulatory element;DREAM,downstream-regulatory-element antagonistic modulator;DRG,dorsal root ganglion;DSCR1,Down syndrome critical region gene 1;DYRKIA,dual-specificity tyrosine-phosphorylation-regulated kinase 1A;ERK,extracellular-signal-regulated kinase;ES,embryonic stem;ESET,ERG (ets-related gene)-associated protein with SET (suppressor of variegation,enhancer of zest and trithorax)domain;FGF ,fibroblast growth factor;FMRP ,fragile X mental retardation protein;FOXO1,forkhead box O1;GABA,γ-aminobutyric acid;Gadd45a,growth arrest and DNA-damage-inducible 45alpha;GluR2,glutamate receptor subunit 2;Gria2,glutamate receptor,ionotropic,AMPA2;GSK3,glycogen synthase kinase 3;H3K4,histone H3Lys 4(etc.);HAT,histone acetyltransferase;HDAC,histone deacetylase;HEK-293,human embryonic kidney-293;ICER,inducible cAMP repressor;IP 3R1,Ins(1,4,5)P 3receptor type 1;JARID1C,jumonji,AT -rich interactive domain 1C;Lmtk1,lemur tyrosine kinase 1;LSD,lysine-specific demethylase;LTP ,long-term potentiation;MBD2,methyl-CpG-binding domain protein 2;MeCP2,methyl-CpG binding protein 2;mEPSC,miniature excitatory postsynaptic current;MKP ,MAPK (mitogen-activated protein kinase)phosphatase;MSK,mitogen-and stress-activated kinase;MLL,mixed-lineage leukaemia;MOR1,μ-opiod receptor;NFAT,nuclear factor of activated T -cells;NMDA,N -methyl-D -aspartate;KChIP ,potassium channel-interacting protein;PI3K,phosphoinositide 3-kinase;PP1,protein phosphatase 1;PTBP ,polypyrimidine-tract-binding protein;Ptf1a,pancreas-specific transcription factor 1a;REST,repressor element 1-silencing transcription factor;SCA,spinocerebellar ataxia;TDG,thymine-DNA glycosylase;TORC,transducer of regulated CREB activity;VMN,visceral motor neuron.1To whom correspondence should be addressed (email i.c.wood@).c The Authors Journal compilation c 2008Biochemical SocietyB i o c h e m i c a l J o u r n a l328L.Ooi and I.C.WoodDEVELOPMENT AND DIFFERENTIATIONCells in the nervous system arise from ES(embryonic stem)cells that develop into neural stem cells that,in turn,differentiate into neurons,astrocytes or oligodendrocytes.Many groups have now identified a core regulatory network of the transcription factors Nanog/Sox2/Oct4that is important for controlling pluripotency and self-renewal of ES cells(reviewed in[2]).Combinatorially these transcription factors regulate their own expression levels in addition to those of many other genes,and disruption of any component of the network is sufficient to disrupt the whole system.Once initiated,this circuit is self-maintaining, and differentiation of ES cells toward specific cell fates requires external signalling via secreted molecules such as BMP(bone morphogenetic protein).Nanog is critical for maintaining the ES cell state.The expression of Nanogfluctuates in ES cells over time and,when levels of Nanog are low,ES cells are predisposed to cell differentiation[3].Thus low levels of Nanog provide a window of opportunity for ES cell differentiation that is lost when the levels of Nanog subsequently rise[3].Sox2expression is important for maintaining Oct4levels.In the absence of Sox2, other Sox proteins expressed in ES cells,such as Sox4,11and 15,can co-operate with Oct4to activate expression of target genes,including Oct4itself[4].However,loss of Sox2results in reduced expression of the gene Nr5a2(nuclear receptor subfamily 5,group A,member2),which encodes a steroid hormone receptor that activates Oct4expression,and increased expression of Nr2f2 (nuclear receptor subfamily2,group F,member2),which encodes a steroid hormone receptor that represses Oct4expression.Thus despite the ability of Sox4,11and15to substitute for Sox2and directly enhance Oct4expression,loss of Sox2leads to reduced Oct4levels and ES cell differentiation.In ES cells that lack Sox2, it is the effect on Oct4levels that are crucial,as highlighted by the fact that ectopic expression of Oct4is sufficient to prevent ES-cell differentiation[4].Oct4appears to play a more general role than either Sox2or Nanog.In co-operation with Sox2and Nanog, Oct4binds to and regulates the expression of many genes.Unlike Nanog,Oct4levels do not appear tofluctuate,and redundancy among Oct factors has not been identified.ES cells become committed to neural cell lineages and differentiate toward specific neuronal or glial cell fates in response to a range of signals, including retinoic acid,FGF(fibroblast growth factor),inhibition of BMP signalling,and Notch and Wnt signalling[5].Cell fate specification towards either neural-stem-cell maintenance or differentiation toward post-mitotic neurons or glia is achieved through a balance of antagonistic transcription factors[6,7]. Transcription factors play a key role in specifying neuronal identity upon neuronal differentiation,and much work has been undertaken in an attempt to understand the transcription-factor network that defines specific neuronal binations of transcription factors result in different,but specific,cell fates.The co-ordinated function of homoeodomain and bHLH (basic helix–loop–helix)transcription factors,including Mash1, neurogenin and Math1,are involved in differentiation of neural progenitors into neurons and specification of neuronal subtype [6].Most neurons differentiate toward either a glutamatergic (excitatory neuron)or GABAergic(inhibitory neuron;GABA is γ-aminobutyric acid)phenotype,and the actions of a range of transcription factors have been implicated in implementing this decision in different regions of the nervous system.Expression of the bHLH gene Mash1promotes generation of GABAergic neurons from neural stem cells of the subependymal zone[8], whereas the bHLH transcription factor Ptf1a(pancreas-specific transcription factor1a)[as a heterodimer with RBJ(rab-and DnaJ-domain containing)protein]defines GABAergic neurons in the cerebellum[9].In the absence of Ptf1a,only glutamatergic neurons are formed in the cerebellum,whereas ectopic expression of Ptf1a in glutamatergic precursors is sufficient to switch neurons to the GABAergic phenotype[10].The homoeobox containing transcription factors Tlx1and Tlx3promote specification of glutamatergic neurons,inhibit GABAergic differentiation in spinal-cord neurons[11]and antagonize the functions of Lbx1,which promotes GABAergic differentiation[12].Lbx1is expressed in glutamatergic neurons,but its actions to promote GABAergic differentiation are inhibited by the expression of Tlx1and Tlx3.Mice lacking Tlx3show increased GABAergic differentiation,which is due to the presence of Lbx1,but interestingly,normal glutamatergic differentiation is restored in mice that lack both Tlx3and Lbx1[12].Transcription factors operate combinatorially and can promote different cell fates as a result of interactions with other transcription factors in specific cells.Neurogenin2expression in the forebrain promotes the generation of glutamatergic neurons [13]and in the spinal cord in association with Olig2,promotes motor-neuron differentiation[14].Combinations of Pax6,Olig2 and Nkx2.2and their inhibitors,Id and Hes,define both neuronal,and then glial,differentiation[7],while Dlx1and Dlx2 promote neurogenesis by inhibiting Olig2in mouse forebrain progenitors[15].Transcription factors such as Pax6,Olig2and Nkx2.2can also act combinatorially in specifying cell fate towards motor neurons,oligodendrocytes and dopaminergic neurons [16–18].Nkx2.2promotes differentiation toward serotonergic neurons[19],whereas Olig2promotes a motor-neuron fate [20].In fact Nkx2.2and Olig2function antagonistically,and both transcription factors repress each other’s expression during the differentiation process[16].Though many such mutually antagonistic relationships between individual transcription factors are known,the complete identification of all target genes for a particular factor during development has not been carried out.Such information,though technically challenging to obtain, would provide thefirst steps towards really understanding the transcription-factor networks that specify the multitude of neuronal cell types.The timing of transcription-factor activity is important during the generation of many cell types;neuronal subtypes are often produced sequentially from the same pool of multipotent progeni-tors.One such example is found in the hindbrain,where VMNs (visceral motor neurons)and serotonergic neurons are gene-rated sequentially from the same set of progenitor cells[21]. This mechanism requires the actions of two transcription factors, Phox2b and Mash1,that are required for VMN and seroto-nergic differentiation respectively.Foxa2is also required for serotonergic specification and,as with Nkx2.2and Olig2,Foxa2 and Phox2b mutually repress each other’s expression[21].The activity of Phox2b dominates that of Foxa2,meaning that initially VMNs are produced and a switch to serotonergic differentiation is initiated only by increased expression of Foxa2[21]through an unidentified mechanism.The increased levels of Foxa2repress expression of Phox2b and activate serotonergic differentiation. Foxa2therefore acts as a key molecular switch and in its absence, serotonergic neurons are not produced.In addition to roles in guiding neuronal progenitors toward specific neuronal fates,other transcription factors are important for regulating more general aspects of neuronal phenotype. One such transcription factor is the REST[repressor element 1-silencing transcription factor,also known as NRSF(neural restrictive silencer factor)].Reduced expression of the transcrip-tional repressor REST is an important step in neuronal differ-entiation.REST is expressed in ES cells[22–24],and down-regulation of REST is required prior to neuronal differentiationc The Authors Journal compilation c 2008Biochemical SocietyRegulation of gene expression in the nervous system329[22,24,25].Removal of mitogens and addition of retinoic acid to cultures of ES cells results in loss of REST expression both by reduced mRNA levels and enhanced targeting of REST protein for degradation and a concomitant differentiation of cells into neurons[22,24,26].REST can recruit multiple chromatin-modifying enzymes via interactions with at least two independent co-repressor complexes containing mSin3and CoREST[27–29] (for a recent review on chromatin and REST,see[30]),which are utilized to repress its predicted1800target genes[31,32].Many of these genes are normally expressed in differentiated neurons and are important for neuronal functions such as neurotransmitter release[24,33–35]and axon guidance[36].The exact role of REST repression in defining neuronal gene expression is still not entirely clear,though it is able to contribute to the deposition of epigenetic marks in neuronal genes,and these effects persist even after REST expression is lost.Indeed, loss of REST from promoters of some genes during neuronal differentiation does not lead to their immediate de-repression and the CoREST–MeCP2(methyl-CpG binding protein2)complex recruited by REST may remain bound to the promoter[22].In this way repression of Calb1(calbindin)and Bdnf[BDNF(brain-derived neurotrophic factor)]genes is maintained until released by other events,such as membrane depolarization,which results in MeCP2phosphorylation and/or DNA demethylation,loss of MeCP2binding and gene activation[22].Thus,during development and differentiation,combinatorial actions of transcription factors regulate specification of neural-cell type as well as the acquisition of general features associated with the neuronal phenotype.Appropriate control of gene regulation remains important in the mature nervous system,the difference being that the outcomes of gene regulation switch from specification and differentiation to regulating gene expression in response to neuronal activity.GENE EXPRESSION CHANGES IN RESPONSE TO NEURONAL ACTIVITYNeuronal activity results in the influx of calcium and a rise in intracellular calcium levels in neurons.Influx of calcium and changes in the intracellular calcium levels influence the function of several transcription factors(Figure1).CREB(cAMP response element binding protein)CREB is a key modulator in regulating gene expression programs in response to neuronal activity and is pivotal in mediating long-term memory and synaptic plasticity[37].Neuronal activity and calcium entry through synaptic NMDA(N-methyl-D-aspartate) receptors results in phosphorylation of CREB at Ser133and recruitment of the transcriptional co-activator CBP(CREB-binding protein)[38].Transcriptional activation is also regulated by phosphorylation of CBP by CaMKIV(calmodulin kinase IV), and CBP activates transcription via its intrinsic HAT(histone acetyltransferase)activity[39].Another co-activator of CREB is TORC(transducer of regulated CREB activity).There are three members of the TORC family encoded by individual genes, one of which(TORC1)is expressed in neurons[40].TORC1 can interact with CREB independently of the phosphorylation status of Ser133and potentiates CREB-mediated transcriptional activation and is required for LTP(long-term potentiation)in hippocampal neurons[40](LTP is the process whereby communi-cation between two neurons is strengthened as a result of both neurons being active at the same time.The effects are long-lasting and are mediated by increased neurotransmittersignalling Figure1Neuronal activity and calcium influx regulate the function of several transcription factors in neuronsDepolarization of neurons results in the entry of Ca2+through voltage-gated calcium channels such as Cav1.2,whereas glutamate stimulates NMDA receptor activation and Ca2+influx through NMDA receptors of depolarized neurons.Increased intracellular calcium has many effects and underlies many neuronal responses to synaptic activity.Of particular relevance to the present review are:(i)the phosphorylation of CREB,which results in recruitment of CBP and activation of CREB responsive genes;(ii)the activation of the protein phosphatase calcineurin,which dephosphorylates NFAT,allowing NFAT to enter the nucleus,bind to DNA and regulate transcription;(iii)the inhibition of the cleavage of Cav1.2,which prevents the cleaved C-terminal region CCAT from moving to the nucleus to regulate transcription. An animated version of this Figure can be found at /bj/414/0327/ bj4140327add.htm.between the two neurons.LTP is thought to be a good candidate for the molecular mechanism that underlies memory formation).The requirement for TORC1in LTP provides another layer of CREB regulation,because TORC1is normally located in the cytoplasm and translocates to the nucleus in response to phosphorylation by an unidentified kinase.Both calcium entry(through NMDA receptors or voltage-gated calcium channels)and stimulation of cAMP is required for phosphorylation of TORC1and transcrip-tional activation by CREB[41].Thus TORC1acts as a coincid-ence detector for activity,neither calcium entry nor increased cAMP levels alone being sufficient to stimulate activity.One of the most well-studied targets of CREB transcriptional activation is BDNF,whose identification provided thefirst insights into the mechanisms by which CREB activation could modulate synaptic activity and neuronal survival[42].The subsequent availability of complete genome sequences has allowed a more holistic approach in trying to predict the response to CREB activ-ity.Although a genome-wide analysis to identify CREB target genes in neurons has not yet been performed,data from several other cell types can shed light on the gene targets and mechan-isms of action of ing a genome-wide ChIP–SACO (chromatin immunoprecipitation–serial analysis of chromatin occupancy)technique,Impey et al.[43]identified6302sites that were bound by CREB in the rat pheochromocytoma cell line PC12.In a separate study,Zhang et al.[44]used a ChIP microarray analysis in HEK-293(human embryonic kidney-293) cells and predicted that CREB is bound at approx.4000sequences, on the basis of their observations of2811bound promoters from a selection of16000genes.Binding of CREB to genes does not appear to be regulated by its phosphorylation status.It would appear that at most promoters CREB is phosphorylatedc The Authors Journal compilation c 2008Biochemical Society330L.Ooi and I.C.Woodin response to increased cAMP;however,CBP is recruited,and transcription is activated,at only a subset of those genes[44]. Given the wide-ranging roles of CREB outside as well as within the nervous system,it is not surprising that it has the potential to regulate such a large number of genes.The evidence would suggest that it is the recruitment of CBP that dictates which CREB-regulated genes are responsive in any particular cell type. Although CBP recruitment is known to require phosphorylation of Ser133in CREB there must be additional mechanisms(perhaps further cofactor interactions)that are also required for CBP recruitment in vivo,as increased cAMP can increase Ser133 phosphorylation but not CBP recruitment to all CREB-bound genes[44].CCAT(calcium-channel-associated transcription regulator)As previously highlighted,influx of calcium is pivotal for activ-ity-dependent changes in neuronal gene expression.The L-type voltage-gated calcium channel Cav1.2contributes to this mechan-ism by its ability to allow calcium into the cell but,in addition,a region of its C-terminus can also be cleaved to create a transcrip-tion factor(Figure1).CCAT is a75kDa protein which is the product of cleavage of the C-terminus of the Cav1.2channel[45]. The cleavage has been characterized in a subset of inhibitory (GABAergic,GAD65-positive)neurons in the rat cortex and the peptide produced translocates to the nucleus.Within the nucleus,CCAT binds to the nuclear protein p54(nrb)/NonO, associates with gene promoters and activates the expression of some target genes such as Gjb5(connexin31.1)and Ntn4 (netrin4),while it repress the expression of other genes such as Trpv4(transient receptor potential vanilloid-4)and Kcnn3 (potassium intermediate/small conductance calcium-activated channel,subfamily N,member3).In fact many of the genes regu-lated by CCAT encode proteins that play a role in neuronal excit-ability,thereby providing another mechanism by which neuronal depolarization results in a remodelling event that impacts upon future activity[45].Calcium entry through Cav1.2or NMDA, but not AMPA(α-amino-3-hydroxy-5-methyl-4-isoxazoleprop-ionic acid)receptors,was shown to promote relocalization of CCAT from the nucleus to the cytoplasm.Such relocalization of CCAT is inhibitory,as it prevents its ability to influence transcription.The in vivo function of CCAT is not clear,though there is some evidence to support a role for regulation of the neuronal cytoskeleton,suggesting it may play a role in modulating neuronal connectivity.When expressed in HEK-293cells[46]and in Purkinje neurons[47],the P/Q voltage-gated calcium channel Cav2.1is also cleaved and produces a74kDa proteolytic fragment constitutively,which accumulates in the nucleus.The truncated fragment contains the polymorphic CAG repeat region,expansion of which is responsible for SCA6(spinocerebellar ataxia type6), and fragments containing expanded CAG repeats are toxic and result in cell death.Though there is no evidence that toxicity is mediated via changes in gene expression,such a mechanism would be consistent with proposed mechanisms of toxicity of CAG expansions in other types of SCAs such as SCA1[48,49] and neurodegenerative diseases such as Huntington’s disease[50]. Cleavage of another L-type calcium channel,Cav1.3,is stimulated by calcium entry through NMDA receptors and appears to produce a channel with increasedflow of calcium[51].Whether the proteolytic fragment may also function as a transcription factor,or whether its loss is only required for altering Cav1.3properties,has not been studied.Other examples of membrane proteins that are cleaved to produce transcription factors include PKD1(polycystic kidney disease1)[52]and the APP(β-amyloid precursor protein) [53].NFAT(nuclear factor of activated T-cells)NFAT proteins were originally identified as transcription factors that promote expression of target genes in response to activation of immune cells[54].However,in actuality they are expressed in many cell types,including neurons.NFATc proteins shuttle to and from the nucleus as a consequence of their phosphorylation status, which is altered in response to changes in intracellular calcium levels.Phosphorylated NFATc is retained in the cytoplasm and is inactive.Increases in intracellular calcium activate the calcium binding phosphatase calcineurin,which dephosphorylates and activates NFATc,causing it to translocate to the nucleus,where-upon it up-regulates target gene expression(Figure1).However, activation of NFATc can also repress expression of target genes in other systems.For example,in mouse cardiomyocytes,activated NFAT results in reduced expression of the potassium channel gene Kcnd2that encodes Kv4.2[55].However,whether this is a direct effect of NFATc is not clear.Nuclear NFAT is inactivated by phosphorylation by the kinases CK1(casein kinase1)[56]and GSK3(glycogen synthase kinase3)[57].There are four members of the family,NFATc1–NFATc4,which have functions in many regions of the brain.NFATc is activated in hippocampal neurons in response to neuronal signalling[57],in superior cervical ganglion neurons by repetitive action potentials[58],in spinal neurons by substance P or neurotrophins[59,60]and in developing cochlear neurons by de-afferentation[61]or cocaine[62].Just as the stimuli for NFAT activation are varied,so are the functional outcomes, which include neuronal survival[60],neuronal death[61]and pain-sensitivity[63].In mouse striatal neurons,stimulation of dopamine D1receptors activates NFATc4via calcium entry through L-type calcium channels[62].Two potential targets for activated NFATc4are IP3R1[Ins(1,4,5)P3receptor type1]and GluR2(glutamate receptor subunit2),both of which can be activated by nuclear NFATc4and show increased mRNA levels after dopamine-receptor activation[62].The dopamine receptor signalling path-way is important in the development of addiction,particularly the addiction to some drugs of abuse,such as cocaine[64]. Exposure of mice to repeated,but not a single,injection of cocaine over a5-day period resulted in enhanced nuclear localization of NFATc4in striatal neurons and an approx.2-fold increased expression of IP3R1and GluR2mRNA[62].Increased expression of these genes should result in enhanced synaptic transmission, and thus NFATc4activation may underlie some of the gene-expression changes that play a role in the remodelling of neuronal transmission mediated by repeated exposure to addictive drugs. Interestingly,although increased IP3R1expression is mediated via NFAT activation in striatal neurons[62]and hippocampal neurons[60],in spinal neurons increased IP3IR expression occurs independently of NFAT activation[63].NFATc1–NFATc4genes are all expressed in rat DRG(dorsal root ganglion)neurons,and NFAT activation induced the expression of the CCR2and CCR5(C–C chemokine receptors type2and5)in DRG neurons in response to depolarising stimuli [65].Increased expression of CCR2in neurons has been linked to the development of allodynia and neuropathic pain[66](allodynia is a painful response to a normally non-painful stimulus.It is the result of the increased sensitivity and excitability of sensory neurons.Neuropathic pain is a chronic pain resulting from some problem within the neuron.Unlike nociceptive pain,in which neurons send pain signals to the brain in response to tissue damage,in neuropathic pain the signal is generated within the neurons themselves).The stimulation of CCR2expression by NFAT activation is likely to be direct,as the CCR2promoter region contains an evolutionary conserved NFAT-binding sitec The Authors Journal compilation c 2008Biochemical Society。

