Ras-Raf-MAPK-mTOR-PI3K-AKT信号通路详解

1、Signal Transduction Pathways & the Clinical Implications Lixin KAN, Ph DProfessor Anhui Medical University, China Distinguished professor Bengbu Medical College, China Adjunct professor Northwestern University, USA Overview: Concept, components, common features of cell signal tr

2、ansduction pathwaysRas-Raf-MAPK pathwayPI3K/AKT/mTOR pathwayJak-STAT pathway NF-kB pathwayNotch pathway Abnormal Cell signaling and diseasesCONTENTOVERVIEWSignal transduction refers to the process that the extracellular stimuli regulate the biological functions, through membranous or intracellular r

3、eceptors mediated signal cascade. Definition Proliferation Differentiation Motility Death Transformation Signals Biochemical processesGrowth factorsCytokinesExtracellular matrix proteinsCell stressesSchematic view of Cell signalingCell signaling affects virtually EVERY ASPECT of cell structures and

4、functions. 1. Extracellular signal molecules: Ligand/Agonist.2. Receptor are specific proteins, which are able to recognize and bind to corresponding ligands. Binding is specific, but not always in 1-1 fashion. 3. Transducers carries the message from the receptor to the effector. Transducers could b

5、e enzymes, adaptor, or others. Transduction is a multi-step process. 4. Effector proteins produces a 2nd messenger such as cAMP. COMPONENTSBind to membrane receptorsBind to intracellular- receptorsExtracellular moleculesChemical SignalProteins and peptides:Hormones, cytokinesAmino acid derivatives:C

6、atecholamine Fatty acid derivatives:ProstaglandinsSteroid hormones, Thyroxine , VD3 Physical Signal light, wave, voice, pressure & temperature Signal molecules Ligand/AgonistExtracellular signal molecules: Ligand/Agonist are normally released by signal-producing cells, reach and bind to the receptor

7、 protein of target cells.Types of signal molecules Lipid-soluble molecules Water-soluble molecules Gaseous molecules (NO)Properties of signal moleculesSpecificity, highly efficient, competitiveDosage-dependent, saturableReversible, can be deactivated2. Receptor proteins are specific proteins, which

8、are able to recognize and bind to corresponding ligands, and transduce signal to next signaling molecules inside of the cell. act as cells letter boxes, and receive messagesTransmit a message into the cell, which normally leads to a specific cellular effectEach cell has multiple specific receptors,

9、making it responsive to different chemicals. Distribution: Cell surface receptorsIntracellular receptorsDomains of cell surface receptors: The extracellular segment-binding siteThe hydrophobic membrane spanning segment- Trans-membrane （ highly conserved）The cytoplasmic segment-enzyme domain（ highly

10、conserved），transferring a phosphate group from ATP to tyrosine or serine/threonine on a substrate protein.Receptors Properties: SpecificitySaturabilityHigh affinityReversibilityLigand-Receptor binding is an reversible equilibrium process, obeying the law of massDifferent Kinds of intracellular prote

11、ins serve different purposesRelay proteinsMessenger proteinsAdaptor proteinsAmplifier proteinsTransducer proteinsBifurcation proteinsIntegrator proteinsLatent gene regulatory proteinsIntracellular proteins Effector protein produces a mobile second messenger such as cAMP. In the case of cAMP, the eff

12、ector protein is adenylate cyclase (also called adenylyl cyclase).Effector protein1. Receptors could either be cell membrane or nuclear receptors. 2. a relatively small number of mechanisms have been employed. 3. conserved intracellular proteins play key roles in different signaling pathways. 4. dif

13、ferent signaling pathways are re-used in different contexts.5. Amplification of external signals is often necessary.6. Convergence, divergence, and crosstalk among different signaling pathways.7. Desensitization of receptors.8. External signals induce two direct cellular responses: changes the activ

14、ity of pre-existed proteins changes in gene expression. common features of cell signalingcell membrane receptorsmost water-soluble molecules/factors bind to cell membrane receptors and activate the signaling transduction pathway.Testosterone, and other lipid soluble factors, such as RA, travels thro

