細(xì)胞生物學(xué)-7細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)課件

1、細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) cellular signal transduction pathway 黃 辰生物醫(yī)學(xué)基礎(chǔ)研究中心遺傳學(xué)與分子生物學(xué)系胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)一、細(xì)胞膜受體的結(jié)構(gòu)和特性 膜受體（membrane receptor）是可選擇性地識(shí)別和結(jié)合外來信號(hào)，并引起相應(yīng)的細(xì)胞生物效應(yīng)的一類膜蛋白。其化學(xué)成分多為跨膜糖蛋白，約占膜總蛋白的。不同的受體有不同的結(jié)構(gòu)，一般包括三個(gè)部分：、識(shí)別部（discriminator）也稱調(diào)節(jié)亞單位，是受體蛋白的胞外部分。依賴其糖鏈的多樣性，識(shí)別不同的化學(xué)信號(hào)。、效應(yīng)部（effector）或稱催化亞單位。是受體的膜內(nèi)胞質(zhì)部分，通常具有酶

2、活性。在受體與化學(xué)信號(hào)結(jié)合后被激活，產(chǎn)生相應(yīng)的生物學(xué)效應(yīng)。、轉(zhuǎn)化部（transducer）亦稱之為傳導(dǎo)部（inducer），是受體與效應(yīng)部之間的偶聯(lián)成分。它可將識(shí)別部所接受的信息經(jīng)過轉(zhuǎn)換傳遞給效應(yīng)部。細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)二、膜受體的特性識(shí)別外來信號(hào)并通過構(gòu)象變化引發(fā)胞內(nèi)繼發(fā)效應(yīng)是受體的兩個(gè)基本作用。它一般具有以下特性：、特異性受體與化學(xué)信號(hào)的專一性結(jié)合是依靠分子之間的立體構(gòu)象互補(bǔ)的非共價(jià)鍵結(jié)合。這種特異性結(jié)合不是絕對(duì)的。、可飽和性一個(gè)細(xì)胞或一定量組織內(nèi)受體數(shù)目是有限的，各類細(xì)胞中不同受體的濃度是相對(duì)恒定的。因此，受體與配體的結(jié)合有一個(gè)飽和度。、高親和性受體與配體具有很強(qiáng)的親和力，表現(xiàn)為：當(dāng)溶

3、液中只有相當(dāng)?shù)蜐舛鹊呐潴w時(shí)，就足以使二者結(jié)合達(dá)到飽和。細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、可逆性由于受體與配體之間屬于非共價(jià)鍵結(jié)合，所以，分子間的識(shí)別反應(yīng)往往是可逆的。當(dāng)結(jié)合引發(fā)出生物效應(yīng)后，受體配體復(fù)合物便解離，受體又恢復(fù)到原來狀態(tài)，能再與配體結(jié)合。、特定的組織定位受體的分布，無論在種類，還是數(shù)量上，均呈現(xiàn)特定的模式，即受體之存在于靶細(xì)胞。細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)三、膜受體類型 根據(jù)膜受體蛋白的類型及相應(yīng)的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制之不同，可將膜受體歸納為以下三種類型： 離子通道偶聯(lián)受體 G蛋白偶聯(lián)受體 酶偶聯(lián)受體 核受體 細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) G蛋白耦聯(lián)型受體 三聚體GTP結(jié)合調(diào)節(jié)蛋白（trimeric GTP-b

4、inding regulatory protein）簡(jiǎn)稱G蛋白，位于質(zhì)膜胞質(zhì)側(cè)，由、三個(gè)亞基組成， 和亞基通過共價(jià)結(jié)合的脂肪酸鏈尾結(jié)合在膜上，G蛋白在信號(hào)轉(zhuǎn)導(dǎo)過程中起著分子開關(guān)的作用，當(dāng)亞基與GDP結(jié)合時(shí)處于關(guān)閉狀態(tài)，與GTP結(jié)合時(shí)處于開啟狀態(tài)，亞基具有GTP酶活性，能催化所結(jié)合的ATP水解，恢復(fù)無活性的三聚體狀態(tài)，其GTP酶的活性能被RGS（regulator of G protein signaling）增強(qiáng)。RGS也屬于GAP（GTPase activating protein）。細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)GEffectorSignal細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) 4 superfamilies