Prognostic serum miRNA biomarkers associated with Alzheimer's disease shows concordan ce withneuropsychological and neuroimaging assessment.Cheng L1, Doecke JD2, Sharples RA1, Villemagne VL3, Fowler CJ4, Rembach A4, Martins RN5, Rowe CC6, Macaulay SL7, Masters CL4, Hill AF1.Author informationAbstractThere is no consensus for a blood-based test for the early diagnosis of Alzheimer's disease (AD). Expression profiling of small non-coding RNA's, microRNA (miRNA), has revealed diagnostic potential in human diseases. Circulating miRNA are found in small vesicles known as exosomes within biological fluids such as human serum. The aim of this work was to determine a set of differential exosomal miRNA biomarkers between healthy and AD patients, which may aid in diagnosis. Using next-generation deep sequencing, we profiledexosomal miRNA from serum (N=49) collected from the Australian Imaging, Biomarkers and Lifestyle Flagship Study (AIBL). Sequencing results were validated using quantitative reverse transcription PCR (qRT-PCR; N=60), with predictions performed using the Random Forest method. Additional risk factors collected during the 4.5-year AIBL Study including clinical, medical and cognitive assessments, and amyloid neuroimaging with positron emission tomography were assessed. An AD-specific 16-miRNA signature was selected and adding established risk factors including age, sex and apolipoprotein ɛ4 (APOE ɛ4) allele status to the panel of deregulated miRNA resulted in a sensitivity and specificity of 87% and 77%, respectively, for predicting AD. Furthermore, amyloid neuroimaginginformation for those healthy control subjects incorrectly classified with AD-suggested progression in these participants towards AD. These data suggest that an exosomal miRNA signature may have potential to be developed as a suitable peripheral screening tool for AD.Molecular Psychiatry advance online publication, 28 October 2014; doi:10.1038/mp.2014.127.有用于基于血液的测试的早期诊断没有共识阿尔茨海默氏病（AD）。

