细胞通讯-信号转导.ppt

单击此处编辑母版标题样式,*,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,第一节 细胞通讯的分子基础,一、细胞间通讯方式,二、化学信号分子的种类及特性,三、受体,四、细胞信号转导的基本方式,五、细胞内关键信号转导分子,一、细胞间通讯方式,细胞间隙连接(,gap junction,)：使相邻的细胞可以共享一些具有特殊功能的小分子物质，使细胞群成为整体。,膜表面分子接触通讯：细胞膜表面的糖蛋白、脂蛋白分子作为细胞的触角，可以与其他细胞的膜表面分子特异性的识别和相互作用。,化学信号通讯：一些细胞分泌化学物质至细胞外（化学信号）作用于其它细胞（靶细胞），调节其功能。,二、化学信号分子的种类及特性,内分泌(,endocrine,)系统的化学信号称为激素，作用距离最远。,神经系统的化学信号神经递质（,neural transmitter,），作用距离最近。,旁分泌(,paracrine,)系统的化学信号包括各种细胞因子，作用距离介于以上两类之间。,三、受 体,受体：细胞膜上或细胞内能特异识别生物活性分子并与之结合，进而引起生物学效应的特殊蛋白质。个别是糖脂。,配体：能与受体呈特异性结合的生物活性分子，包括细胞间信息物质、药物、维生素、毒物等。,三、受 体,1、受体作用的特点,2、分类,膜受体,细胞内受体,1、受体作用的特点,高度专一性,高度亲和力,可饱和性,可逆性,特定作用模式,细胞膜表面受体,特性,离子通道,G蛋白偶联,单次跨膜,配体,神经递质,激素、味、光,细胞因子,结构,寡聚体,单体,单体,跨膜区段数目,4个,7个,1个,功能,离子通道,激活G蛋白,激活蛋白酪氨酸激酶,细胞应答,去极化与超极化,去极化与超极化，调节蛋白功能和表达,调节蛋白功能和表达，调节细胞增值和分化,（一）膜受体,1、环状受体：配体依赖性离子通道,2、七个跨膜受体,3、单个跨膜螺旋受体,细胞信号转导的基本方式,细胞信号转导网络由一些关键的蛋白分子和一些小分子活性物质，即信号转导分子构成。,信号在细胞内转换和传递的基本方式是改变信号转导分子、效应分子及靶分子的结构、含量及细胞内分布。,cAMP,的生成,cGMP,的生成,磷脂酰肌醇4,5-二磷酸(PIP2),三磷酸肌醇(IP3),二脂酰甘油(DAG),磷脂酰肌醇特异的,磷脂酶,C(PI-PLC),+,IP3和DAG的生成,3、单个跨膜螺旋受体,酪氨酸蛋白激酶受体型,非酪氨酸蛋白激酶受体型,（二）胞内受体,包含四个区,高度可变区,DNA结合区,激素结合区,铰链区,二、细胞内信息物质,在细胞内传递细胞调控信号的化学物质，称为细胞内信息物质，其中的小分子物质如Ca,2+,、DAG、IP,3,、Cer、cAMP、cGMP称为第二信使。,细胞内关键信号转导分子,GTP,结合蛋白,蛋白激酶和蛋白磷酸酶,蛋白质相互作用的调控结合元件,GTP,结合蛋白,GTP,结合蛋白三聚体：与七次 跨膜受体相结合的G蛋白，以,（G ）,亚基和,(G),亚基三聚体的形式存在于细胞膜内侧。,低分子量,G,蛋白：与,G,蛋白三聚体比，分子量要小得多，主要位于,MAPK,系统的上游，非活化状态时结合,GDP,，在外源信号的作用下释放,GDP,，结合,GTP,而被活化。,Extracellular,Cytoplasmic,COOH,-,-NH2,i1,i2,i3,e1,e2,e3,TM1,TM2,TM3,TM4,TM5,TM6,TM7,D,R,Y,G,蛋白偶联受体（400-600个氨基酸残基组成的多肽）,G-protein-coupled receptors,-S-S-,与配体结合,G蛋白作用部位,G,i/o,G,s,G,q,G,12/13,inhibition of cAMP production,inhibition of Ca,2+,channels,activation of GIRK K,+,channels,G-protein subtypes,increased synthesis of cAMP,activation of Ca,2+,and K,+,channels,activation of PLC,leading to,activation of PKC(DAG),intracellular Ca,2+,release(IP,3,),mediates signalling between GPCRs and RhoA(GTPase),function under investigation,GTP binding protein-G protein,Hundreds of cell-surface receptors for hormones and other ligands use G proteins to transduce intracellular signalling pathways.Dramatic recent advances by several laboratories have provided exciting details of the structure of the heterotrimeric G proteins.With mating receptor proteins that act like molecular levers,a trio of switch domains that can adopt open or closed conformations,and a beta subunit that resembles a propeller,these proteins may be thought of as molecular nanomachines.,GTP binding protein-G protein,The Heterotrimeric G Protein