1、細胞信號轉導 cellular signal transduction pathway 黃 辰生物醫學基礎研究中心遺傳學與分子生物學系胞生物學細胞信號轉導一、細胞膜受體的結構和特性 膜受體（membrane receptor）是可選擇性地識別和結合外來信號，并引起相應的細胞生物效應的一類膜蛋白。其化學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構，一般包括三個部分：、識別部（discriminator）也稱調節亞單位，是受體蛋白的胞外部分。依賴其糖鏈的多樣性，識別不同的化學信號。、效應部（effector）或稱催化亞單位。是受體的膜內胞質部分，通常具有酶

2、活性。在受體與化學信號結合后被激活，產生相應的生物學效應。、轉化部（transducer）亦稱之為傳導部（inducer），是受體與效應部之間的偶聯成分。它可將識別部所接受的信息經過轉換傳遞給效應部。細胞生物學細胞信號轉導二、膜受體的特性識別外來信號并通過構象變化引發胞內繼發效應是受體的兩個基本作用。它一般具有以下特性：、特異性受體與化學信號的專一性結合是依靠分子之間的立體構象互補的非共價鍵結合。這種特異性結合不是絕對的。、可飽和性一個細胞或一定量組織內受體數目是有限的，各類細胞中不同受體的濃度是相對恒定的。因此，受體與配體的結合有一個飽和度。、高親和性受體與配體具有很強的親和力，表現為：當溶

3、液中只有相當低濃度的配體時，就足以使二者結合達到飽和。細胞生物學細胞信號轉導、可逆性由于受體與配體之間屬于非共價鍵結合，所以，分子間的識別反應往往是可逆的。當結合引發出生物效應后，受體配體復合物便解離，受體又恢復到原來狀態，能再與配體結合。、特定的組織定位受體的分布，無論在種類，還是數量上，均呈現特定的模式，即受體之存在于靶細胞。細胞生物學細胞信號轉導三、膜受體類型 根據膜受體蛋白的類型及相應的信號轉導機制之不同，可將膜受體歸納為以下三種類型： 離子通道偶聯受體 G蛋白偶聯受體 酶偶聯受體 核受體 細胞生物學細胞信號轉導 G蛋白耦聯型受體 三聚體GTP結合調節蛋白（trimeric GTP-b

4、inding regulatory protein）簡稱G蛋白，位于質膜胞質側，由、三個亞基組成， 和亞基通過共價結合的脂肪酸鏈尾結合在膜上，G蛋白在信號轉導過程中起著分子開關的作用，當亞基與GDP結合時處于關閉狀態，與GTP結合時處于開啟狀態，亞基具有GTP酶活性，能催化所結合的ATP水解，恢復無活性的三聚體狀態，其GTP酶的活性能被RGS（regulator of G protein signaling）增強。RGS也屬于GAP（GTPase activating protein）。細胞生物學細胞信號轉導GEffectorSignal細胞生物學細胞信號轉導 4 superfamilies

5、binds GTPThree subunits細胞生物學細胞信號轉導細胞生物學細胞信號轉導 cAMP信號通路 cAMP信號通路的組成：膜刺激型受體（stimulatary receptor，Rs）；抑制型受體（inhibitory receptor，Ri）；刺激型調節蛋白（sitimulatary regulatory protein，Gs）；抑制型調節蛋白（inhibitory regulatory protein，Gi）；腺苷酸環化酶（adenylate cyclase，AC）；蛋白激酶A（protein Kinase A，PKA）；環腺苷酸磷酸二酯酶（cAMP phosphodieste

6、-rase）。細胞生物學細胞信號轉導蛋白激酶A細胞生物學細胞信號轉導環腺苷酸磷酸二酯酶細胞生物學細胞信號轉導細胞生物學細胞信號轉導細胞生物學細胞信號轉導細胞生物學細胞信號轉導Effects of PKA Ser/Thr殘基磷酸化（1）對代謝的調節作用 腎上腺素調節糖原分解（2）對基因表達的調節作用 cAMP應答元件（CRE）基因調控區 cAMP應答元件結合蛋白（CREB）反式作用因子 PKA的催化亞基進入細胞后使CERB特定的Ser/Thr磷酸化，形成同源二聚體，結合DNA， 激活轉錄細胞生物學細胞信號轉導 cAMP信號通路的傳遞過程Gi調節模型細胞生物學細胞信號轉導 磷脂酰肌醇信號通路 胞外