Scaffolding and Docking Proteins of the Heart14-3-3Proteins—a focus on cancer and human diseaseErik Wilker,Michael B.Yaffe *Center for Cancer Research,Massachusetts Institute of Technology,E18-580,77Massachusetts Avenue,Cambridge,MA 02139,USAReceived and revised 10March 2004;accepted 23April 2004Abstract14-3-3Proteins are a ubiquitous family of molecules that participate in protein kinase signaling pathways within all eukaryotic cells.Functioning as phosphoserine/phosphothreonine-binding modules,14-3-3proteins participate in phosphorylation-dependent protein–protein interactions that control progression through the cell cycle,initiation and maintenance of DNA damage checkpoints,activation of MAP kinases,prevention of apoptosis,and coordination of integrin signaling and cytoskeletal dynamics.In this review,we discuss the regulation of 14-3-3structure and ligand binding,with a focus on the role of 14-3-3proteins in human disease,particularly cancer.We discuss the latest data on the role of different 14-3-3isotypes,the interaction of 14-3-3proteins with Raf,Cdc25,and various integrin family members,and the likelihood that 14-3-3proteins could be useful therapeutic targets in the treatment of human disease.©2004Elsevier Ltd.All rights reserved.Keywords:14-3-3Proteins;Cancer;Human disease;Phosphoserine/threonine;Modular signaling domains1.Introduction14-3-3Proteins are among the most abundant proteins within the cell,having been initially identified in 1967as a family of acidic proteins within the mammalian brain [1].Despite this,the actual function of 14-3-3proteins remained obscure until 1995,when Muslin et al.[2],building on studies of Raf phosphorylation and 14-3-3-binding by Mor-rison et al.[3],demonstrated for the first time that 14-3-3proteins could bind specifically to phosphoserine-containing peptides.Thus,14-3-3proteins became the first example of a specific phosphoserine/threonine-binding molecule or do-main.At that time,it was well appreciated that modular signaling domains capable of binding to short phosphotyrosine-containing sequences,such as SH2do-mains and PTB domains,existed [4–6],but it was thought that protein phosphorylation followed by substrate binding to modular phospho-binding domains was unique to signaling events mediated by tyrosine kinases.In contrast,cell signal-ing by serine and threonine kinases,which constitute ~92%of all protein kinases in humans,was thought to proceed by other mechanisms such as phosphorylation-induced confor-mational changes in the substrate.It is increasingly evident,however,eukaryotic cells contain a diverse assortment of phosphoserine/threonine-binding domains in addition to 14-3-3proteins.These domains,like 14-3-3proteins them-selves,play critical roles in the global control of cell prolif-eration,in the response of cells to DNA damage,in regulation of chromatin structure and gene expression,and in orchestrating the mechanics of mitosis itself [7].Intriguingly,mutations within,or altered expression of,a number of these phosphoserine/threonine-binding domains have been impli-cated in a variety of human diseases (c.f.[8–16]).This article focuses on the roles of a subset of 14-3-3proteins and their interacting proteins in cell proliferation and differentiation as relevant to human diseases,especially cancer.Excellent gen-eral reviews of 14-3-3proteins are available elsewhere [17–20].The term 14-3-3refers to a family of acidic dimeric proteins that are expressed in all eukaryotic cells.This family of highly conserved proteins consisting of seven isotypes in human cells (b ,c ,f ,r ,e ,g ,s )plays crucial roles in regulating multiple cellular processes including the maintenance of cell cycle checkpoints and DNA repair,the prevention of apoptosis,the onset of cell differentiation and senescence,and the coordina-tion of cell adhesion and motility.All 14-3-3proteins bind to phosphoserine/phosphothreonine-containing peptide motifs*Corresponding author.Tel.:+1-617-452-2103;fax:+1-617-452-4978.E-mail address:myaffe@ (M.B.Yaffe).Journal of Molecular and Cellular Cardiology 37(2004)633–642/locate/yjmcc0022-2828/$-see front matter ©2004Elsevier Ltd.All rights reserved.doi:10.1016/j.yjmcc.2004.04.015corresponding to the sequences RSXpSXP or RXXXpSXP [21].Many 14-3-3-binding proteins contain sequences that closely match these motifs,although a number of ligands bind to 14-3-3in a phospho-independent manner using alter-native sequences that do not closely resemble these motifs.Pozuelo Rubio et al.[22]recently used 14-3-3-affinity chro-matography and mass spectrometry to identify over 20014-3-3-binding ligands.All of these ligands lost their ability to bind to 14-3-3upon dephosphorylation by the serine/threonine phosphatase PP2A in vitro.Some of the proteins bound to the 14-3-3-affinity column contained se-quences that closely matched the optimal binding motifs,while others diverged significantly from the consensus se-quences,despite their apparent phospho-specific binding [22].All 14-3-3proteins are thought to share a similar tertiary structure as revealed by the crystal structures for f and s isoforms [23,24],consisting of nine a -helices for each 14-3-3monomer,with the biologically functional dimer resembling a somewhat squat,flattened “U”shaped structure (Fig.1).Four of the a -helices directly participate in dimer formation,while the remainder constitute the side walls and top of the “U”.14-3-3-Binding to phosphoserine/threonine-containing sequence motifs involves direct interactions between the phosphate and Lys-49and Arg-56in helix a C,and Arg-127and Tyr-128in helix a E.These residues form a basic pocket in an otherwise acidic molecule,explaining the ability of substrate serine/threonine phosphorylation to act as a mo-lecular switch controlling ligand binding.Wang and Shakes [25]compared all the known 14-3-3sequences available in 1996to identify five conserved sequence blocks within the 14-3-3family.These regions turn out to comprise the helices involved in ligand binding (a C,a G,a E and a I),along with the end of helix a A,the a A/a B-connecting loop,and the start of helix a B,which form an important part of the dimer interface [26].Despite of their conserved sequence and struc-ture,not all isotypes of 14-3-3bind equivalently to particularligands.Although only a few isotype-specific or isotype enhanced interactions have been well characterized to date,this phenomenon is likely to be biologically important,and merits further study.Recent reports have shown that many 14-3-3family mem-bers,including b and f ,can undergo post-translational modi-fication by phosphorylation [27–31](Fig.1).Aitken et al.[27]and Dubois et al.[28]identified several sites on 14-3-3proteins that are targets for proline targeted kinases (Ser-185),or other kinases (Thr-233),resulting in their phospho-rylation in vivo and in vitro.Structural modeling and direct experimental investigation using Raf suggested that phos-phorylation of these sites could alter the phospho-binding ability of 14-3-3[28].Interestingly,Obsilova et al.[32]reported that 14-3-3f phosphorylation on Thr-233by casein kinase 1caused a conformational change in the C-terminus as measured by time-resolved fluorescence,resulting in de-creased phospho-dependent ligand binding and suppression of 14-3-3-stimulated serotonin-N -acetyltransferase activity in vitro.Powell et al.[30]demonstrated that MAPKAP kinase 2could interact with and phosphorylate 14-3-3f on Ser-58in puter-modeling studies along with ex-periments utilizing a 14-3-3S58D mutant showed that phos-phorylation at this site would compromise 14-3-3’s ability to dimerize.In a series of important experiments,Shen et al.[33]investigated the susceptibility of 14-3-3f to undergo phos-phorylation and concluded that the regulation of 14-3-3by phosphorylation occurs in the monomeric form rather than after dimer assembly.Furthermore,these authors studied the binding of dimeric (wild-type)and monomeric (mutant)14-3-3proteins bind to phosphorylated and non-phosphorylated endogenous proteins from COS-7and HEK cells by using an antibody against one of the 14-3-3phospho-binding motifs (RSXpSXP)and [35]-S-methionine-labeled extracts.Al-though many [35]-S-labeled proteins bound to the mutant GST-14-3-3(monomeric)form,this interaction was phospho-independent,since only the proteins that bound to the GST-14-3-3dimeric (wild-type form)could be detected by the phospho-specific 14-3-3motif antibody.These results suggest that phosphorylation-induced inhibition of 14-3-3dimerization directly alters the subset of bound ligands in vivo,and indirectly influences binding of ligands to the remaining 14-3-3proteins by decreasing the amount of par-ticular 14-3-3isotypes remaining in the dimeric pool.In this regard,an additional mechanism for the regulation of specific 14-3-3:ligand complexes may involve the tran-scriptional regulation of individual family members.The promoter regions and 5′untranslated segments of the differ-ent 14-3-3isoforms are divergent,suggesting that individual 14-3-3isoforms may have distinct transcriptional regulatory mechanisms.Once transcribed and translated,Aitken [34]showed that 14-3-3isoforms differ in their specificities for homo-and heterodimerization,with particular heterodimeric pairs forming at the expense of others.Thus,dramatic alter-ations in the expression of specific 14-3-3isoformswouldFig.1.The structure of 14-3-3.The X-ray structure of the 14-3-3dimer is shown in cartoon representation,with the monomeric subunits in green and purple.Helices a A through a I are indicated.Ligand binding occurs in the central cavity.The side chains of two of the known phosphorylation sites,Ser-58and Ser-185are shown in stick representation,colored red.Phospho-rylation of Ser-58disrupts the dimer interface,while phosphorylation of Ser-185affects ligand binding.The third phosphorylation site,Thr-233,lies beyond the region of helix a I seen in the crystal structure.634 E.Wilker,M.B.Yaffe /Journal of Molecular and Cellular Cardiology 37(2004)633–642alter the resulting population of14-3-3heterodimers.Since different heterodimers likely bind to different target ligands, or bind to the same target ligand with different affinities, these changes in the expression of specific14-3-3isoforms may result in longer term alterations in cell signaling.In addition,high level expression of one isotype might seques-ter a second isotype,preventing it from dimerizing with a third less abundant isotype,in a process that could be called “isotype interference.”Further investigation will be needed to determine the distinct contributions of particular het-erodimers and the role of isotype interference in14-3-3-mediated signaling.The dimeric nature of14-3-3proteins allows them to bind to more than one site on a single target ligand simultaneously.One model for14-3-3function involves binding to a single dominant“gatekeeper”residue,al-lowing additional secondary sites with lower affinity for 14-3-3to then interact in a dynamic way by virtue of their proximity and high local concentration[26].In this way, the rigid14-3-3structure might act as a“molecular anvil”to stabilize conformations of the bound ligand that are sampled less frequently in the ligand’s unbound state.These14-3-3-bound conformations could increase the bound protein’s catalytic activity(c.f.Raf[35],serotonin-N-acetyl trans-ferase[36],Chk1[37],and Wee1[38–40]),stabilize and sequester the phosphorylated protein(c.f.Cdc25C[41,103–107]and NUDEL[42]),or facilitate additional phosphoryla-tion events(c.f.BAD[43]).In addition,14-3-3-binding might expose or mask cellular targeting sequences,leading to alterations in nuclear or other organelle import or export by “molecular interference”[44].It is interesting that although14-3-3proteins are ex-pressed in all eukaryotic cells,including plants,yeast, and protozoa,there is no clear prokaryotic ancestor.De-spite the high sequence homology and structure of all 14-3-3proteins,the pathways in which these molecules participate have diverged considerably.This suggests that eukaryotic evolution has created new signaling compo-nents and pathways to take advantage of14-3-3function. The variations in isoform number in higher organisms might indicate the necessity offine-tuning these interac-tions to achieve distinct functions within a complex sig-naling network.Some of this complexity and isoform specificity in14-3-3signaling has become apparent in recent reports of human diseases-associated alterations in specific14-3-3isoform expression or function.2.Roles of14-3-3in human cancer2.1.14-3-3rAlterations of the expression of many14-3-3proteins have been associated with several human cancers.The down-regulation of14-3-3r has been associated with a multitude of human epithelial cancers.Vercoutter-Edouart et al.[45]dem-onstrated through a proteomics approach that14-3-3r was easily detectable in Coomassie stained2D gels of normal breast epithelial cells,but was undetectable in the breast cancer protein samples as identified by MALDI-TOF and MS/MS after trypsin digestion.Ferguson et al.[15]demon-strated through SAGE analysis that mRNA levels of14-3-3r were down-regulated to undetectable levels in45out of 48primary breast carcinomas and further investigation re-vealed that there was a high frequency of hypermethylation in75out of82primary breast carcinomas in CpG islands at the14-3-3r locus,leading to gene silencing.Furthermore, treatment of these breast cell lines with5-aza-2′-deoxycytidine resulted in the demethylation of the gene and increased synthesis of14-3-3r mRNA.Umbricht et al.[46] reported that in patients with breast cancer,the promoter region of14-3-3r was hypermethylated even in the histologi-cally normal adjacent tissue surrounding the tumors(i.e.“field cancerization”).This data suggests that gene silencing of14-3-3r may be an early event during breast carcinogen-esis,since none of the control tissue from cancer-free pa-tients exhibited this trend.Recently,Urano et al.[16]identified another mechanism by which breast cancer cells reduced14-3-3r protein levels, in this case through upregulation of the protein Efp,a ring-finger-dependent ubiquitin ligase(E3),which targets14-3-3r for ubiquin-mediated degradation by the proteosome. MCF7cells treated with anti-sense Efp constructs had in-creased levels of14-3-3r,and reduced tumor burden when these cells were transplanted into athymic mice.Similar trends of cancer-associated down-regulation of14-3-3r have been observed in human lung carcinomas,vulva squamous neoplasias,bladder carcinomas,hepatocellular carcinomas, oral carcinomas,and head-and-neck squamous cell carcino-mas[47–51],suggesting a general role for14-3-3r as a tumor suppressor gene.Intriguingly,14-3-3r appears to play an important role in the sustaining the G2/M checkpoint in epithelial cells following DNA damage,based on a series of experiments by the V ogelstein laboratory.Hermeking et al.[52]dem-onstrated that14-3-3r was a major p53response gene in HCT116cells following exposure to DNA damaging agents, and suggested a role for14-3-3r in the maintenance of the G2/M checkpoint.This did not seem to be surprising at the time since14-3-3s had been implicated in the G2/M checkpoint through their ability to sequester Cdc25C. However,14-3-3r seems to play a unique role in this process since it has now been demonstrated not to inter-act with Cdc25C.Chan et al.[53]demonstrated in14-3-3r-targeted ho-mologous recombinant knockout HCT116cells that14-3-3r plays a crucial role in the maintenance of the G2/M check-point following exposure to adriamycin.14-3-3r-/-HCT116 cells initiated,but failed to maintain,a G2/M checkpoint following exposure to adriamycin,and subsequently under-went an alternative cell death termed mitotic catastrophe. Chan et al.showed that14-3-3r-/-HCT116cells failed to635E.Wilker,M.B.Yaffe/Journal of Molecular and Cellular Cardiology37(2004)633–642sequester Cdc2/cyclin B1in the cytoplasm following treat-ment with adriamycin,resulting in G2/M checkpoint pro-gression.An interaction between14-3-3r and Cdc2has been reported in several additional studies[16,53,54],but many questions still exist regarding the extent to which this inter-action is direct,DNA damage-dependent,and modulated by cell signaling.Additional confirmatory information is needed regarding this proposed mechanism for the regulation of the G2/M checkpoint by14-3-3r.Several publications suggest that activation of p53follow-ing DNA damage directly upregulates the levels14-3-3r, and that this upregulation is critical for maintaining the G2/M checkpoint[52,53].The interactions between p53and14-3-3 are complex,and likely to be at least partially cell type specific.For example,14-3-3r and other14-3-3isotypes have been reported to directly bind to p53itself,increasing the DNA-binding function and activity of p53as a transcrip-tion factor[55–57].Waterman et al.[56]reported that this interaction was between a suboptimal14-3-3-binding site (Ser-378)on p53that was formed in cells following exposure to DNA damaging agents,resulting in the simultaneous de-phosphorylation of Ser-376to create a14-3-3-binding site. Stavridi et al.[55]reported that,in mutant p53cells unable to bind14-3-3on Ser-378,the transcriptional activity of p53is lower.Furthermore,these authors showed that in non-epithelial cells,14-3-3c,e,and s isoforms,rather than14-3-3r,interacted with p53in a DNA damage-dependent man-ner.While most14-3-3proteins are expressed in many cell types,14-3-3r is expressed primarily in epithelial cell lines. Subsequently,Yang et al.[57]were able to detect a DNA damage-dependent interaction between14-3-3r and p53in a human epithelial lung cell line.As expected,the interaction between14-3-3r and p53resulted in p53stabilization and increased transcriptional activity.Intriguingly,the overall levels of MDM2,an E3-ubiquitin ligase that targets p53for degradation,decreased as exogenous levels of14-3-3r in-creased.Despite the importance of p53in14-3-3r expression, p53-/-epithelial cells maintain basal14-3-3r expression levels.In this regard,p63(and perhaps p73)can also bind to the sigma promoter,suggesting alternative forms of regula-tion[58,59].Weber et al.demonstrated that p53-/-HCT116 cells still express substantial basal levels of14-3-3r protein (c.f.Fig1in Weber et al.[60]),and these levels appeared to increase following exposure to adriamycin similar to p53-wild-type HCT116cells.In contrast,Chan et al.[53]demon-strated a more robust induction of14-3-3r protein levels in the same HCT116cell line following adriamycin treatment. Other transcriptionally regulated targets of p53besides14-3-3r in epithelial cells also participate in the G2/M check-point,even in the absence of14-3-3r’s stimulatory action on p53activity.For example,while p53-/-HCT116cells cannot upregulate p21levels,HCT11614-3-3r-/-cells can(c.f. Figure2in Weber et al.[60]),suggesting that the p53’s transcriptional regulation of p21is not fully dependent on the presence of14-3-3r.De Laurenzi et al.[61]demonstrated that p63and p73can bind to,and transcriptionally modulate p21levels,however,their exact roles in DNA damage events remain unresolved.Since studies by Bunz et al.[62]and Chan et al.[53]demonstrated that p53-/-cells,14-3-3r-/-cells and p21-/-cells are all capable of initiating,but not maintaining,a G2/M checkpoint,it seems likely that14-3-3r and p21,both targets of p53,function in parallel pathways that are critical for maintenance of G2/M arrest.3.Other14-3-3family members3.1.14-3-3Modulation of Raf activationThe de-regulation of the Ras family of proteins is consid-ered an important step in the tumorigenesis process.It has been demonstrated that members of the14-3-3family of proteins,including b and f can modulate the ability of Ras to activate Raf,and thereby activate the Erk1/2MAP kinase pathway.Several in vitro models have suggested that14-3-3b’s ability to interact with and modulate c-Raf-1function may play a role in oncogenic transformation[63–65],but further studies are needed to determine the extent to which mis-regulation of the14-3-3b/Raf interaction participates in tumorigenesis.14-3-3Proteins also bind to A-Raf and B-Raf, and activating mutations in the B-Raf gene have recently been identified in isolated melanomas and colon cancers [66].These reports are thefirst time that any Raf isoform has directly been linked to tumorigenesis in human cancers. Because our understanding of the14-3-3:Raf interaction has been most extensively investigated for the c-Raf-1isoform, we discuss that interaction in some detail.Interactions be-tween14-3-3and B-Raf,while similar,may not function in an analogous way[67–70],and therefore,deserve additional study.Since its initial identification as a Raf-binding protein, numerous studies have investigated14-3-3’s ability to bind to Raf-1in a phospho-dependent manner in the CR-2and CR-3domains.Freed et al.[71]and Irie et al.[72]demon-strated in a yeast-two hybrid system that the association between14-3-3b,f,or the yeast14-3-3Bmh1with c-Raf-1 was as strong as the interaction between c-Raf-1and H-Ras. Irie et al.[72]reported that MBP-14-3-3potentiated the activation of c-Raf-1in vitro.This interaction was further explored by Fu et al.[73]using baculoviruses in an insect cell system,where it was shown that the interaction of c-Raf-1 with14-3-3was not dependent on the c-Raf-1activation state.In a small proportion of samples tested,they observed an increase in c-Raf-1activity in the presence of14-3-3. Similar increases in c-Raf-1(and B-Raf)activity upon inter-action14-3-3were observed in model organisms including Xenopus[74,75],and Drosophila[76–78],as well as in several in vitro systems[35,70,79–83].Other data,however,appeared to contradict the proposi-tion that the interaction of14-3-3with c-Raf-1led to en-hanced c-Raf-1activation.Resolution of these conflicting636 E.Wilker,M.B.Yaffe/Journal of Molecular and Cellular Cardiology37(2004)633–642results emerged with the recognition that c-Raf-1binds to 14-3-3at multiple sites,some of which are stimulatory and some of which are inhibitory.For example,mutations con-tained in the c-Raf-1CRD[63]and CR-2domains result in decreased14-3-3-binding as well as an increased activity of Raf-1,while mutations in the CR-3domain result in dimin-ished Raf-1activity[3,63]demonstrated a decreased interac-tion between14-3-3and c-Raf-1containing a CRD domain deletion in vitro.This decreased binding of14-3-3to Raf-1 mutants resulted in the increased susceptibility of Raf-CRD mutant-expressing NIH3T3cells to Ras-dependent transfor-mation.c-Raf-1possesses at least four14-3-3-binding sites, namely Ser-259,Ser-621[2,3]),Ser-233[84]and a fourth site contained in the CRD between residues136and187 [63].The interaction of14-3-3with c-Raf-1via the Ser-259 site appears to negatively regulate c-Raf-1by constraining c-Raf-1in an inactive state.Both14-3-3and Ras compete for binding to the Ser-259region,so that Roy et al.[81]and Light et al.[85]were able to observe displacement of14-3-3-bound to cytosolic c-Raf-1when c-Raf-1was recruited to the plasma membrane by an activated Ras.Michaud et al.[86]found that loss of the Ser-259:14-3-3interaction by a Ser-259A c-Raf-1mutant resulted in higher c-Raf-1activity in vitro in a reconstituted insect cell system,and conferred an increased ability of c-Raf-1to induce meiotic maturation of Xenopus oocytes.Studies from Ory et al.[87]and Jaumot and Hancock[88]suggest that PP2A can effectively dephos-phorylate c-Raf-1on Ser-259,thereby allowing release of 14-3-3-binding and facilitating Ras binding to c-Raf-1at this site.In contrast,interaction of14-3-3with Ser-621on c-Raf-1 appears to stimulate c-Raf-1activity,either by stabilizing the kinase domain’s active conformation,or by the association of this complex with other factors.Thorson et al.[80]demon-strated that14-3-3-binding to Raf-1on serine621results in stabilization of the phosphorylation of Raf-1on this critical activation site and subsequently enables Raf-1to remain in an activation competent conformation.Morrison et al.have now identified another critical component of the Raf-14-3-3 complex—the scaffolding protein KSR that brings MEK to Raf to stimulate the next step in the MAP kinase activation cascade[87].Like c-Raf-1,KSR also appears to interact with 14-3-3through multiple phosphorylation sites.Their data suggest that the critical events in c-Raf-1activation involve dephosphorylation of c-Raf-1on Ser-259and KSR on Ser-342,releasing both of these sites from14-3-3,while preserv-ing the c-Raf-1Ser-621:14-3-3interaction[87].The net effect of this partial dephosphorylation is to bring MEK to c-Raf-1while stabilizing c-Raf-1’s kinase activity.The rel-evant phosphatase appears to be PP2A,another component of the c-Raf-1:KSR:14-3-3complex[87].While this model appears to capture the essential features of c-Raf-1activa-tion,additional levels of regulation are likely involved as well,since other protein kinases including PKA and PKC f appear to influence c-Raf-1activation in a14-3-3-dependent manner[84,89].3.2.14-3-3regulation of Cdc25activityThe Cdc25family of phosphatases is comprised of three isoforms—Cdc25A,B and C.While Cdc25A is required for efficient S-phase entry,it also appears to play a role late in the cell cycle where increased expression can facilitate the tran-sition between the G1/S and G2/M checkpoints[90–93].The other two family members,Cdc25B and C play important roles in governing entry into mitosis and the G2/M check-point[94]).However,it appears as though Cdc25B plays a more prominent role during the early portion of the G2/M checkpoint(possibly late S-phase),while Cdc25C governs the later portions of the G2/M checkpoint,as reviewed by Donzelli and Draetta[95].It has been demonstrated that all three of these isoforms can interact with14-3-3and these interactions play important roles in controlling cell cycle transitions[41,96,97].Mechanisms for regulation of Cdc25 activity by14-3-3-binding have been best explored and char-acterized in the interactions between14-3-3and Cdc25C.The molecular“motor”that drives cells into mitosis is the cyclin B/Cdc2protein kinase.Throughout the cell cycle in animal cells,this kinase shuttles between the nucleus and cytoplasm,being primarily cytoplasmic except during M-phase.In interphase cells,this kinase is maintained in an inactive state by phosphorylation on Thr-14and Tyr-15by the nuclear kinase Wee1and the cytoplasmic kinase Myt1 [98–101].In addition,Cdc25C is phosphorylated on Ser-216 and bound to14-3-3proteins at this time[102],which ap-pears to both inhibit the phosphatase activity,and facilitate nuclear exclusion[103–107].Peng et al.[41]demonstrated that Cdc25C was phosphorylated on Ser-216in synchronized HeLa cells following release from the double thymidine block.This phosphorylation remained constant until10–12h post thymidine release,which corresponded to the cells en-tering mitosis.Cells that over-express a S216A mutant form of Ccd25C that was unable to bind to14-3-3hadfive times the number of mitotic nuclei and increased amounts of cyclin B1-associated histone H1kinase activity.In response to DNA damage during G2,Cdc25C remains phosphorylated on Ser-216,establishing a cell cycle check-point and preventing cells from entering M-phase[41,108]. In addition,DNA damage stimulates Chk1-depenent Wee1 binding to14-3-3,thereby increasing Wee1activity to ensure a“double-lock”on Cdc25C inhibition[38–40,109].The ki-nases responsible for Cdc25C Ser-216phosphorylation in-clude Cds1/Chk2and c-TAK-1[110–112].A number of publications indicated that Chk1was the critical Cdc25C Ser-216kinase responsible for Cdc25C:14-3-3-binding fol-lowing DNA damage[41,108,111,113].Recent experiments using Chk1siRNA,however,indicate that,at least in mam-malian cells,another as-yet-unknown kinase is responsible for the majority of Ser-216phosphorylation under these conditions[114].Entry into mitosis is accomplished by release of Cdc25C from14-3-3and dephosphorylation of the Ser-216site by PP1[115].Intriguingly,mitotic entry is also accompanied by637E.Wilker,M.B.Yaffe/Journal of Molecular and Cellular Cardiology37(2004)633–642。