Complex,A Molecular Nanomachine,The typical heterotrimeric G protein consists of,an alpha subunit of 45-47 kD,a beta subunit of 35 kD,a gamma subunit of 7-9 kD,GTP binding protein-G protein,The Alpha Subunit,The alpha subunit of heterotrimeric G proteins consists of two domains,a GTPase domain and an alpha-helical domain.The GTPase domain is similar in structure to p21ras and other members of the GTPase superfamily of proteins.It consists of five helices surrounding a six-stranded beta sheet with five strands running parallel and one strand antiparallel to the others.The second of the five helices is a 3(10)helix,rather than an alpha helix.The alpha-helical domain,G,种类,效应分子,细胞内信使,靶分子,s,AC活化,cAMP,PKA,i,AC抑制,cAMP,PKA,q,磷脂酶c活化,Ca2+,IP3,DAG,PKC,t,cGMP磷酸二酯酶活化,cGMP,Na+通道关闭,G蛋白的,亚基的效应分子及其功能,蛋白激酶和蛋白磷酸酶,蛋白激酶（,protein kinase,）能将,磷酸基团从供体转移至底物蛋白的氨基酸残基的一大类酶。,分类：,丝,/,苏氨酸蛋白激酶,酪氨酸蛋白激酶,组,/,赖,/,精氨酸蛋白激酶,半胱氨酸蛋白激酶,天冬,/,谷氨酸蛋白激酶,MAPKmitogen activated protein kinase,丝/苏氨酸蛋白激酶。,作用途径：被MAPKK磷酸化激活，转移至核内，可使一些转录因子磷酸化，从而改变基因表达状态。,作用：接受膜受体转换和传递的信号并将其带入细胞核内，参与细胞增殖、分化及调亡等多种细胞功能的调控。,蛋 白 酪 氨 酸 激 酶,蛋白酪氨酸激酶受体,位于胞浆部分的蛋白酪氨酸激酶,Src家族，包括Src,Fyn,Lck,Lyn等常与受体结合存在。,ZAP70家族，包括ZAP70和Syk等。,Tec,家族，包括Btk,Itk,Tec等。,JAK家族，包括JAK1,JAK2,JAK3等。,位于核内的蛋白酪氨酸激酶,Abl：存在于核内和胞浆，参与转录和细胞周期调节。,Wee：调节cyclin2，调节细胞有丝分裂。,Mechanism of Tyrosine Kinase Receptors,When the receptors aggregate,the tyrosine kinase domains phosphorylate the C terminal tyrosine residues.,This phosphorylation produces binding sites for proteins with SH2 domains.GRB2 is one of these proteins.GRB2,with SOS bound to it,then binds to the receptor complex.This causes the activation of SOS.,SOS is a guanyl nucleotide-release protein(GNRP).When this is activated,it causes certain G proteins to release GDP and exchange it for GTP.Ras is one of these proteins.When ras has GTP bound to it,it becomes active.,Activated ras then causes the activation of a cellular kinase called raf-1.,Raf-1 kinase then phosphorylates another cellular kinase called MEK.This cause the activation of MEK.,Activated MEK then phosphorylates another protein kinase called MAPK causing its activation.This series of phosphylating activations is called a kinase cascade.It results in amplification of the signal.,Among the final targets of the kinase cascade are transcriptions factors(fos and jun showed here).Phosphorylation of these proteins causes them to become active and bind to the DNA,causing changes in gene transcription.,蛋白质相互作用的调控结合元件,SH2结构域（src homology 2 domain），介导信号分子与含磷酸酪氨酸蛋白分子的结合。