15、ugh the blood and enters cells throughout the body.In the cytoplasm, they bind and activate receptor proteins.These activated proteins enter the nucleus and turn on target genes .1 The steroid hormone testosterone passes through the plasma membrane. The bound proteinstimulates thetranscription ofthe

16、 gene into mRNA.4 The mRNA istranslated into aspecific protein.5 Testosterone bindsto a receptor proteinin the cytoplasm,activating it.2 The hormone-receptor complexenters the nucleusand binds to specific genes.3Signaling pathway via nuclear receptorOther features: Signal Flow is not always in 1-1 f

17、ashion Signal Receptor Transducer TargetsResponseEach cell is programmed to respond to a specific combinations of extracellular signal molecules, but the final read-outs are context-dependentSIGNALS MAY HAVE CONTEXT-DEPENDENT EFFECTSHCell-surface Receptors Belong to Different ClassesRas-Raf-MAPK, mT

18、OR-PI3K-AKT, Jak-STAT, NF-kBNotchRTK-Ras-Raf-MAPK signaling pathwayOverview of RTK-Ras-Raf-MAPK signaling pathwayRTK-induced Ras activationRas-MAP kinase cascade MAPK phosphorylates transcription factors or other proteinsThe signal starts when a signaling molecule binds to the receptor on the cell s

19、urface and ends when the DNA in the nucleus expresses a protein and produces some change in the cell, such as cell division. The pathway includes many proteins, including MAPK (mitogen-activated protein kinases, originally called ERK, extracellular signal-regulated kinases), which communicate by add

20、ing phosphate groups to a neighboring protein, which acts as an on or off switch.The RTK-Ras-Raf-MAPK pathway consists of at least 8 main components:a ligand a receptor (RTK) Adaptor proteins (Grb2, sos) Ras Raf(MAPKKK)Mek(MAPKK)Erk(MAPK)Transcription factor Receptor tyrosine kinases (RTKs)RTKs is a

21、 important type of cell-surface receptors, which can phosphorylate tyrosine residues on itself and on cytoplasmic substrates:Single transmembrane;Dimerization after ligand binding. RTKs are activated by autophosphorylation, RTK-induced Ras activationAdaptor protein, such as Grb2, function as a link

22、between an activated RTK and Sos, a guanine nucleotide exchange factor(GEF). Binding of Sos to inactive Ras causes a large conformational change that permits release of GDP and binding of GTP, forming active Ras.Ras acts as a molecular switchRas is a small GTPase that is held at the inner surface of

23、 plasma membrane, being functionally similar to G subunit of G protein. GTPase activating protein (GAP)Guanine nucleotide dissociation Inhibitor(GDI)Ras-MAP kinase cascadeRas initiates three-step phosphorylation that is characteristic of all MAPK kinase cascades.Once activated, MAPK phosphorylates t

24、ranscription factors or other proteins.MAPK regulates activity of transcription factors and gene expressionMAPK phosphorylates the kinase p90RSK, which then moves into the nucleus and phosphorylates the SRF. MAPK translocates to nucleus and directly phosphorylates the TCF. Together, the phosphorylat

25、ion of these transcription factors stimulate transcription of genes (e.g., c-fos) that contain an SRE sequence in their promoter.T-cell factor(TCF)serum response factor (SRF) serum response element(SRE) RTK-Ras signaling pathwaySUMMARY PI3K/AKT/mTOR pathwayThe PI3K/AKT/mTOR pathway consists of at le

26、ast 6 main components:a ligand/ ligands a receptor (RTK or other receptors) PI3K Akt mTORTranscription factor The PI3K/AKT/mTOR pathway is an intracellular signaling pathway important in regulating the cell cycle and apoptosis. Multiple Membrane receptors PI3K activation activates AKT in the plasma

27、membrane activating multiple transducers . mTOR proliferation apoptosisinhibiting multiple BAD, FOXO1, p27, Factors that enhance the PI3K/AKT pathway: EGF, shh, IGF-1, insulin, CaM. factors that inhibit the PI3K/AKT pathway: PTEN, GSK3B, HB9. Regulation of mTOR1 signaling in T-ALL cells by Notch1, A