ORIGINAL ARTICLESoybean 14-3-3gene family:identification and molecular characterizationXuyan Li •Sangeeta DhaubhadelReceived:31August 2010/Accepted:3November 2010/Published online:26November 2010ÓHer Majesty the Queen in Rights of Canada 2010Abstract The 14-3-3s are a group of proteins that are ubiquitously found in eukaryotes.Plant 14-3-3proteins are encoded by a large multigene family and are involved in signaling pathways to regulate plant development and protection from stress.Recent studies in Arabidopsis and rice have demonstrated the isoform specificity in 14-3-3s and their client protein interactions.However,detailed characterization of 14-3-3gene family in legumes has not been reported.In this study,soybean 14-3-3proteins were identified and their molecular characterization performed.Data mining of soybean genome and expressed sequence tag databases identified 1814-3-3genes,of them 16are transcribed.All 16SGF14s have higher expression in embryo tissues suggesting their potential role in seed development.Subcellular localization of all transcribed SGF14s demonstrated that 14-3-3proteins in soybean have isoform specificity,however,some overlaps were also observed between closely related isoforms.A comparative analysis of SGF14s with Arabidopsis and rice 14-3-3s indicated that SGF14s also group into epsilon and non-epsilon classes.However,unlike Arabidopsis and rice 14-3-3s,SGF14s contained only one kind of gene structure belonging to each class.Overall,soybean consists of the largest family of 14-3-3proteins characterized to date.Our results provide a solid framework for further investigationsinto the role of SGF14s and their involvement in legume-specific functions.Keywords 14-3-3proteins ÁGene regulation ÁMultigene family ÁProtein–protein interaction ÁSoybean ÁPhylogeny Abbreviations DAP Days after pollination EST Expressed sequence tag TC Tentative contig BiFC Bimolecular fluorescence complementationassayBLAST Basic local alignment search tool YFP Yellow fluorescent protein YN N-terminal half of yellow fluorescent protein YC C-terminal half of yellow fluorescent protein MYA Million years agoIntroductionThe 14-3-3s are a group of proteins that are ubiquitously found in eukaryotes.Originally isolated from brain tissue,14-3-3s are abundant,soluble acidic proteins (reviewed in Moore and Perez 1967)that are thought to be brain specific for a long time.They regulate activities of a wide array of target proteins via protein–protein interactions which involves binding with phosphoserine/phosphothreonine residues in the target proteins (Muslin et al.1996;Yaffe et al.1997).The targets for 14-3-3s range from proteins involved in signal transduction to gene regulation with which they interact as a dimer with native dimeric size of *60kDa.Each monomer in the dimer is capable ofElectronic supplementary material The online version of this article (doi:10.1007/s00425-010-1315-6)contains supplementary material,which is available to authorized users.X.Li ÁS.Dhaubhadel (&)Southern Crop Protection and Food Research Center,Agriculture and Agri-Food Canada,1391Sandford Street,London,ON N5V 4T3,Canadae-mail:sangeeta.dhaubhadel@agr.gc.caPlanta (2011)233:569–582DOI 10.1007/s00425-010-1315-6interacting with a separate target protein.The dimeric property of14-3-3s allows them to serve as scaffolds by bringing two different regions of the same protein into proximity within a single complex or two different proteins together.Two consensus14-3-3binding phosphopeptide motifs,RSXpSXP(mode I)and RXY/FXpSXP(mode II), where X is any amino acid and pS is the phosphoserine, have been reported(Yaffe et al.1997).However,many 14-3-3-binding sites do not conform to these consensus motifs(Aitken2006).In addition,some targets do not require phosphorylation status to bind with14-3-3s(Fu et al.2000;Sumioka et al.2005).Although it is possible that the unphosphorylated target protein interacts with an intermediary protein that is phosphorylated and binds to 14-3-3s(Johnson et al.2010).Plant14-3-3proteins were identified about two decades after theirfirst discovery in animals.They werefirst reported from four different plant species,Hordeum vulgare(Brandt et al.1992),Arabidopsis thaliana(Lu et al.1992),Spinacia oleracea and Oenothera hookeri(Hirsch et al.1992).Sub-sequently,more14-3-3s have been isolated and character-ized from several plants(reviewed in Chevalier et al.2009). As in animal system,plant14-3-3proteins have been found to regulate a variety of biological processes such as meta-bolic,growth and developmental or signaling pathways via interactions with their target proteins(reviewed in Sehnke et al.2002).They have been involved in regulation of gene expression,protein synthesis,protein folding,primary metabolism including plasma membrane-localized proton pump,organellar/nucleocytoplasmic shuttling,hormone metabolism,and chromatin remodeling(Huber et al.2002; Paul et al.2008).Some examples of the target proteins for plant14-3-3s include RSG(for repression of shoot growth), that controls gibberellin biosynthesis(Igarashi et al.2001), ABA-responsive element binding factors ABF(Schoonheim et al.2009)as well as the key transcription factors BZR1and BZR2involved in brassinosteroid signal transduction(Bai et al.2007;Gampala et al.2007;Ryu et al.2007).Several studies have also established a role for14-3-3in signaling pathways and environmental stress response(Aksamit et al. 2005;Lapointe et al.2001;Seehaus and Tenhaken1998;Xu and Shi2006).Recently the whole genome sequencing of plants has assisted for a survey of plant14-3-3proteins and possible implications for their role in plant growth and develop-mental processes.Data mining has identified15and8 14-3-3genes in Arabidopsis and rice genome,respectively (DeLille et al.2001;Rosenquist et al.2001;Sehnke et al. 2006;Yao et al.2007b).Of those14-3-3genes identified, only13genes in Arabidopsis and6in rice are transcribed. Presence of large number of isoforms in these plants sug-gests that isoform-specific interactions with specific targets might be an important element in regulation of14-3-3function.These14-3-3isoforms are encoded by different genes with small differences in their sequences but perform similar function by physically interacting with specific client proteins and bringing a modification in clients. Arabidopsis14-3-3isoforms display distinct and differen-tial patterns of subcellular distribution and their localiza-tion is both isoform specific together with their interaction with cellular clients(Alsterfjord et al.2004;Paul et al. 2005).The isoform-specific interaction of14-3-3proteins has also been found in rice where different isoform showed different binding specificity towards ACC synthase(Yao et al.2007a).Therefore,it is important to address the implications of14-3-3family diversity within organisms as their genome becomes available.The involvement of spe-cific14-3-3isoforms in a given physiological condition and their temporal and spatial expression may elucidate their role in plant development and/or resistance to stress.Soybean(Glycine max[L.]Merr.)is an important cash crop that contains world’s major supply of both vegetable oil and protein with variety of uses in human food and animal feed.Despite that much is learnt about14-3-3s in non-legumes,the diversity of this group of proteins in legume plants is not yet known.A proteomic study of seed filling in soybean identified a number of14-3-3proteins from soybean seeds(Hajduch et al.2005)suggesting their role in seed development.The expression of a soybean 14-3-3gene was also upregulated in response to plant–microbe interaction(Seehaus and Tenhaken1998).Avail-ability of full genome sequence of soybean provides an excellent opportunity to explore diversity of legume14-3-3s in general.Furthermore,a comparative analysis of the 14-3-3proteins from different plant species may provide new insights on the phylogeny and functional conservation and diversification of the14-3-3s in plants.In this study, data mining of soybean genome was performed and18 14-3-3genes were identified.By using reverse transcriptase (RT)-PCR,it was confirmed that out of1814-3-3s,only16 genes(SGF14a-SGF14p)are expressed in soybean.This study demonstrates that(1)soybean SGF14isoforms were produced by tandem duplication as well as whole genome duplication events,(2)the expression patterns of SGF14 genes are diverse and vary depending on tissue type and developmental stage,and(3)SGF14s are localized in dif-ferent subcellular compartments and the localization pattern is isoform specific with some overlaps between closely related isoforms.Identification of all the members of SGF14 family in soybean and understanding their diversity as well as specificity towards their client proteins may provide key information for further study of this important gene family in soybean.Our study presents the foundation for an in-depth and detailed analysis of14-3-3gene family in legumes.The results may facilitate cross-utilization of genetic resources in agricultural important legume crops.Materials and methodsPlant materialsSoybean(G.max L.Merr.)cv Harosoy63seeds were planted at Agriculture and Agri-Food Canada experimental station in southern Ontario,London,in2008.Regular agronomic practices and planting dates were followed.The pods were tagged on thefirst day of pollination and har-vested at30,40,and50days after pollination(DAP). Soybean tissues were randomly collected from5–7plants and were frozen in liquid nitrogen,and stored at-80°C.Tobacco(Nicotiana benthamiana)seeds were grown in pots under the condition of a12-h light/dark cycle at25°C and70–80%relative humidity.Identification of soybean14-3-3ESTs and isolationof full length cDNAsThe DFCI Soybean Gene Index database(http://compbio. /tgi/cgi-bin/tgi/gireport.pl?gudb=soybean) was searched by using‘14-3-3’as a key word.Candidate expressed sequence tags(ESTs)or tentative contigs(TCs) were utilized to search against the soybean genome data-base(/search.php?show=blast). PCR amplification was carried out using pooled cDNA from different soybean tissues as template with gene-spe-cific primers designed to amplify complete open reading frame of each soybean14-3-3gene.In each forward pri-mer,CACC was added before ATG to facilitate the target sequence into Gateway pENTR/D-TOPO entry vector (Invitrogen,Carlsbad,CA,USA)in correct orientation. The PCR product was gel purified and recombined into pENTR/D-TOPO entry vector.The resulting recombinant plasmid contained target14-3-3sequenceflanked by attL region.The full length cDNA sequence for each14-3-3 clone was confirmed by sequencing and primer specificity to amplify specific14-3-3gene was verified.The soybean 14-3-3isoforms were named(SGF14a–SGF14r)following existing nomenclature for constancy.The gene-specific primer sequences for isolating soybean14-3-3genes are shown in supplementary Table S1.Gene structure and phylogenetic analysisComparison and analysis of soybean14-3-3sequences were performed using BLAST(Altschul et al.1990)at the National Center for Biotechnology Information(http:// /).Multiple sequence alignments were obtained using ClustalW2(/ Tools/clustalw2/index.html).The phylogenetic tree was built by neighbor-joining method using MEGA4software (Tamura et al.2007).Bootstrap values were calculated from1,000trials.The evolutionary distances were com-puted using the Dayhoff matrix based method(Schwarz and Dayhoff1979).Gene structure was constructed by SIM4(http://pbil.univ-lyon1.fr/members/duret/cours/ inserm210604/exercise4/sim4.html).The accession num-bers of Arabidopsis and rice14-3-3proteins used in phy-logenetic analysis are:AtGF14j(AAD51783),AtGF14k (AAD51781),AtGF14v(AAA96254),AtGF14u(AAB 62224),AtGF14x(AAA96253),AtGF14m(AAD51782), AtGF14t(AAB62225),AtGF14w(AAA96252),AtGF14l (AAD51784),AtGF14i(AAK11271),AtGF14o(AAG 47840),AtGF14e(AAD51785),AtGF14p(NP_565174), OsGF14a(AAO72553),OsGF14b(AAB07456),OsGF14c (AAB07457),OsGF14d(AAB07458),OsGF14e(CAB 77673),OsGF14f(AAX95656),OsGF14g(BAD73105), OsGF14h(ABA94733)and human theta(NP_006817). The cDNA accession numbers of soybean14-3-3proteins are shown in Table1.cDNA and predicted protein sequences for transcribed soybean SGF14s were used for sequence alignment and phylogenetic analysis. Quantitative RT-PCRTotal RNA was isolated from soybean tissues following the procedure of Wang and Vodkin(1994),quantified using a nanodrop1000(Bio-Rad Laboratories,Inc.),and concen-tration and integrity of RNA checked.RNA sample from each tissue was treated with RQ1RNase-Free DNase I (Promega,Madison,WI)at37°C for30min prior to RT-PCR.DNase I-treated RNA samples were extracted with phenol:chloroform,precipitated with ethanol and re-quan-tified using nanodrop1000.Total RNA(4l g)from each sample was used for reverse transcription using Thermo-Script TM RT-PCR system(Invitrogen)according to manu-facturer’s instruction.Quantitative PCR was conducted using QuantiTectÒSYBR Green PCR(Qiagen Inc.,Miss-issauga,ON,Canada)in a standard PCR reaction according to manufacturer’s instructions using CFX96real-time PCR detection system(Bio-Rad Laboratories,Inc.).A negative control without cDNA template was included for each pri-mer combination.Soybean ubiquitin-3(SUBI-3)gene transcript does not vary among different tissues and was used as a reference gene for data normalization and to calculate the relative mRNA levels(Fig.S1,Kim et al. 2005;Trevaskis et al.2002).The primers used to amplify SGF14genes and their amplicon sizes are shown in sup-plementary Table S2.PCR efficiency for each primer set was determined by standard curve method(Table S2).At least two biological replicates and three technical replicates for each biological replicate were used.Data analysis was performed by using Bio-Rad CFX manager(Bio-Rad Laboratories,Inc.).A melt curve analysis was performed where a single peak was detected for each SGF14amplicon.Subcellular localization of soybean14-3-3genesThe subcellular localization study was conducted using Bimolecular Fluorescence Complementation(BiFC)assay where splitfluorescent protein segments are brought together to form a functionalfluorophore as a result of protein–protein interaction.Each SGF14gene cloned into pENTR/D-TOPO vector was recombined with Gateway BiFC vectors pEarly-gate201-YN and pEarlygate202-YC(Lu et al.2010),that contains N-terminal(1–174amino acids)and C-terminal (175–239amino acids)of eYFP,in separate reactions using LR clonase reaction mix(Invitrogen,Carlsbad,CA,USA)and transformed into E.coli strain DH5a.The recombined plas-mids were extracted and sequenced to confirm the sequence integrity.Each plasmid DNA was transformed into Agro-bacterium tumefaciens strain GV3101via electroporation. Cotransformation of A.tumefaciens carrying SGF14gene cloned into each of the pEarlygate201-YN and pEarlygate202-YC vectors and transient expression in N.benthamiana leaves was conducted according to Sparkes et al.