,SH3结构域(src homology 3 domain），介导信号分子与富含脯氨酸的蛋白分子结合。,PH结构域（Pleckstrin homology domain），其功能尚未确定。,PTB结构域(protein tyrosine binding domain),识别一些汉磷酸酪氨酸的基序。,From Src Homology domains to other signaling modules:proposal of the protein recognition code,Marius Sudol,Oncogene 1998 Sep 17;17(11 Reviews):1469-74,Abstract,The study of oncogenes has illuminated many aspects of cellular signaling.The delineation and characterization of protein modules exemplified by Src Homology domains has revolutionized our understanding of the molecular events underlying signal transduction pathways.Several well characterized intracellular modules which mediate protein-protein interactions,namely SH2,SH3,PH,PTB,EH,PDZ,EVH1 and WW domains,are directly involved in the multitude of membrane,cytoplasmic and nuclear processes in multicellular and/or unicellular organisms.,The modular character of these protein domains and their cognate motifs,the universality of their molecular function,their widespread occurrence,and the specificity as well as the degeneracy of their interactions have prompted us to propose the concept of the protein recognition code.By a parallel analogy to the universal genetic code,we propose here that there will be a finite set of precise rules to govern and predict protein-protein interactions mediated by modules.Several rules of the protein recognition code have already emerged.,第二节 主要细胞信号转导途径及其作用机制,细胞内受体的信号转导过程,离子通道型受体及其信号转导,G蛋白偶联型受体及其信号转导,单次跨膜受体介导的信号转导,细胞信号转导过程的基本规律,细胞信号转导过程的基本规律,细胞信号转导过程中信号的发生和终止,细胞信号转导过程中信号的级联放大效应,细胞信号转导途径的通用性和特异性,不同信号转导通路的交叉联系,细胞内受体（转录因子）,激素受体复合物,DNA激素反应元件（HRE）,开放或关闭下游基因,细胞内受体的信号转导过程,激素,基本转录因子,热休克蛋白,离子通道型受体及其信号转导,配体主要为神经递质,作用：导致细胞膜电位改变,乙酰胆碱受体,组成：5个高度同源的亚基构成，即,2每个亚基都是四次跨膜蛋白。,乙酰胆碱的结合部位位于亚基。,Nippon Rinsho 2002 Feb;60(2):222-9,Signal transduction mechanisms of hormones through membrane receptors,Yasufuku-Takano J,Takano K.,Hormones exert their effect on cells either via membrane receptors or intracellular receptors.This paper aims to review membrane receptors and the intracellular signal transduction mechanisms.Membrane receptors could be classified according to their structural characteristics and the way they initiate the intracellular signal transduction.These include 1)Seven transmembrane(or G-protein coupled)receptors-heterotrimeric G-proteins-effector,system,2)Receptor tyrosine kinases-protein-protein interaction through SH2,SH3,and PTB domain-MAP kinase cascades and PI3-kinase pathways,3)Cytokine receptors-JAK-STAT pathways,4)Receptors of the TGF-beta