28、KT, and AMPKJAK-STAT signaling pathway The JAK-STAT system is a major signaling alternative to the second messenger system.The JAK-STAT pathway consists of 4 main components:a ligand a receptor (2) Janus kinase (JAK) (3) Signal Transducer and Activator of Transcription (STAT)JAK-STAT signaling pathw

29、ay STAT is a transcription factor, which contains an SH2 domain(binding to the receptor) and a nuclear-localization signal (NLS) .STAT monomers bound to receptors are phosphorylated by receptor-associated JAK kinases, then dimerize and move to the nucleus, where they activate transcription.JAK: janu

30、s kinaseSTAT: signal transducers and activators of transcriptionThe canonical Jak-STAT signaling Non-redundant JAK/STAT signaling in miceThe Jak-STAT signaling pathway activated by interferon Other Non-canonical STAT signalingunphosphorylated STAT is localized on heterochromatin in association with

31、HP1 in the nucleus. Increasing STAT phosphorylation (by JAK or others) leads to HP1 displacement and heterochromatin instability, inducing target-gene expression. Nuclear factor kappa-light-chain-enhancer of activated B cells(NF-kB) pathway NF-kB is a protein complex that controls transcription of D

32、NA, cytokine production and cell survival. NF-B is found in almost all animal cell types and is involved in cellular responses to stimuli. Dysregulation of NF-B has been linked to cancer, inflammatory, and autoimmune diseases, septic shock, viral infection, and improper immune development. NF-B has

33、also been implicated in processes.ClassProteinAliasesGeneINF-B1p105 p50NFKB1NF-B2p100 p52NFKB2IIRelAp65RELARelBRELBc-RelRELThere are five key proteins in the mammalian NF-B family:NF-B family members share structural homology with the retroviral oncoprotein v-Rel, resulting in their classification a

34、s NF-B/Rel proteins.NF-B (green) heterodimerizes with RelB (cyan) to form a ternary complex with DNA (orange) that promotes gene transcription.In its inactive form, NF-kB (p50) is sequestered in the cytoplasm, bound by members of the IkB family of inhibitor proteins. The various stimuli that activat

35、e NF-kB cause phosphorylation of IkB, which is followed by its ubiquitination and subsequent degradation. This results in the exposure of the nuclear localization signals (NLS) on NF-kB subunits and the subsequent translocation of the molecule to the nucleus. In the nucleus, NF-kB binds to the promo

36、ter of various genes and thus activates their transcription. Non-canonical NF-kB pathway lymphotoxin-, BAFF or RANKL, activate the non-canonical NF-B pathway to induce NF-B/RelB:p52 dimer in the nucleus. NF-B kinase (NIK) phosphorylation and proteasomal processing of the NF-B2 precursor protein p100

37、 mature p52 subunit p52 dimerizes with RelB to form RelB:p52 DNA binding activity regulate a distinct class of genes.non-canonical signaling critically depends on NIK mediated processing of p100 into p52. however, an integrated NF-B system network underlies activation of both RelA and RelB containin

38、g dimer and that a malfunctioning canonical pathway will lead to an aberrant cellular response also through the non-canonical pathway.A model of noncanonical NF-B regulationNotch signaling Notch signaling as a regulator for cell differentiation was first identified in 1937. It is now recognized that

39、 Notch signaling plays an important role in determining cell fate and maintaining progenitor cell population as well as the balance between cell proliferation, differentiation and apoptosis .Key components in the Canonical Notch pathwayNotch ligand DSL family, Delta-like 1, Delta-like 3, Delta-like

40、4, Jagged 1, Jagged 2Notch (receptor) : Notch1Notch4Transcriptional factors that transduse the signal: CBF1/RBP-JFactors that process Notch: Presenilin1, Presenilin2, Metallo-protease, Furin-like proteaseModifying factors, Lunatic, Radical, Manic Fringe, Numb, Numb-like, Deltex13, Mastermind-like 13