(2006).Epidermal cell layers of N.benthamiana leaves were assayed forfluo-rescence2–3days after infiltration.Imaging of YFP was performed by a Leica TCS SP2inverted confocal microscope using a639water immersion objective and Leica Confocal software at an excitation wavelength of514nm,and emis-sions were collected between530and560nm.The experi-ment was repeated at least three to four times for each gene.ResultsIdentification of soybean14-3-3gene family and their phylogenetic relationshipsThe soybean EST database at DFCI gene index containing 1315705ESTs represents72042tentative contig(TC) sequences and62990singleton ESTs.These TC and single-ton sequences were used as an initial source to identify potential14-3-3genes by keyword search.This process identified94sequences including40singletons which were then used as query sequences to perform BLAST search against soybean genome database to obtain14-3-3genes.The search revealed that many of the TCs were highly similar and/ or corresponded to a different region of the same gene.Our exhaustive data mining identified1814-3-3genes in soybean genome which were located in12different chromosomes as shown in Table1.Out of18,1614-3-3s(SGF14a to SGF14p) have ESTs/TCs with high sequence identity ranging from96 to100%at nucleotide level.Even though SGF14q(in chro-mosome7)and SGF14r(in chromosome20)showed85% similarity with TC302191(Table1),the identical residues were dispersed through out the sequence as revealed by their sequence alignment(Fig.1).Table1provides the detail information on all soybean SGF14genes.The SGF14genes in soybean encode proteins with calculated molecular masses ranging from28.2to30.5kDTable1Soybean14-3-3gene informationGene name cDNAaccession#Protein size(amino acids)Chromosome number:gene location aCorresponding TC#(%identity)bSGF14a HM004359257Gm18:61885459–61888618(?strand)TC336378(100) SGF14b AK285530250Gm04:8085999–8088679(?strand)TC296072(97) SGF14c HM004361259Gm05:34754666–34758625(?strand)TC321441(98) SGF14d AK285774/U70536261Gm13:37943326–37946655(?strand)TC325659(96) SGF14e HM004360259Gm01:7614430–7618123(?strand)TC302191(100) SGF14f HM004358259Gm02:11188826–11193147(?strand)TC279559(100) SGF14g BT096871262Gm02:42460325–42462765(?strand)TC289097(99) SGF14h HM004357259Gm04:9057040–9059334(-strand)TC299321(100) SGF14i AK286671259Gm06:8046858–8049172(-strand)TC299321(97) SGF14j AK285891250Gm06:7426323–7428626(?strand)TC298321(100) SGF14k AK286798261Gm14:44375153–44377652(?strand)TC298759(99) SGF14l AK286414259Gm08:8884778–8887873(?strand)TC321441(97) SGF14m AK286318257Gm08:46677649–46680782(-strand)TC331886(100) SGF14n HM004356265Gm12:38944080–38948239(-strand)TC284291(99) SGF14o AK286943/AK244317261Gm12:36987099–36990513(-strand)TC325659(100) SGF14p AF228501263Gm13:36124809–36128694(?strand)TC277945(100) SGF14q–261Gm07:40343251–40346828(-strand)TC302191(85) SGF14r–261Gm20:2852621–2859029(-strand)TC302191(85)a Chromosome location indicates the position of each gene in chromosomeb The tentative contig(TC)number with the highest homology to target SGF14gene,the identity is shown in the bracketand estimated pI ranging from4.56to4.83.An alignment of deduced SGF14isoforms from soybean indicated that the amino acid sequences are highly conserved except at the N-terminal and C-terminal regions(Fig.2).The sequence conservation among SGF14proteins was also supported by the percentage identity at amino acid and nucleotide level that vary from58to99%and58to97%, respectively(Table S3).Fig.1Nucleotide sequence alignment of SGF14q,SGF14r and TC302191.Identical residues are shown in asterisks. Hyphen indicates a gapA phylogenetic analysis was conducted on all tran-scribed14-3-3s from soybean,Arabidopsis and rice using both protein and cDNA matrices.As shown in Fig.3, SGF14b,SGF14j SGF14m,SGF14a,SGF14g,SGF14k, SGF14h and SGF14i grouped together with the Arabid-opsis and rice non-epsilon isoforms while SGF14o, SGF14d,SGF14c,SGF14l,SGF14n,SGF14p,SGF14e and SGF14f formed a branch together with the Arabidopsis and rice epsilon-like isoforms.The analysis also grouped two SGF14s with sequence identity96%or higher into a dis-crete clade for example SGF14p and SGF14n(Fig.3). Further,inclusion of all14-3-3s from Arabidopsis and rice in the phylogenetic tree of soybean14-3-3s,established the evolutionary relationships of14-3-3proteins from these three different species that are consistent with the species evolutionary history.The presence of highly similar14-3-3s in these plant species could have resulted from their whole genome duplication event.The comparativeanalysisof14-3-3s in Arabidopsis,rice and soybean suggested that Arabidopsis14-3-3s experienced independent gene duplication(e.g.,AtGF14m and AtGF14t,AtGF14v and AtGF14u,AtGF14j and AtGF14k)after its separation from soybean(Fig.3).One such14-3-3gene duplication occurred in rice which generated OsGF14b and OsGF14e. Soybean14-3-3gene structure comprised4and6exons in non-epsilon and in epsilon group,respectively(Fig.4).The gene structure analysis revealed that the two SGF14genes that formed a discrete clade in the phyogenetic tree(such as SGF14p and SGF14n)consisted of mostly similar size of exons but differed in the size of their introns.Soybean14-3-3isoforms are regulated differentiallyTo determine if all1814-3-3genes are expressed in soy-bean,we designed gene-specific primers for each SGF14s and performed RT-PCR using pooled cDNAsamplesprepared from different soybean tissues at various stages of development.The results showed that except for SGF14q and SGF14r,all other SGF14genes were transcribed in soybean.Search for ESTs corresponding to SGF14q and SGF14r in the EST database identified TC302191with 85%identity with both SGF14q and SGF14r(Table1). TC302191also displays100%identity with SGF14e. Therefore,we conclude that despite1814-3-3genes being present in soybean genome,only16isoforms are transcribed.To evaluate the tissue-specific expression of14-3-3 genes in soybean tissues during development,we per-formed quantitative RT-PCR using gene-specific primers. As shown in Fig.5,SGF14genes were expressed ubiqui-tously in all the tissues except for SGF14p which was not expressed in root tissue.The transcript levels and expres-sion profiles of SGF14isoforms varied depending on tissue type and developmental stage in soybean.All the SGF14 genes were expressed to higher level in embryo during seed development compared to other tissues with the exception of SGF14p,SGF14n and SGF14j whose expression levels were higher in other tissues.Furthermore,expression level of majority of soybean SGF14genes were higher during early seed development(30DAP)followed by a gradual decrease in the level as the embryos approached maturity excluding SGF14m(Fig.5).Comparison of relative tran-script levels of SGF14genes in different tissues showed that SGF14b,SGF14n,SGF14j,SGF14d,SGF14i and SGF14g were expressed at higher levels in leaf compared to other vegetative tissues with SGF14j level being the highest.Soybean14-3-3transcripts accumulated to rela-tively lower level in roots compared to other tissues.The qPCR analysis detected relatively higher levels of SGF14n and SGF14p transcripts in pod wall and SGF14m,SGF14p and SGF14k transcripts in seed coat tissues.However,no expression was detected for SGF14n and SGF14p tran-scripts in soybean root.Apart from SGF14m and SGF14d, the expression of other SGF14s was relatively lower level in stem compared to other tissues included in the study. The expression patterns for those SGF14s that formed same clade in phylogenetic tree(Fig.3)generally showed sim-ilar tissue-specific expression pattern except for SGF14a and SGF14m.Subcellular localization of soybean14-3-3proteinsThe principle of BiFC is based upon bringing together split fluorescent protein to form a functionalfluorophore.Theassociation of the splitfluorescent protein molecule does not producefluorescence spontaneously and requires interaction between proteins that are fused to each of the fluorophore fragments.The protein–protein interaction of fused proteins results in the association between the split fluorophore fragments leading to formation of afluorescent protein that has the same spectral properties as the unsplit fluorescent protein(Ohad et al.2007).To investigate the subcellular localization of soybean SGF14s and to ensure that the observed localization is a result of their homodi-merization and not due to the interference from tobacco 14-3-3s,we performed BiFC assay using split yellow fluorescent protein(YFP).A translational fusion of each SGF14gene from soybean was created with N-terminal (YN)and C-terminal(YC)half of YFP under the control of CaMV35S promoter.YN and YC constructs for each SGF14were coexpressed transiently in tobacco epidermal cells by Agrobacterium-mediated transformation and pro-tein expression was monitored by confocal microscopy. Control experiments were performed by coexpressing SGF14-YN or YC fusion for each14-3-3protein with non-fusion half of YFP or coexpressing vector only YN and YC.Thefluorescence detection parameters for YFP observation were kept consistent for the entire SGF14 localization experiment.No signals were detected for negative controls(data not shown).Our results indicated that all SGF14s can form homodimer.The14-3-3s with highest sequence identity that form separate clade in the phylogenetic tree(Fig.3)showed similar subcellular localization pattern(Fig.6).The assay confirmed cyto-plasmic localization of all soybean14-3-3s.In tobacco epidermal cell,the cytoplasmicfluorescence typically looks like thin area between the cell wall and the turgescent vacuole(Walter et al.2004).All the SGF14s were also localized in nucleus.However,the pattern and intensity of SGF14expression in the nucleus varied in different iso-forms.SGF14p and SGF14n YFP signals were strong in nucleus compared to other14soybean SGF14s.No SGF14 were found in nucleolus.There was no overlap of YFP signal with chloroplast autofluorescence suggesting that SGF14s may not be present in chloroplast.Thefluorescent protein usually shows network structure when targeted to endoplasmic reticulum(ER)(Dhaubhadel et al.2008). Absence of such network structure of SGF14fused YFP in the cell suggests that SGF14s may not be localized in ER. Soybean14-3-3proteins can form heterodimerIt has been well documented that14-3-3proteins form both homo-and heterodimers(reviewed in MacKintosh2004). The presence of16expressed14-3-3s in soybean could generate a large number of heterodimers.In order to investigate if soybean SGF14proteins form heterodimer and how the heterodimerization affects their subcellular localization,we selected representative SGF14genes that are either closely related such as SGF14m and SGF14a or distantly related such as SGF14m and SGF14n and per-formed BiFC assay using the translational fusion of YN with YC with different SGF14s.The resultsdemonstratedthat SGF14s hereodimerizes with its isoforms,irrespective of the sequence similarity (Fig.7).However,their sub-cellular localization differed depending on each monomer involved in dimerization.No change in subcellular location was observed for the closely related SGF14heterodimers (Fig.7,SGF14c/SGF14l,SGF14n/SGF14p and SGF14m/SGF14a).Although,the YFP signal for the heterodimers was detected in both cytoplasm and nucleus for the SGF14s belonging to two different classes that lie far apart in the phylogenetic tree (SGF14m/SGF14n and SGF14m/SGF14p),the intensity of signal differed from their homodimer status (compare Figs.6and 7).Discussion14-3-3proteins are encoded by a large multigene family in plants.Arabidopsis and rice genome consist of 15and 814-3-3genes,respectively (Rosenquist et al.2001;Yao et al.2007b ).A large number of 14-3-3s have also been identi-fied in tomato and tobacco and additional 14-3-3genes are expected to be identified as full genome sequence of other organisms becomes available.We have identified 1814-3-3genes,SGF14a -SGF14r ,in soybean genome (Schmutz et al.2010),of which 16SGF14s are transcribed.Soybean is an allotetraploid and its genome is much larger (1,115Mb)compared to Arabidopsis (145Mb)and rice (420Mb)(Arumuganathan and Earle 1991).Soybean genome has undergone at least two whole genome dupli-cations events approximately 14and 42million years ago (MYA)(Blanc and Wolfe 2004;Gill et al.2009;Schlueter et al.2004;Shoemaker et al.1996)while the whole gen-ome duplications in Arabidopsis and rice genomes occur-red about 100–200and 60–70MYA,respectively (Blanc et al.2000;Yu et al.2005).The genome duplication event usually results in rearrangement and reshuffling of chro-mosomal regions creating the potential for new diversity and possibility of subfunctionalization (Force et al.1999).Analysis of soybean SGF14gene sequences show very high sequence identity between two subgroups of SGF14s that form a distinct clade in the phylogenetic tree,however,these genes are found to be located in different chromo-somes (Table 1;Fig.3).The observation of two distinct subgroups of closely related SGF14family members such as SGF14n and SGF14p,SGF14o and SGF14d supportstheFig.6Subcellular localization of SGF14using BiFC assay.N.benth-amiana leaves were co-transformed with SGF14gene constructs fused N-and C-terminally to YFP followed by confocal microscopy.The YFP signal in nucleus and/or cytoplasm resulted from homodi-merization of same SGF14isoform.Scale bars are shown in l m.The arrowhead with a ,b and c indicate nucleus,cytoplasmic streaming and cell periphery,respectivelyc。