superfamily-SMAD pathways,5)Apoptosis-related receptors-caspase pathways,and 6)ligand-gated ion channels.There are growing knowledge of cross-talks between these pathways.It is being recognized that steroid hormones have distinct membrane receptors,which mediate rapid,nongenomic effect.,G蛋白偶联型受体及其信号转导,配体与受体结合,激活G蛋白,激活或抑制效应分子,效应分子改变细胞内小分子物质的含量和分布,（小分子物质不位于能量代谢的中心，浓度和分布改变迅速）,小分子物质作用于靶分子,（,主要为各种蛋白激酶,）,改变代谢过程及基因表达,靶分子蛋白激酶,蛋白激酶A：cAMP依赖的蛋白激酶，作用于糖脂代谢相关的酶、离子通道和某些转录因子。,蛋白激酶G：cGMP依赖的蛋白激酶，在脑和平滑肌中含量较丰富。,蛋白激酶C：是DAG、IP3、Ca,2+,等的靶分子，在细胞生长分化的调控中起到重要作用。,钙调蛋白依赖性蛋白激酶：钙离子的重要靶分子钙调蛋白（calmodulin,CaM）,对于平滑肌的运动、神经递质的合成、细胞分裂等多种功能有调节作用。,G,种类,效应分子,细胞内信使,靶分子,s,AC活化,cAMP,PKA,i,AC抑制,cAMP,PKA,q,磷脂酶c活化,Ca2+,IP3,DAG,PKC,t,cGMP磷酸二酯酶活化,cGMP,Na+通道关闭,G蛋白的,亚基的效应分子及其功能,第三节 信息的传递途径,一、膜受体介导的信息传递,cAMP,-,蛋白激酶,A,途径：,Ca,2+,-,依赖性蛋白激酶途径,cGMP,-,蛋白激酶途径,酪氨酸蛋白激酶（,TPK,）,途径,核因子,-B,途径,二、胞内受体介导的信息传递,cAMP-蛋白激酶A途径,G,蛋白的调节,cAMP,的生成与降解,cAMP,的作用机制,PKA,的作用,肾上腺素能受体,胰高血糖素受体,激活Gs增加AC活性,cAMP,PKA,促进心肌钙转运,心肌收缩性增强,增加肝脏,糖原分解,进入核内PKA,激活靶基因转录,(一),腺苷酸环化酶途径,肾上腺素+受体,肾上腺素-受体复合物,PKA,cAMP,磷酸化酶b激酶,磷酸化酶a,糖原合成酶,ATP,磷酸化酶b,磷蛋白磷酸酶,糖原合成,糖原分解,血糖,肾上腺素对糖代谢的调节,G蛋白,AC,(促肾上腺皮质激素),(胰高血糖素),(嗅觉）,（前列腺素）,（5-羟色氨）,（生长激素抑制素）,Ca,2+,-依赖性蛋白激酶途径,在收缩、运动、分泌和分裂等复杂的生命活动中，需要Ca,2+,参与，胞浆内Ca,2+,浓度在0.01-1mol/L比细胞外Ca,2+,浓度（约2.5mmol/L）低得多。,IP,3,、Ca,2+,钙调蛋白激酶途径,与Gq,结合,PLC,质膜上的磷脂酶肌醇二磷酸（PIP,2,）,IP,3,DG,肌浆网上的IP,3,操纵的钙通道开放,释放钙离子,作为第二信使调,节细胞多种功能,与钙调蛋白结合,发挥生物学效应,1肾上腺素能受体,内皮素受体,血管紧张素受体,(嗅觉的),DG-蛋白激酶C途径,DG+Ca,2+,PKC,活化,细胞膜Na,+,/H,+,交换蛋白磷酸化,磷酸化转录因子,（AP-1、NF-,B,）,H,+,外流增加、,Na,+,内流增加,促进靶基,因转录,鸟苷酸环化酶信号转导途径,(此途径多存在于心血管系统与脑组织内),激活胞浆可溶性,心钠素,脑钠素,激活膜结合性,GTP cGMP,激活PKG,磷酸化靶蛋白,引起血管舒张等生物学效应,鸟苷酸环化酶（GC）,膜结合型,GC,胞浆可溶型,GC,单次跨膜受体介导的信号转导,单次跨膜受体的分类：,细胞因子受体,I类细胞因子受体,II类细胞因子受体,TNF受体家族,抗原受体,蛋白丝氨酸激酶,蛋白酪氨酸激酶受体,PDGF受体家族（platelet-derived growth factor,stem cell factor,colony-stimulating factor）,EGF受体家族（epidermal growth factor）,FGF受体家族（fibroblast growth factor）,IGF受体家族（insulin-like growth factor-1）,HGF受体家族（hepatocyte growth factor）,VEGF受体家族（vascular endothelial growth factor）,几种单次跨膜受体介导的信号转导,表皮生长因子受体介导的信号转导途径,淋巴细胞抗原受体介导的信号转导途径,干扰素受体介导的,信号转导途径,转化生长因子,受体介导的,信号转导途径,活化受体TPK,结合生长因子后，受体TPK 二聚化 导致自身磷酸化,Grb2,含有SH2区的生长因子连接蛋白,SH3,SOS吸引到细胞膜,RasGDP,活化Raf(MAPK、K、K),激活MEK(MAPKK),胞浆蛋白磷酸化,进入核促进靶基因转录,EGF、PDGF等生长因子,RasGTP(有活性),1.经Ras蛋白活化丝裂原蛋白激酶,(一)受体酪氨酸蛋白激酶途径,2.经PLC,激活蛋白酶C,活化受体TPK,激活PLC,PIP,2,IP,3,DG,Ca,2+,PKC,Raf(MAPK、K、K),ERK,靶基因转录,(一)受体酪氨酸蛋白激酶途径,以干扰素为例说明,INF,+受体,胞浆内的酪氨酸蛋白激酶,（,JAK,激酶）,结合并磷酸化JAK激酶,结合并磷酸化STAT(信号转导和转录激活因子),入核,诱导靶基因表达促进多种蛋白合成，进而增强细胞抵御病毒感染能力。,(二)非受体酪氨酸蛋白激酶信号转导途径,(non-receptor tyrosine kinases passway),受体本身无酪氨酸蛋白激酶活性，常见的配体为一些细胞因子，如白介素（,IL),、,干扰素（,INF,）、,红细胞生成素等,。