7、信號分子與細胞表面G蛋白耦聯型受體結合，激活質膜上的磷脂酶C（PLC-），使質膜上4，5-二磷酸磷脂酰肌醇（PIP2）水解成1，4，5-三磷酸肌醇（IP3）和二酰基甘油（DG）兩個第二信使，胞外信號轉換為胞內信號。細胞生物學細胞信號轉導細胞生物學細胞信號轉導細胞生物學細胞信號轉導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細胞生物學細胞信號轉導 細胞生物學細胞信號轉導 細胞生物學細胞信號轉導細胞生物學細胞信號轉導細胞生物學細胞信號轉導細胞生物學細胞信號轉導細胞生物學細胞信號轉導細胞生物學細胞信號轉導細胞生物學細胞信號轉導細胞生物學細胞信號轉導細胞

9、生物學細胞信號轉導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.細胞生物學細胞信號轉導Most GTP-binding proteins depend on helper proteins GAPsGTPase Activating Proteins, promote GTP hydrolysis.細胞生物學細胞信號轉導GEFs, Guanine Nucleotide Exchange Factors, promote GDP/

11、GTP exchange.The activated receptor (GPCR) serves as GEF for a heterotrimeric G protein. 細胞生物學細胞信號轉導鳥苷酸環化酶（GC）GC的激活間接地依靠Ca2+效應蛋白磷酸化NO作用機理：肌肉細胞中激活GC蛋白激酶G HRGCGTPcGMPcGMP-依賴性蛋白激酶蛋白激酶G （一個cGMP結合位點）cGMP-蛋白激酶途徑細胞生物學細胞信號轉導Cyclic Nucleotide Metabolism - cGMP 細胞生物學細胞信號轉導 酶偶聯受體及其介導的信號轉導通路 酶偶聯受體的一般特征 酶偶聯受體（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。其共同的特點是： 該類受體均為一次跨膜肽鏈，胞外區是配體結合位點；胞內區是蛋白激酶催化部位。 無需G蛋白的介導作用，僅由受體自身酶蛋白的激活來直接完成信號的跨膜轉導。 受體的二聚化（dimerization）是該類受體激活的普遍機制。細胞生物學細胞信號轉導類型 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 受體鳥苷酸環化酶 組氨酸激酶連接的受體細胞生物學細胞信號轉導酪氨酸激酶 細胞生物學細胞信號轉導信號轉導過程 配體（主要是各種生長因子

13、）與受體結合； 受體胞外區構象變化，聚合成二聚體； 受體胞內區酪氨酸殘基自磷酸化（受體激活）； 活化的受體結合，并與胞質中帶有蛋白SH（Src homolog region）或PH結構域（Pleckstrin Homology）的信號轉導分子結合，形成受體胞內信號蛋白復合體； 傳遞信號并引發細胞相應的生物效應。細胞生物學細胞信號轉導 SH2結構域（Src Homology 2 結構域）：約100個氨基酸組成，介導信號分子與含磷酸酪氨酸的蛋白分子結合。 SH3結構域（Src Homology 3 結構域）：約50-100個氨基酸組成，介導信號分子與富含脯氨酸的蛋白分子結合。 PH結構域（Plec

14、kstrin Homology 結構域）：約100-120個氨基酸組成，可以與膜上磷脂類分子PIP2、PIP3、IP3等結合，使含PH結構域蛋白由細胞質中轉位到細胞膜上。 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細胞生物學細胞信號轉導細胞生物學細胞信號轉導 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.細胞生物學細胞信號轉導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細胞生物學細胞信號轉導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.細胞生物學細胞信號轉導 JAK(just another kinase, Janus protein tyrosine kin