转录组综述一. 引言:基因的表达分为转录和翻译过程，对同一生物体而言，虽然每个细胞具有相同的基因，但不同的细胞在特定的时空条件下表达不同的基因，转录出不同的RNA分子。

例如，人类基因组包含有30亿个碱基对，大约有5万个基因转录成mRNA分子，转录后的mRNA能被翻译生成蛋白质只占整个转录组的40%左右，通过转录组谱数据研究可以得到什么条件下什么基因表达的信息［1］，这是基因功能及结构研究的基本出发点，随着生物学研究已经跨入后基因组时代，高通量测序技术的出现，大规模的基因表达水平研究的序幕已经拉开，转录组学作为一门新技术开始在生物学前沿研究中绽露头角，已经成为生命科学研究的热点，并逐渐走向应用。

二. 转录组概念：转录组学(transcriptomics)，是一门在整体水平上研究细胞中基因转录的情况及转录调控机制的学科，主要从RNA水平研究基因表达的情况。

一般来说，把转录组学分为广义和狭义转录组学［2］，广义转录组指从一种细胞或者组织的基因组所转录出来的RNA的总和，包括编码蛋白质的mRNA和各种非编码RNA (rRNA, tRNA, snoRNA, snRNA,microRNA和其他非编码RNA等)，狭义转录组是特定组织或细胞在某一发育阶段或功能状态下转录出来的所有RNA的总和［3］。

三. 转录组研究内容：转录组学的研究内容包括：对所有的转录产物进行分类，确定基因的转录结构,通过对转录谱的分析，推断相应某一基因的功能，揭示特定调节基因的作用机制，辨别细胞的表型归属等［4］。

四. 棉花转录组研究的意义棉花纤维转录组研究起步较晚，但近年来大量高质量棉花胚珠、纤维cDNA文库的构建,EST数据库的丰富，以及高通量基因芯片的应用和转录组测序工作的开展,在涉及纤维起始分化、伸长及次生壁加厚等的各个发育阶段均取得了不小的成果。

从整体的转录组水平上对棉纤维复杂的多基因遗传机制进行深入研究以及了解整个纤维发育的分子调控机制，结合分子标记技术定位的大量与纤维产量和纤维品质相关的QTLs，非常有助于分子标记辅助选择(MAS )育种和纤维品质的改良。