5、binds GTPThree subunits細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) cAMP信號(hào)通路 cAMP信號(hào)通路的組成：膜刺激型受體（stimulatary receptor，Rs）；抑制型受體（inhibitory receptor，Ri）；刺激型調(diào)節(jié)蛋白（sitimulatary regulatory protein，Gs）；抑制型調(diào)節(jié)蛋白（inhibitory regulatory protein，Gi）；腺苷酸環(huán)化酶（adenylate cyclase，AC）；蛋白激酶A（protein Kinase A，PKA）；環(huán)腺苷酸磷酸二酯酶（cAMP phosphodieste

6、-rase）。細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)蛋白激酶A細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)環(huán)腺苷酸磷酸二酯酶細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)Effects of PKA Ser/Thr殘基磷酸化（1）對(duì)代謝的調(diào)節(jié)作用 腎上腺素調(diào)節(jié)糖原分解（2）對(duì)基因表達(dá)的調(diào)節(jié)作用 cAMP應(yīng)答元件（CRE）基因調(diào)控區(qū) cAMP應(yīng)答元件結(jié)合蛋白（CREB）反式作用因子 PKA的催化亞基進(jìn)入細(xì)胞后使CERB特定的Ser/Thr磷酸化，形成同源二聚體，結(jié)合DNA， 激活轉(zhuǎn)錄細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) cAMP信號(hào)通路的傳遞過程Gi調(diào)節(jié)模型細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) 磷脂酰肌醇信號(hào)通路 胞外

7、信號(hào)分子與細(xì)胞表面G蛋白耦聯(lián)型受體結(jié)合，激活質(zhì)膜上的磷脂酶C（PLC-），使質(zhì)膜上4，5-二磷酸磷脂酰肌醇（PIP2）水解成1，4，5-三磷酸肌醇（IP3）和二?；视停―G）兩個(gè)第二信使，胞外信號(hào)轉(zhuǎn)換為胞內(nèi)信號(hào)。細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)Gi/oGsGqG12/13 inhibition of cAMP production inhibition of Ca2+ channelsactivation of GIRK K+ channelsG-protein subtypes increased synthesis of cAMPactivation o

8、f Ca2+ and K+ channelsactivation of PLC leading toactivation of PKC (DAG)intracellular Ca2+ release (IP3) mediates signalling between GPCRs and RhoA (GTPase) function under investigation細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) 細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) 細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞

9、生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)Small GTP-binding proteins includeRas (growth factor signal cascades).Rho (regulation of actin cytoskeleton)Rab (vesicle targeting and fusion).ARF (forming vesicle coatomer coats).Ran (transport of proteins into & out of the nucleus).All GTP-binding proteins differ in conformation depending

10、 on whether GDP or GTP is present at their nucleotide binding site. Generally, GTP binding induces the active state.細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)Most GTP-binding proteins depend on helper proteins GAPsGTPase Activating Proteins, promote GTP hydrolysis.細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)GEFs, Guanine Nucleotide Exchange Factors, promote GDP/

11、GTP exchange.The activated receptor (GPCR) serves as GEF for a heterotrimeric G protein. 細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)鳥苷酸環(huán)化酶（GC）GC的激活間接地依靠Ca2+效應(yīng)蛋白磷酸化NO作用機(jī)理：肌肉細(xì)胞中激活GC蛋白激酶G HRGCGTPcGMPcGMP-依賴性蛋白激酶蛋白激酶G （一個(gè)cGMP結(jié)合位點(diǎn)）cGMP-蛋白激酶途徑細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)Cyclic Nucleotide Metabolism - cGMP 細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) 酶偶聯(lián)受體及其介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)通路 酶偶聯(lián)受體的一般特征 酶偶聯(lián)受體（en

12、zyme-linked receptor），又稱催化性受體（catalytic receptor）。是一大類重要的膜受體家族。其共同的特點(diǎn)是： 該類受體均為一次跨膜肽鏈，胞外區(qū)是配體結(jié)合位點(diǎn)；胞內(nèi)區(qū)是蛋白激酶催化部位。 無需G蛋白的介導(dǎo)作用，僅由受體自身酶蛋白的激活來直接完成信號(hào)的跨膜轉(zhuǎn)導(dǎo)。 受體的二聚化（dimerization）是該類受體激活的普遍機(jī)制。細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)類型 受體酪氨酸激酶 酪氨酸激酶連接的受體 受體酪氨酸磷脂酶 受體絲氨酸/蘇氨酸激酶 受體鳥苷酸環(huán)化酶 組氨酸激酶連接的受體細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)酪氨酸激酶 細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)信號(hào)轉(zhuǎn)導(dǎo)過程 配體（主要是各種生長(zhǎng)因子