41、Target genes: Hes1, Hes 5Notch receptorsCanonical Notch SignalingRegulation of PI3KAKT signaling in T-ALL cells by Notch1Canonical vs. Non-Canonical Notch SignalingNon-Canonical Notch Signaling: -secretase regulated activation of the Notch pathway that occurs independently of ligand interaction; NIC

42、D activity independent of RBPJ/CSL; membrane bound Notch signaling in the absence of cleavage by the -secretase complex and, in some cases, independent of ligand interactionNon-canonical Notch signaling pathwaysPost-Translational Regulation of -Catenin Protein by Notch60Hanahan & Weinberg 2000Summar

43、y of 30 years of research (1971-2001)Clinical implications Rasopathies theRASopathiesare a clinically defined group of medical genetic syndromes caused by germline mutations in genes that encode components or regulators of the Ras/mitogen-activated protein kinase (MAPK) pathway.Ras-Raf-MAPK pathway

44、and diseases Genetic syndromes of theRAS-MAPKpathway:“Rasopathies” Neurofibromatosistype I(NF-1) .Capillary malformation-arteriovenous malformation (CM-AVM).Noonan syndrome with multiple lentigines (NSML, also called LEOPARD syndrome)cardiofaciocutaneous (CFC) Costello syndrome (CS)Clinical images o

45、f patients with RASopathies. (a) A young boy who has a clinical diagnosis of neurofibromatosis type 1. (b) A young girl with Noonan syndrome who has aPTPN11mutation. (c) A young adult woman with Costello syndrome who has the common p.G12SHRASmutation. (d) A school-age boy with cardio-facio-cutaneous

46、 syndrome who has aMEK2mutation.TheRASsignaling pathway and genes mutated in juvenile myelomonocytic leukemia (in black)Mosaic RASopathiesMosaic RASopathyGeneReported mosaic mutationsKeratinocytic epidermal nevusHRASKRASNRAS(FGFR3)G12C, G12V, G13R, Q61LG12DG12D, P34L, Q61RR248C, S249C, G372CKeratino

47、cytic epidermal nevus syndromeHRASKRAS(FGFR3)G12SG12DR248C, S249CSebaceous nevusHRASKRASA11S, G12C, G12D, G12S, G13RG12D, G12VSchimmelpenning syndromeHRASKRASG13RG12DMosaic Costello syndromeHRASG12SSegmental neurofibromatosis type INF1Various point mutations, deletions, insertionsFarnesyltransferase

48、 inhibitorsCurrent Status of Ras/MAPK Small Molecules for Clinical TrialsAgentTargetCompany/partnerStatusRAS modulationLonafarnib (Sarasar)Farnesyl transferaseMerck/Schering-PloughPhase IITipifarnib (Zarnestra)Farnesyl transferaseJohnson & JohnsonPhase I/IISalirasibRAS antagonistConcordiaPhase I/IIR

49、AF inhibitionSorafenib (Nexavar)RAF/multi-targetedOnyx/Bayer PharmaceuticalsApprovedRAF265RAF/multi-targetedNovartisPhase I/IIPLX4032BRAFPlexxikon/RochePhase IIIPLX5568BRAFPlexxikon/RochePhase I/IIGSK2118436BRAFGlaxoSmithKlinePhase I/IIXL281 (BMS908662)BRAFExelixis/Bristol-Myers SquibbPhase I/IIMEK

50、inhibitionCI-1040MEKPfizerTerminatedPD0325901MEKPfizerOn HoldAZD6244 (ARRY-886)MEKArray/AstraZenecaPhase I/IIAZD8330 (ARRY-704)MEKArray/AstraZenecaPhase I/IIARRY-162MEKArray/NovartisPhase I/IIARRY-300MEKArray/NovartisPhase I/IIRDEA119MEKArdea/BayerPhase I/IIRDEA436MEKArdea/BayerPhase I/IIE6201MEKEis