,以干扰素为例说明,IFN Signal Passway,核因子-B途径(NFB),核因子-B（nuclear factor-B,NF-B）途径体系主要涉及机体防御、组织损伤和应激、细胞分化和凋亡及肿瘤生长抑制过程的信息传递。,二、胞内受体介导的信息传递,信号转导的抑制和终止,信号的终止,1、受体与配体的解离,2、膜受体的内化降解：由于膜受体的内化降解可导致受体的数量减少，称为受体的向下调节（down-regulation）,3、激活的G蛋白或小G蛋白转为失活型,4、在蛋白磷酸酶作用下失活,信号转导的抑制和终止,信号转导蛋白的抑制和信号通路间的拮抗,1、存在具有抑制性作用的受体和信号转导成分。,2、信号转导通路的负反馈调节,第三节 细胞信号转导研究的方法和医学中的意义,细胞信号转导异常与疾病,研究方法,信号转导药物,细胞损伤和应激反应中的信号转导,应激激活的信号转导通路：,1、应激原：物理性因素，化学性因素，生物性因素,2、应激激活的信号转导通路：MAPK 家族（SAPK/JNK，P38）,应激激活的信号转导通路的靶蛋白,胞浆蛋白，PLC,PLA，表皮生长因子受体，转录因子,应激激活的信号转导通路的效应,1、产生非特异性防御反应,2、促进细胞增殖分化,3、产生特异性防御反应,4、在细胞凋亡中起作用,血管平滑肌增殖性疾病,正常VSMC：非增殖性的收缩表型,病理VSMC：合成表型，分泌激素，自身肥大、增殖,（一）作用于VSMC的胞外信息分子,1 生物化学性因素：激素、细胞因子、生长因子,2 机械性因素：切应力、牵张力,（二）机械性因素引起的信号转导,1 整合素（intergrins)介导的信号转导,机械刺激,细胞外蛋白与整合素相连,改变细胞骨架结构,激活细胞内信息分子,2 离子通道,3 MAPKS,机械刺激同时激VSMC中ERK和SAPK/JNK途径，引起VSMC肥大与增殖。,心肌肥大,机械刺激的感受器,1、离子通道,2、整合素,3、PTK,机械刺激的信号转导途径,1、磷脂酶类,2、MAPKs联级反应,3、生长因子分泌,Alterations in G protein and MAP kinase signaling pathways during cardiac remodeling in hypertension and heart failure.,Hypertension NLM-MEDLINE,;Apr 2003;,Kacimi R,;,Gerdes AM,;,The present study was undertaken to elucidate the G-protein and mitogen-activated kinase(MAP kinase)coupled signaling profile in a genetic model of hypertension and congestive heart failure(CHF)that mimics similar disease in humans.At the receptor level,Ang II type 1 receptor(AT1R)increased in left ventricular hypertrophy(LVH)and reverted to normal in CHF,whereas there was a downregulation of the Ang II type 2 receptor(AT2R)in CHF.At the transducer level,Galphaq and Galpha12 protein levels were unchanged during LVH but decreased significantly in CHF.In contrast,Gbeta and Galpha13 protein content were markedly upregulated in CHF.Furthermore,using phospho-specific antibodies in Western blots and in vitro kinase assays,we found at the effector level an upregulation of the small G-protein Rac1 activity during LVH but a decrease during CHF.In parallel,small G-protein Rho activity was significantly increased during LVH but was unchanged in failure.We found at the downstream level that MAP kinase isoforms extracellular signal regulated-kinase(ERK1/2),big mitogen-activated kinase(BMK1/ERK5),C-jun N-terminal-activated kinase(JNKs/SAPKs),and stress-activated kinase(p38)bioactivities were increased during LVH.During CHF,ERK1/2 and JNK1/2 kinase activities were decreased,whereas BMK1/ERK5 kinase activity reverted to normal values.In conclusion,this study demonstrates,for the first time,multistep alterations of G-protein and MAP kinase signaling pathways in LVH and progression to failure in a genetic model of hypertension and failure.,磷酸化的信号转导分子的鉴定,在信号转导过程中可发生多种蛋白底物的磷酸化，包括酪氨酸磷酸化、苏氨酸残基磷酸化及丝氨酸残基磷酸化。