13、）與受體結(jié)合； 受體胞外區(qū)構(gòu)象變化，聚合成二聚體； 受體胞內(nèi)區(qū)酪氨酸殘基自磷酸化（受體激活）； 活化的受體結(jié)合，并與胞質(zhì)中帶有蛋白SH（Src homolog region）或PH結(jié)構(gòu)域（Pleckstrin Homology）的信號(hào)轉(zhuǎn)導(dǎo)分子結(jié)合，形成受體胞內(nèi)信號(hào)蛋白復(fù)合體； 傳遞信號(hào)并引發(fā)細(xì)胞相應(yīng)的生物效應(yīng)。細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) SH2結(jié)構(gòu)域（Src Homology 2 結(jié)構(gòu)域）：約100個(gè)氨基酸組成，介導(dǎo)信號(hào)分子與含磷酸酪氨酸的蛋白分子結(jié)合。 SH3結(jié)構(gòu)域（Src Homology 3 結(jié)構(gòu)域）：約50-100個(gè)氨基酸組成，介導(dǎo)信號(hào)分子與富含脯氨酸的蛋白分子結(jié)合。 PH結(jié)構(gòu)域（Plec

14、kstrin Homology 結(jié)構(gòu)域）：約100-120個(gè)氨基酸組成，可以與膜上磷脂類分子PIP2、PIP3、IP3等結(jié)合，使含PH結(jié)構(gòu)域蛋白由細(xì)胞質(zhì)中轉(zhuǎn)位到細(xì)胞膜上。 Ligand binds receptor PTK Ligand binds receptor PTK Autophosphorylation on tyrosine PPPP Ligand binds receptor PTK Autophosphorylation on tyrosine GRB2 (a SH2- and SH3-containing protein) binds to the receptor phos

15、photyrosine motif Y-V/L-N-X via its SH2 domainPPPPSH2SH3GRB2SOS Ligand binds receptor PTK Autophosphorylation on tyrosine GRB2 (a SH2- and SH3-containing protein) binds to the receptor phosphotyrosine motif Y-V/L-N-X via its SH2 domain The SH3 of GRB2 binds constitutively to the proline-rich sequenc

16、e in the C-terminus of SOS (a guanine nucleotide exchange factor for RAS). PPPPSH2SH3GRB2SOSPPPPSH2SH3GRB2SOSGRB2 24 kDa adaptor molecule. Only contains an SH2 domain between two SH3 domainsPPPPSH2SH3GRB2SOSRASGTPRAS Oncogenic forms of RAS often have point mutations that lock RAS in the active GTP-b

17、ound form.Evidence: Cells were induced to proliferate by + PDGF and EGF. Microinjection of anti-RAS antibodies into the cells blocked the cell proliferation. Microinjection of a constitutively active mutant of RAS caused cells to proliferate in the absence of PDGF and EGF. The RAS-GTP effector domai

18、n interacts with the N-terminal regulatory region of the RAF (serine/threonine protein kinase), hence recruiting RAF to the membranePPPPSH2SH3GRB2SOSRASGTPRAFPPPPSH2SH3GRB2SOSRASGTPRAFRAF RAF is a serine/ threonine protein kinase RAF is a MAPK kinase kinase (MAPKKK). Can transform cells when constit

19、utively active or when overexpressed.14-3-3PPPPSH2SH3GRB2SOSRASGTPRAFRAF The 14-3-3 family of scaffold proteins interacts constitutively with RAF via the phosphorylated Ser621 in RAF Both 14-3-3 and RAS may be required for activation of RAF.14-3-3PPPPSH2SH3GRB2SOSRASGTPRAFRAF RAS recruits RAF to the

20、 membrane. Membrane targeting of RAF is necessary to fully activate RAF. (Expression of mutant RAF that cannot bind RAS no stimulation of cell proliferation by a constitutively active RAS).14-3-3 Activation of RAF (most likely by phosphorylation of RAF and binding to the scaffold protein 14-3-3)PPPP

21、SH2SH3GRB2SOSRASGTPRAF14-3-3 Activated RAF in turn activates MEK (also called MAPK kinase) by phosphorylation on two conserved serine residues in MEK.PPPPSH2SH3GRB2SOSRASGTPRAFMEKPP14-3-3 Activated RAF in turn activates MEK (also called MAPK kinase) by phosphorylation on two conserved serine residue