51、aiPhase I/IIGSK1120212MEKGlaxoSmithKlinePhase I/IIWX554MEKWilex AGPhase I/IIGDC-0973MEKGenentechPhase I/IIAS703026MEKEMD SeronoPhase I/IIRG7168 (CH4987655)MEKChugai/RochePhase I/IITAK-733MEKTakeda/MilleniumPhase I/IIPI3K/AKT/mTOR in disease Since the PI3K/AKT/mTOR pathway is an intracellular signali

52、ng pathway important in regulating the cell cycle and apoptosis, dysregulated PI3K/AKT/mTOR signaling may play key role in diseases, especially in tumors. The PI3K/AKT/mTOR signaling pathway in cancerClasses of PI3K/AKT/mTOR pathway inhibitorsOngoing clinical trials in breast cancer, NSCLC, and glio

53、blastoma that are evaluating the potential for PI3K/AKT/mTOR pathway inhibitors to overcome resistance to standard anticancer therapyClassAgentTargetAdministrationDevelopmentRapalogsSirolimusmTORC1OralFDA approvedaEverolimusmTORC1OralFDA approvedaTemsirolimusmTORC1ParenteralFDA approvedaRidaforolimu

54、smTORC1Oral and parenteralClinical trialsmTOR kinase inhibitorsPP242mTORC1/2ParenteralPre-clinicalINK128mTORC1/2OralClinical trialsAZD8055mTORC1/2OralClinical trialsOSI-027mTORC1/2OralClinical trialsPI3K and PI3K/mTOR inhibitorsBKM120PI3KOralClinical trialsSAR245408PI3KOralClinical trialsGS-1101PI3K

55、 (p110)OralClinical trialsWortmanninPI3K, mTORC1, PI4KNot for clinical usePre-clinicalLY294002PI3K, mTORC1, PI4K, PIM1, PLK1, ATM, CK2, DNA-PKNot for clinical usePre-clinicalPX-866PI3K, mTORC1OralClinical trialsSF1126PI3K, PIM1, mTORC1/2, PLK1, DNA-PK, CK2, ATMOralClinical trialsSAR245409,PI3K, mTOR

56、C1OralClinical trialsPictrelisibPI3K, mTORC1, DNA-PKOralClinical trialsGDC-0980PI3K, mTORC1OralClinical trialsPI-103PI3K, mTORC1ParenteralPre-clinicalPF-4691502,PI3K, mTORC1OralClinical trialsBGT226PI3K, mTORC1OralClinical trialsGSK2126458PI3K, mTORC1OralClinical trialsZSTK474PI3K, mTORC1OralClinica

57、l trialsXL-499PI3K (p110), mTORC1Not publishedPre-clinicalBEZ235PI3K, mTORC1/2, DNA-PKOralClinical trialsAKT inhibitorsPerifosineAKTOralClinical trialsbTriciribineAKTParenteralClinical trialsMK-2206AKTOralClinical trialsGSK690693AKT, PAK6, PKC, PrkXParenteralPre-clinicalGSK2141795AKTOralClinical tri

58、alsGSK2110183AKTOralClinical trialsGDC-0068AKTOralClinical trialsLY2780301AKT, p70S6KOralClinical trialsPDK-1 inhibitorsUCN-01PDK-1ParenteralClinical trialsGSK2334470PDK-1Not publishedPre-clinicalAR12PDK-1OralPre-clinicalOther4EGI-1elF4FNot publishedPre-clinicalRibavirinelF4EOral, parenteralFDA appr

59、ovedaInhibitors of PI3K/AKT/mTOR pathwayMutations in JAKsJAKsSubstitutionDisease associationHopscotch (D. melanogaster)E695KLymphoproliferative diseasesG341ELeukemiaJAK2 (human)V617F(myeloproliferative disorders)MPDsK539L (and the region F537E543)PV(polycythemia vera)T875NAcute megakaryoblastic myel

60、oid leukemiaDeletion of IREED (position 682686)Acute lymphoblastic leukemiaJAK3 (human)A572VAcute megakaryoblastic myeloid leukemiaV722IAcute megakaryoblastic myeloid leukemiaP132TAcute megakaryoblastic myeloid leukemiaJAK1 (human)G871EaUterine leiomyosarcomasJak-STAT pathway and diseaseDeleted gene