它们分别由不同的蛋白激酶所催化。这些磷酸化蛋白通常都为信号转导分子，在信号传递过程中发挥重要的作用。一些信号蛋白结构中含SH-2结构域可同某些磷酸化的酪氨酸残基相结合，使信号得以逐级传递。含酪氨酸磷酸化的蛋白鉴定通常是首先分离粗提蛋白，采用针对含这些氨基酸残基磷酸化蛋白的单克隆抗体进行免疫沉淀(immunoprecipitation),SDS-PAGE电泳，然而采用Western blotting及immunoblotting鉴定其分子量，进一步利用分子生物学技术克隆信号蛋白，搞清其基因结构和氨基酸序列。,信号转导中第二信使含量的测定,Ca2+i的测定 细胞内Ca2+的测定方法有原子吸收光谱法、离子微电极测定法、放射示踪法及标记示踪法等。目前常用标记示踪法，即用荧光探针标记靶细胞。常用的荧光探针有quin-2/Am、Fura-2/Am及Indo-1等。其中Fura-2/Am和Indo-1较为敏感。,IP3的测定 采用3H-TdR标记的肌醇标记靶细胞后，用不同的刺激剂刺激细胞，分离磷脂酰肌醇混合物，通过阴离子交换层析柱(Serva公司 Dowex 18)分离洗脱，收集IP3洗脱峰后进行液闪测定。此外，还可以使用Amersham公司生产的D-myo-IP33H分析系统直接测定粗提物中的IP3含量，此方法简易、敏感。,信号转导中第二信使含量的测定,DAG的测定 首先提取含DAG的样品，然后采用Amersham公司生产的DAG分析系统进行测定。此系统测定的原理是用DAG激酶催化底物DAG，使之发生磷酸化，外源加入32-ATP，最后将反应产物进行分离后测定放射性含量，根据标准品计算出样品中DAG的含量。,cAMP的测定 可选择不同的方法：(1)cAMP3H分析系统，其优点是放射性半衰期长,可用于大量样品的分析；(2)cAMP125I分析系统，用于小量样品的分析；(3)cAMP ELISA测定方法，此方法简便、敏感。,激酶活性的测定,信号转导过程中往往涉及多种激酶的活化，因而对这些激酶活性的测定可以作为信号转导研究中的一种重要指标。,常见激酶活性的测定有PTK、PKC及PI-3K等。均有商品化的试剂盒。,检测原理是根据激酶可以催化特定底物发生磷酸化，外源加入32-ATP，通过分离反应产物后测定放射活性，从标准曲线推算出样品酶的活性。前已提及，PKC从胞质向胞膜的转位是PKC活化的一个重要指标，因而分离胞质和胞膜中的PKC并分别测定其活性，计算胞膜上PKC活性占总PKC活性的比例，从而判定PKC的活化程度。,G-Proteins and Related Tools,ADP-Ribosylating andADP-Ribosylation Factor Reagents and Related Products,Cell Lysates,Functional G-Proteins and Receptors,G-Protein Activators,G-Protein Antagonists,G-Protein Assay Kit,G-Protein Primer Sets,G-Protein Subunit Antibodies and Blocking Peptides,G-Protein Subunit Immunoblot Standards,Other Related Products,Post-Translational Modification Reagents,Ras Proteins and Related Products,Substrates for Carboxy Methylation,Protein tyrosine kinases(PTK)play a role in many cellular processes,including differentiation,growth,metabolism,and apoptosis.,PanVeras Tyrosine Kinase Assay Kits contain the materials necessary to analyze the products of a protein tyrosine kinase reaction using fluorescence polarization as the detection method.,The principle behind this assay is competition:a fluorescent phosphopeptide tracer and the non-fluorescent phosphopeptides generated during a tyrosine kinase assay compete for binding to an antiphosphotyrosine antibody.In a reaction mixture containing no phosphopeptide product,the fluorescent tracer is bound by the antibody and the emission signal is polarized;however,in a reaction mixture containing phosphopeptide product,the tracer is displaced from the antibo