22、s in MEK.PPPPSH2SH3GRB2SOSRASGTPRAFMEKPP14-3-3PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPMEK Also called MAPK kinase (MAPKK). Phosphorylated on Ser218 and Ser222 by activated RAF. Mutation of MEK that leads to constitutive activity (by replacing the two Ser with glutamic acids or aspartic acids - by mimic phosp

23、horylation) MAPK activation, mitogenicity, and cellular transformation.14-3-3 Activated MEK activates MAPK (a serine/threonine protein kinase) by phosphorylation of conserved threonine and tyrosine residues.PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPMAPKPP14-3-3 Activated MEK activates MAPK (a serine/threonine

24、protein kinase) by phosphorylation of conserved threonine and tyrosine residues.PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPMAPKPP14-3-3PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPMAPKPPMAPK Five isoforms of ERK have been identified, but ERK1 and ERK2 have been most studied.14-3-3PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPMAPKPPMAPK MAP

25、K activation is biphasic: a transient peak within 5-10 min, and a sustained peak lasting several hours. The different activation kinetics different cellular response (EGF induces only the transient response and stimulate cell growth; but NGF (nerve growth factor) induces the sustained response and s

26、timulate differentiation).14-3-3PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPMAPKPPMAPK Inactivation of MAPK is achieved by several phosphatases: the serine/threonine phosphatase PP2A14-3-3 Activated MAPK phosphorylates a number of substrates in the cytoplasm PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPMAPKPPSubstratesSubstra

27、tesPP14-3-3 Activated MAPK phosphorylates a number of substrates in the cytoplasm it also translocated into the nucleus(within min) where it phosphorylates nuclear transcription factors. PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPSubstratesMAPKPPMAPKPP14-3-3 Activated MAPK phosphorylates a number of substrates

28、in the cytoplasm it also translocated into the nucleus(within min) where it phosphorylates nuclear transcription factors. PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPSubstratesMAPKPPMAPKPP14-3-3 Activated MAPK phosphorylates a number of substrates in the cytoplasm it also translocated into the nucleus(within min

29、) where it phosphorylates nuclear transcription factors. PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPSubstratesMAPKPPMAPKPPP14-3-3 Activated MAPK phosphorylates a number of substrates in the cytoplasm it also translocated into the nucleus(within minutes) where it phosphorylates nuclear transcription factors. Tra

30、nscription of genes important for cell proliferation. PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPSubstratesMAPKPPMAPKPPP細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) Transcription Factor Regulation (CREB, Elk-1 and c-Fos)The transcription factor CREB binds to the cAMP response element (CRE) and activates gene transcription in respons

31、e to a wide variety of extracellular signals (including growth factors, hormones, and neurotransmitters). Transcriptional activation of CREB is controlled through phosphorylation at Ser133 by p90Rsk and the p44/42 MAP kinase. The transcriptional activity of the proto-oncogene c-Fos has been implicat

32、ed in cell growth, differentiation, and development. Fos is induced by many stimuli, ranging from mitogens to pharmacological agents. c-Fos has been shown to be associated with another proto-oncogene, c-Jun, and together they bind to the AP-1 binding site to regulate gene transcription. Like CREB, c

33、-Fos is regulated by p90Rsk. Elk-1 is a transcription factor that is activated by the MAP kinase-mediated phosphorylation of a Ser/Thr cluster at the carboxyl terminus. Activated Elk-1 binds to the serum response element (SRE) to induce gene transcription in response to serum and growth factors. Rec

34、ent studies have also demonstrated that Elk-1 is a target for the stress-activated kinase SAPK/JNK.細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)JNK pathway JNK translocation into the nucleus phosphorylation of the transcription factor c-JUN at the N-terminal residues (Ser63 and Ser73) activation of transcription by c-JUNRac1/Cdc42GT

35、PSTRESSPAKMEKK1-3PPMEK4PPJNKPPc-JUNPRac1/Cdc42GTPSTRESSPAKMEKK1-3PP14-3-3PPPPSH2SH3Grb2SosRasGTPRafMEK4PPMEKPPJNKPPMAPKPP細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)Specificity of MAP kinase pathways When cells are treated with mitogenic agents (e.g. growth factors), MAPK (ERK) become strongly activated but JNK is poorly activated. Conversely, when cells are challenged with stress, JNK is activated but MAPK (ERK) is only weakly activated.細(xì)胞生物學(xué)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo) JAK(just another kinase, Janus protein tyrosine kin