《分子生物学常用实验技术》课堂练习题参考答案学号：姓名：一、填空题。

1.在用SDS分离DNA时，要注意SDS浓度，0.1%和1%的SDS作用是不同的，前者，后者。

2.SDS是一种提取DNA时常用的阴离子去污剂，它可以溶解膜蛋白和脂肪，使细胞膜和核膜破裂，使核小体和核糖体解聚，释放出。

它还可以使蛋白质变性沉淀，也能抑制。

3.在分离DNA时，需要带手套操作，是因为。

4.用酚-氯仿抽提DNA时，通常在氯仿或酚-氯仿中加入少许异戊醇，这是因为有机溶剂异戊醇可以。

另外，异戊醇有助于分相，使离心后的上层含DNA的水相、中间的变性蛋白质相和下层有机溶剂相维持稳定。

5.在分离DNA时，要用金属离子螯合剂，如EDTA和柠檬酸等，其目的是：。

6.在DNA分离过程中，造成DNA分子断裂的因素很多，主要有：、和。

7.在分离DNA时，常用酸变性、碱变性、热变性和来去除蛋白质。

8.用乙醇沉淀DNA的原理是：。

9.用乙醇沉淀DNA时，通常在DNA溶液中加入单价阳离子，如氯化钠和乙酸钠，其目的是。

10.通常可在3种温度下保存DNA：4～5℃、-20℃和-70℃，其中以℃为好。

11.浓缩DNA的方法通常有：包埋吸水法、蒸发法、膜过滤法和。

12.碱裂解法和清亮裂解法是分离质粒的两种常用方法，二者的原理不同，前者是根据，后者是根据。

13.在技术中，DNA限制性酶切片段经凝胶电泳分离后，被转移到硝酸纤维素薄膜上，然后与放射性标记的DNA14.SSC是氯化钠和柠檬酸钠组成的试剂，其中前者作用是：，后者的作用是：。

15.在southern杂交中，DNA转移的速度取决于、和。

16.在重蒸酚中加入1%的8-羟基喹啉及少量β-巯基乙醇，不仅可以防止酚氧化，还可以抑制活性以及。

17.RNA分子经凝胶电泳后按大小不同分开，然后被转移到一张硝酸纤维素膜（尼龙膜）上，同一放射性DNA探针杂交的技术称__。

18.根据Northern杂交结果可以说明__。

RADIATION RESEARCH160,217–223(2003)0033-7587/03$5.00᭧2003by Radiation Research Society.All rights of reproduction in any form reserved.Reduction of14-3-3Proteins Correlates with Increased Sensitivity to Killing of Human Lung Cancer Cells by Ionizing RadiationWenqing Qi and Jesse D.Martinez1Department of Radiation Oncology,The University of Arizona,and Arizona Cancer Center,Tucson,Arizona85724Qi,W.and Martinez J.D.Reduction of14-3-3ProteinsCorrelates with Increased Sensitivity to Killing of HumanLung Cancer Cells by Ionizing Radiation.Radiat.Res.160,217–223(2003).The14-3-3proteins have a wide range of ligands and areinvolved in a variety of biological pathways.Importantly,14-3-3proteins are known to be overexpressed in some humanlung cancers,suggesting that they may play a role in tumor-igenesis.Here we examined14-3-3expression in several lungcancer-derived cell lines and found that four of the seven14-3-3isoforms,␤,␧,␪and␨,were highly expressed in both lungcancer cell lines and normal lungfibroblasts.Two isoforms,␴and␥,were present only at very low levels.Immunopre-cipitation data showed14-3-3␨could bind to CDC25C in ir-radiated A549cells,and suppression of14-3-3␨in A549cellswith antisense resulted in a decrease in CDC25C localizationin cytoplasm and CDC2phosphorylation on Tyr15.As a con-sequence,CDC2activity remained elevated which resulted inrelease from radiation-induced G2/M-phase arrest.Moreover,16%14-3-3␨antisense-transfected cells underwent apoptosis when exposed to10Gy ionizing radiation.These data indicatethat14-3-3␨is involved in G2checkpoint activation and thatinhibition of14-3-3may be a useful approach to sensitize hu-man lung cancers to ionizing radiation.᭧2003by Radiation Research SocietyINTRODUCTIONThe14-3-3proteins are a family of highly conserved proteins that play important roles in a wide range of cellular processes including signal transduction(1),apoptosis(2), cell cycle progression(3),and checkpoint activation(4). There are seven separate14-3-3genes that encode different isoforms,each designated with a Greek letter(␤,␥,␧,␩,␴,␪,␨).14-3-3proteins have been found to interact with a large number of heterogeneous ligands,and analysis ofthe interaction reveals a consensus motif surrounding a phosphorylated serine(5).Hence the14-3-3proteins are1Address for correspondence:Department of Radiation Oncology,The University of Arizona,1501N.Campbell Avenue,P.O.Box245024, Tucson,AZ85724;e-mail:jmartinez@.thought to regulate the activity of a number of phosphor-ylation-mediated events.Consistent with this,14-3-3proteins have been shown to play a critical role in DNA damage-induced checkpoints by controlling the biological activity of several key cell cycle checkpoint proteins through binding to phosphorylated ser-ine residues(6).In normal cells,DNA damage leads to activation of cell cycle checkpoints and arrest in the G1and G2phases of the cell cycle.This requires inactivation of the CDC25C,a major cell cycle regulator that dephos-phorylates and activates the cyclin-dependent kinase CDC2 to trigger entry into mitosis(6–8).Phosphorylation of CDC25C on serine216creates a binding motif for14-3-3, and this interaction combined with a strong14-3-3nuclear export signal promotes export of CDC25C from the nucleus and suppression of its biological activities(8).Importantly, mutant CDC25C proteins in which serine216was replaced with alanine are unable to DNA damage leads to phos-phorylation of CDC25C on S216,the inactivation of which is a key event in DNA damage-induced G2-phase arrest(9).The14-3-3proteins also have a major role in control of apoptosis.14-3-3tau binds with the BCL2-related protein, BAD,and suppress its pro-apoptosis function by relocal-izing the protein to the cytoplasm(10,11).14-3-3zeta also inhibits the pro-apoptosis effect of ASK1through a mech-anism that may involve disruption of the interaction be-tween ASK1and its death effectors(12).In addition,in-teraction between14-3-3zeta and the pro-survival PI3K stimulates its kinase activity(13).Hence14-3-3proteins act to reduce sensitivity to apoptosis at many levels and by interaction with several different important regulators of ap-optosis.14-3-3proteins bind to the apoptosis-promoting proteins,BAD and BAX,and this interaction prevents BAD and BAX from binding to BCL2or BCL2L1(also known as BCL-xL)and inducing apoptosis(2,14–18).In this study we characterized14-3-3protein expression in lung cancer cells,a tumor type that has been shown to overexpress14-3-3(19),and examined the effect that sup-pression of14-3-3expression had on the response of these cells to ionizing radiation.We found that depressed14-3-3 levels lead to an altered radiation response that resulted in greater sensitivity to killing by radiation.217218QI AND MARTINEZMATERIALS AND METHODSCell Culture and ReagentsCells of the human lung adenocarcinoma A549,non-small cell carci-noma H358and H322,and small cell carcinoma H69as well as human lung normalfibroblast(HEL)cell lines were purchased from American Type Culture Collection(Rockville,MD).The cells were maintained in DMEM supplemented with10%fetal bovine serum at37ЊC in an incu-bator in a humidified atmosphere of95%air/5%CO2.Cells were grown to about70–80%confluence and then treated with ionizing radiation from a60Co source.Cells were then returned to the37ЊC incubator and har-vested at regular intervals as indicated in thefigure legends.The anti-14-3-3␤,14-3-3␥,14-3-3␧,14-3-3␴,14-3-3␪,14-3-3␨and anti-PARP anti-bodies were purchased from Santa Cruz Biotechnology(Santa Cruz,CA). Anti-CDC2/p34was bought from Oncogene Research Products(Cam-bridge,MA).Anti-CDC25C,anti-␤-actin and anti-phospho-CDC2 (Tyr15)were from Sigma(St.Louis,MO)and Cell Signaling(Beverly, MA),respectively.Plasmids and TransfectionUsing total RNA from A549cells,the full14-3-3␨cDNA was made by RT-PCR.The primers specific to14-3-3␨were forward,5Ј-GCCTGTGAGCAGCGAGATCC-3Јand reverse,5Ј-AGCATGGATGA-CAAATGGTC-3Ј.The RT-PCR production was cloned into pGEM-T Easy Vector(Promega)and the insertion was sequenced.Then the14-3-3␨cDNA was subcloned into pLXSN expression vector(20).Inverted insertion clones were selected as antisense of14-3-3␨constructs.A549 cells were transfected with14-3-3␨antisense constructs using Lipofec-tamine reagent(Invitrogen).Twenty-four clones were picked and screened by Western blotting for reduced expression of14-3-3␨.Cell Death AssayFor routine analysis of apoptosis,treated cells were examined for ap-optotic morphology using afluorescence staining technique as described previously(21).Briefly,cells were exposed to0or10Gy ionizing ra-diation as indicated.At regular intervals,cells were harvested by tryp-sinization and stained with mixed dye solution containing100␮g/ml each acridine orange and ethidium bromide.The morphology of the cells was observed byfluorescence microscopy,and the number of apoptotic cells was quantified.In all cases a minimum of200cells were counted for each sample.Cell death was determined by inclusion of trypan blue.At the indicated times after treatment with radiation,cells were detached with trypsin, pelleted and resuspended in medium.After staining with trypan blue, more than500cells infive randomfields of view were counted.The percentage of dead cells was determined as the percentage of total cells.Mitotic IndexCells were grown to70–80%confluence and exposed to10Gy radi-ation.After12h incubation,0.5␮g/ml nocodazole was added and the cells were incubated for another36h or60h.Cells were harvested with trypsin and washed with cold50%PBS.They were washed again in50% PBS and left on ice for10min.After the cells werefixed in cold mitotic indexfixer[50%PBSϩ2%EtOH/acetic acid(3:1)]for30min on ice, they were centrifuged at1000rpm.The cell pellet was resuspended in ethanol/acetic acid(3:1)solution for10min on ice.The cell suspension was dropped onto slides,the slides were air-dried,and the cells were stained with Giemsa solution for2min.Five hundred cells were counted using a light microscope after the slides were rinsed twice in water for 30s.The mitotic index was calculated by dividing the number of mitotic cells by the total number of cells counted,then multiplying by100%.Protein AnalysisTo isolate proteins,the cells were harvested and then lysed with NP-40lysis buffer containing50m M Tris-Cl(pH7.4),0.15M NaCl,0.5% NP-40,1m M DTT,50m M sodiumfluoride,5␮g/ml aprotinin,5␮g/ml leupeptin,and1m M phenylmethylsulfonylfluoride.Protein concentra-tions were determined using the Bio-Rad protein assay kit(Hercules, CA),and50␮g protein was resolved by electrophoresis on a10%SDS-PAGE gel.The proteins were then transferred onto a nitrocellulose mem-brane,and nonspecific binding was blocked by incubating with5%nonfat milk in TBST buffer(0.01M Tris-Cl,pH8.0,0.15M NaCl,0.5%Tween-20)at room temperature for1h.The appropriate antibodies were diluted in TBST with5%nonfat dry milk and incubated with thefilter for1.5 h.Detection was with the ECL detection system.For preparation of cytoplasmic extracts,cells were collected and ho-mogenized with a Dounce homogenizerfitted with a loose pestle in10 m M Hepes,pH7.9,1.5m M MgCl2,10m M KCl,0.2m M phenylmethyl-sulfonylfluoride,and0.5m M dithiothreitol at4ЊC.The nuclear and cy-toplasmic fractions were divided by centrifuging at4500rpm for15min, and protein was extracted as described above.ImmunoprecipitationThe protocol used for immunoprecipitation has been described previ-ously(17).Briefly,cells were collected by scraping9h after irradiation. Lysates(200␮g of protein prepared as above)were incubated with1␮g anti-CDC25C antibody at4ЊC overnight followed by an additional2h of incubation with35␮l of protein A agarose beads(Gibco-BRL,Gai-thersburg,MD)at4ЊC.The beads were then washed four times with lysis buffer,and the proteins were eluted by boiling in SDS-PAGE sample buffer.The proteins were applied to SDS-PAGE gels and14-3-3protein was detected by immunoblotting using the specific different14-3-3iso-form antibodies.Histone H1Kinase AssayCell lysate was prepared using H1lysis buffer(50m M Tris-Cl,pH.7.4, 0.25M NaCl,0.5%Nonidet P-40,50m M sodiumfluoride,1m M phen-ylmethylsulfonylfluoride,10␮g/ml leupeptin,and20␮g/ml aprotinin), and200␮g of protein was incubated with1␮g anti-CDC2antibody and 35␮l protein A agarose beads for1h at4ЊC.The beads containing the immunoprecipitated proteins were washed twice with H1lysis buffer and four times with H1kinase buffer(20m M Tris-Cl,pH7.4,7.5m M MgCl2, and1m M dithiothreitol DTT).The washed protein A beads were resus-pended in50␮l of H1kinase buffer supplemented with15␮M ATP, 0.37MBq of[␥-32P]ATP,and1␮g of histone H1(Gibco-BRL).After30 min of constant agitation at37ЊC,the reactions were terminated with50␮l of SDS-PAGE sample buffer and loaded onto a12%SDS-PAGE gel. After electrophoresis,the gel was dried and autoradiographed.RESULTSIsoform-Specific Expression of14-3-3in Lung CellsIt has been reported that the level of14-3-3proteins is high in lung cancers compared to normal lung tissues,sug-gesting that the14-3-3family of proteins can be an effec-tive marker for use in lung cancer diagnosis and that14-3-3proteins might be involved in the development of lung cancers(19).However,it is unclear which of the seven14-3-3isoforms is expressed.To study this,specific anti-14-3-3␤,␥,␧,␴,␪and␨antibodies were used to detect14-3-3 protein levels in A549,H358,H322,H69and HEL cells. Four isoforms,␤,␧,␪and␨,were present in elevated levels in all of the cell lines examined(Fig.1).On the other hand219REGULATION OF RADIOSENSITIVITY BY14-3-3FIG.1.Isoform-specific expression of 14-3-3proteins in lung cell lines.A549,H358,H322,H69and HEL cells were cultured to 70–80%confluence and harvested.Total proteins were extracted,and 50␮g was loaded on 10%SDS-PAGE gels.Specific 14-3-3␤,␥,␧,␴,␪,and ␨antibodies were used to detect the different 14-3-3isoforms.␤-Actin was used as a loading control.The blot shown is a typical result from four independentexperiments.FIG.2.14-3-3␨binds to CDC25C after irradiation.A549cells either were untreated or were irradiated with 10Gy ionizing radiation and then harvested 9h later.Then 200␮g of cell lysates was incubated with 1␮g anti-CDC25C antibody at 4ЊC overnight followed by an additional 2h of incubation with 35␮l of protein A agarose beads at 4ЊC.After the pellet was washed four times with lysis buffer,it was applied to 10%SDS-PAGE gels,and 14-3-3proteins were detected by immunoblotting with specific 14-3-3antibodies.Fifty micrograms of cell lysate was load-ed as a positive control.14-3-3␴protein could not be detected in any of the cell lines.14-3-3␥was undetectable in H322cells and was pre-sent at low levels in H358and HEL cells,but it was rela-tively abundant in the A549and H69cell lines.These data suggested that most 14-3-3isoforms were abundant in lung carcinoma-derived and normal cells but that two isoforms,␴and ␥,had no or low expression.14-3-3␨Reacts with CDC25C after Exposure to Ionizing RadiationIn response to genotoxic stresses,14-3-3protein binds to and sequesters CDC25in the cytoplasm,making it unable to activate CDC2through dephosphorylation on Tyr-15.However,the specific isoforms that binds with CDC25C is not known.In human cells,CDC25C is involved in control of G 2/M-phase transition and abrogating 14-3-3binding to CDC25C could attenuate the G 2checkpoint.Previously,we reported that 14-3-3proteins bind to CDC25C in A549and that cells were blocked in G 2/M phase after exposure to ionizing radiation (22).Here,we did co-immunoprecipita-tion experiments using an anti-CDC25C antibody to probe extracts from untreated and A549cells irradiated with 10Gy ionizing radiation.Protein complexes were probed for 14-3-3proteins using antibodies specific for six of the 14-3-3proteins (Fig.2).We found that 14-3-3␨could be read-ily detected in complex with CDC25C,whereas 14-3-3␪exhibited only weak binding.None of the other isoforms could be detected in the precipitated protein complexes.Reduction of 14-3-3␨Released G 2-Phase Block Induced by Ionizing RadiationTo determine whether 14-3-3␨had a role in regulating the G 2checkpoint,we designed 14-3-3␨antisense con-structs and stably transfected these into A549cells.Twenty-four clones were selected and screened for 14-3-3protein levels by immunoblotting using specific anti-14-3-3␨anti-body.Figure 3A shows one of three clones in which the 14-3-3␨protein was decreased compared with the vector control.Since 14-3-3␨sequesters CDC25C in cytoplasm and in-hibits CDC2dephosphorylation and G 2/M-phase transition after irradiation,we postulated that decreasing 14-3-3␨would reduce CDC25C localization in cytoplasm and CDC2phosphorylation.To test this,we examined the levels of CDC25C in the cytoplasm and the activity of CDC2in irradiated cells.We found that the levels of CDC25C in the cytoplasm were reduced in cells expressing the 14-3-3␨an-tisense expression vector relative to cells containing the vector only (Fig.3B).Moreover,radiation-induced CDC2Tyr-15phosphorylation was decreased and H1kinase ac-tivity was increased in the 14-3-3␨antisense cells compared with the vector-only transfected cells (Fig.3C and D).Hence CDC2activity was inhibited to a lesser degree by radiation when 14-3-3␨was suppressed,suggesting that cells with reduced 14-3-3␨were less likely to arrest in G 2phase after DNA damage.To confirm this,the mitotic index for vector-only and 14-3-3␨antisense cells were determined after exposure to 10Gy ionizing radiation (Fig.4).The results revealed that at 48h the mitotic index for both cell lines was similar.However,at 72h,the number of mitotic cells among the 14-3-3␨antisense cells was approximately 1.5times that of the vector-only cells.From these experi-ments,we conclude that a reduction in 14-3-3␨resulted in220QI ANDMARTINEZFIG.3.14-3-3␨regulates G 2-phase checkpoints.Panel A:Reduction of 14-3-3␨by antisense construct was detected by immunoblotting with specific anti-14-3-3␨antibody.␤-Actin was used as the loading control.Panel B:CDC25C protein levels in cytoplasm were decreased in irradiated antisense cells.Both vector and antisense cells were treated with 0and 10Gy ionizing radiation.At 12h,cytoplasmic protein extracts were resolved in 10%SDS-PAGE gels and subjected to Western blotting for CDC25C by using anti-CDC25C antibody.␤-Actin was used as the loading control.Panel C:14-3-3␨regulates CDC2activity.Cell extracts were prepared from vector and antisense cells with and without exposure to 10Gy ionizing radiation.CDC2tyrosine-15phosphorylation was detected by immunoblotting using anti-phospho-CDC2(Tyr-15)antibody.Panel D:CDC2-related histone H1kinase activity is elevated in antisense cells after irradiation.Vector and antisense cells were either left untreated or exposed to 10Gy ionizing radiation.Twelve hours later,total cell extracts were prepared,and CDC2was immunoprecipitated using an anti-CDC2specific antibody.H1kinase activity was assayed as described in the Materials and Methods.The band represents phosphorylated histoneH1.FIG.4.Reduction of 14-3-3␨releases G 2-phase block induced by ra-diation.The mitotic index was determined in vector and antisense cells at 48and 72h after irradiation with 0or 10Gy.The results are from three independent assays.Error bars represent standard deviations.an increase in the number of cells that could escape the radiation-induced G 2-phase arrest.Decreased 14-3-3Induces ApoptosisIt has been reported that 14-3-3proteins inhibit apoptosis through binding and modulating the activity of a host of signaling proteins and that inhibition of 14-3-3/ligand in-teractions can induce apoptosis.Importantly,14-3-3protein levels were decreased in radiation-induced apoptotic pros-tate cancer cells,suggesting that 14-3-3mediates apoptosis induced by radiation (23).To determine whether reduction of 14-3-3␨altered the cellular response to radiation,both vector and antisense cells were treated with 0or 10Gy radiation 24,48and 72h later and apoptosis was assayed using fluorescence microscopy (Fig.5A).In vector-only cells,no apoptosis occurs even when cells are treated with radiation.However,in the antisense cells,a low level of constitutive apoptosis,about 8%,was elicited.Notably,ra-221REGULATION OF RADIOSENSITIVITY BY14-3-3FIG.5.Decreased 14-3-3induces apoptosis.Panel A:Vector and antisense cells were untreated or were treated with 10Gy radiation.At 24,48and 72h,cells were harvested and stained using a mixture of ethidium bromide and acridine orange,and apoptosis was quantified using fluorescence microscopy.The results depicted are from three independent experiments.Error bars represent standard deviations.Panel B:Reduction of 14-3-3induces cell death.Vector and antisense cells were treated with 0and 10Gy radiation.At 24,48and 72h,the numbers of dead cells were determined by trypan blue staining and the ratio of dead cells to viable cells was calculated.The results are from three independent experiments.Error bars represent standard deviations.Panel C:PARP was cleaved in antisense cells,especially after irradiation.Both vector and antisense cells were either left untreated or treated with 10Gy radiation.At 48h,PARP was detected by immunoblotting with anti-PARP antibody.␤-Actin was used as the loading control.The blot shown is a typical result from two independent experiments.diation doubled the fraction of apoptotic cells to 16%over that seen in untreated antisense cells.To determine whether the mode of cell death could be attributed exclusively to apoptosis,cells treated as in Fig.5A were also examined by trypan blue dye exclusion (Fig.5B).We found that the kinetics of induction of cell death and the fraction of dying cells as assessed by trypan blue exclusion were similar to those seen in the apoptosis assays,suggesting that the major mode of cell death was apoptosis.To further confirm that apoptosis was occurring,we examined the PARP protein for cleavage by immunoblotting protein extracts from vec-tor and antisense cells treated with radiation (Fig.5C).As can be seen,PARP cleavage was most prominent in the antisense cells treated with radiation,but it was also de-tected at low levels in the untreated antisense cells.No PARP cleavage could be detected in the vector-only cells,which is consistent with the cell death assays indicating that apoptosis could be detected in the antisense cells.Because the sequence of the 14-3-3proteins is very high-ly conserved,we considered that the 14-3-3␨antisense vec-tor might affect the levels of other 14-3-3isoforms.Hence we examined the expression of three other 14-3-3isoforms,␤,␧,and ␪,in vector and antisense cells (Fig.6).We found that 14-3-3␤and ␧proteins were also decreased,whereas the level of 14-3-3␪was not changed.This results sug-gested that reduction of 14-3-3␨also led to the reduction222QI ANDMARTINEZFIG.6.The antisense 14-3-3␨construct also decreased 14-3-3␤and ␧but not ␪protein level.Western blotting was performed to detect 14-3-3␤,␧and ␪isoforms in antisense cells.␤-Actin was used as the loading control.The blot shown is a typical result from two independent exper-iments.of ␤and ␧and that suppression of these isoforms might also contribute to apoptosis induced by radiation in lung cancers.DISCUSSIONThe 14-3-3isoforms are widely distributed in various tis-sues (24).However,there is wide variation in the abundance of these subtypes within a specific tissue or when compari-sons are made between different tissues (25,26).In this study,we found that four 14-3-3isoforms,␤,␧,␪and ␨,were overexpressed in both human lung cancer and normal cell lines.However,14-3-3␴was not detected and 14-3-3␥was present in A549and H69cells only at a low level,which indicated that 14-3-3isoforms were differentially expressed in lung cells.A recent report indicates that 14-3-3proteins are highly expressed in lung cancer tissue compared with the normal lung (19).However,our data showed no difference in 14-3-3expression between cancer and normal cell lines,suggesting that 14-3-3expression is different in cultured cells and tissues from patients.This notion is supported by RT-PCR analysis of normal human lung tissue that demon-strates that only a few 14-3-3isoforms are expressed (un-published data).Nevertheless,the 14-3-3proteins expressed in cancer-derived cell lines are likely to have important roles in biological processes.The 14-3-3proteins play an important role in G 2/M-phase checkpoints (27).After DNA damage,TP53is sta-bilized and transactivates 14-3-3␴(28).14-3-3␴normally sequesters cyclin B1and CDC2in the cytoplasm,keeping CDC2-cyclin B1from entering the nucleus and initiating mitosis (29).Similarly,phosphorylation of CDC25on ser-ine-126by CHK1leads to an interaction with 14-3-3andexport from the nucleus which prevents dephosphorylation and activation of CDC2(30).Here we show using coim-munoprecipitation assays that 14-3-3␨reacted specifically with CDC25C in cells in which DNA damage had been induced by exposure to ionizing radiation.That the inter-action between 14-3-3␨is functionally significant is sup-ported by the fact that repression of 14-3-3␨with antisense resulted in reduced CDC2phosphorylation on tyrosine 15and delayed progression through G 2phase even after ex-posure to ionizing radiation.These data suggest an isoform-specific role for 14-3-3proteins and the function of 14-3-3␨in regulating the G 2-phase checkpoint is conserved among yeast (6,8),Xenopus (9)and human cells.Abrogation of G 2-phase arrest is most consistently as-sociated with increased radiosensitivity because mitotic ca-tastrophes occur if mitosis is induced experimentally in the presence of unreplicated or damaged DNA (29,31).Indeed,in this study,reduction of 14-3-3␨impaired the G 2-phase arrest induced by radiation and also resulted in 16%of the cells undergoing apoptosis.However,our previous study showed that no apoptosis was induced when the G 2block was released by caffeine in the same cell line (22),sug-gesting that abrogation of the G 2checkpoint may not be sufficient to induce apoptosis in cells exposed to ionizing radiation.Considering that 14-3-3␨antisense also resulted in reduction of 14-3-3␤and ␧proteins,it may be that re-ducing the levels of these two isoforms also contributed to apoptosis.Previous studies indicate that overexpression of 14-3-3␤in NIH 3T3cells stimulates cell growth and tumor formation in nude mice (32)and that inhibition of 14-3-3(all isoforms)/ligand interactions can elicit apoptosis (33).Hence the reduction of 14-3-3(especially ␤and ␧)proteins may also be important in the increased apoptosis observed in the antisense cells.In conclusion,four 14-3-3proteins (␤,␧,␪and ␨)were overexpressed in cultured lung cells.One isoform,14-3-3␨,could bind to CDC25C after exposure to ionizing radiation.Reduction of 14-3-3␨decreased CDC25C localization in cytoplasm and CDC2phosphorylation and consequently in-terfered with the radiation-induced G 2-phase arrest.These data suggest that inhibition of 14-3-3may represent a useful therapeutic target for the treatment of lung cancer,espe-cially together with radiotherapy.ACKNOWLEDGMENTSWe thank Dr.D.A.Galloway,Fred Hutchinson Cancer Center,for providing us the retrovirus vector,pLXSN.This work was supported by Arizona Disease Control Research Commission Contract No.10013.Received:November 11,2002;accepted:April 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