Signaltransduktion des T-Zell- Rezeptors (TCR) Intracellular Signal
Werbung
Signaltransduktion des T-ZellRezeptors (TCR) 1) Prinzipien und Komponenten intrazellularer Signaltransduktion 2) Das Immunsystem und T-Zellen Dr. Ingo Schmitz Institut für Molekulare Medizin Universität Düsseldorf Intracellular Signal Transduction 3) Signaltransduktion des TCR 4) TCR und Apoptose Biochemical Reactions in Signal Transduction Kinases Stimulus Receptor Mediator(s) Effector(s) Response -Kinases -Phospholipases -GTPases -Adaptors -Transcription Factors -Structural Proteins -Enzymes -Proliferation -Differentiation -Apoptosis -Cell activation -protein kinases phosphorylate their substrates -phosphorylation changes the activity, subcellular location, or aggregation of the proteins Biochemical Reactions in Signal Transduction Kinases Biochemical Reactions in Signal Transduction Phosphatases Primary structure of a kinase Kinase domain ( approx. 30 kDa) Subdomains I II III IV V AXK VI VII VIII IX X XI Activation loop (T-loop) Tyr Kinases: Lck, Fyn, ZAP70, Tec Ser/Thr Kinases: PKC, Raf, MAPK, Akt Lipid Kinase: PI3K Biochemical Reactions in Signal Transduction Phospholipases Biochemical Reactions in Signal Transduction Phospholipases -phospholipase C (PLC) catabolizes phospholipids (PIP2) generating IP3 -IP3 binds a receptor and increases cytosolic calcium -increased cytosolic calcium activates other signaling mechanisms Biochemical Reactions in Signal Transduction Biochemical Reactions in Signal Transduction GTPases GTPases GTPases: Ras, Rac1, Rho, Cdc42 GEFs: Sos, Ras-GRP, Vav1 GEFs GAPs Signal Vav1 -G proteins are turned “on” and “off” by GTP and GDP, respectively - active G proteins transmit the signal to other “downstream” proteins Biochemical Reactions in Signal Transduction Adaptor proteins Biochemical Reactions in Signal Transduction Adaptor proteins Protein-protein interaction domains SH2 14-3-3 SH3 P -Y-X-X-hyExample: SH2 domains -recognize phosphoryated tyrosines -Recruit signaling proteins to receptor -adaptors have no enzymatic activity of their own -function is to recruit signaling enzymes to the receptor-ligand complex -this concentrates signaling proteins and increases efficiency PTB -P-X-X-P-XWW PH P -hy-X-N-P-X-Y- P R-S-X-S-X-P -P-P-X-Y- PIP3 Biochemical Reactions in Signal Transduction Adaptor proteins 1) Prinzipien und Komponenten intrazellularer Signaltransduktion Grb2 : Growth factor receptor binding protein 2 2) Das Immunsystem und T-Zellen LAT : Linker for activation of T-cells 3) Signaltransduktion des TCR 4) TCR und Apoptose SLP-76 : SH2-domain containing leukocyte phosphoprotein of 76 kDa Die Organe des Immunsystems Die Zellen des Immunsystems Bone Marrow Stem Cell Myeloid Precursor Lymphoid Precursor B Cell Neutrophil, Monocyte Polymorphonuclear leukocyte or PMN Macrophage Dendritic Cell T Cell Plasma Cell Memory B Cell T-Lymphozyten Anzahl reifer T Zellen in der Peripherie: MHC Restriktion 1016 in Elephanten 1011 in Menschen 108 in Mäusen zelluläre Immunantwort --> Erkennung von Antigen nur in Zusammenhang mit MHC (MHC-Restriktion) Entwicklung im Thymus: bis zu 1018 Möglichkeiten, den TCR zu rearrangieren Selektion der „nützlichen“ Zellen: 1) Erkennung von pMHC Komplexen (positive Selektion) 2) Autoreaktivität (negative Selektion) CD4+ : Helfer T-Zellen erkennen „exogene“ Antigene auf MHC II MHC II nur auf professionellen APCs CD8+ : zytotoxische T-Zellen = CTL erkennen „endogene“ Antigene auf MHC I MHC I auf (fast) allen Zellen Phases of T Cell Responses Effektor-T-Zellen 1. Selektion und Differenzierung im Thymus zu CD8+ oder CD4+ T Zellen 2. Polarisierung der CD4+ T Zellen in der Peripherie zu Th1 der Th2 IFN! IL12 IL4 IL5 IL10 Full activation of signaling in T cells depends on two signals 1) APC B7-1/2 Ag-MHC Ag-MHC B7-1/2 Prinzipien und Komponenten intrazellularer Signaltransduktion 2) Das Immunsystem und T-Zellen 3) Signaltransduktion des TCR T cell TCR CD28 TCR signal 2 signal 1 Possible consequences: CD28 no biological effect - antigen desensibilisation - anergy - apoptosis TCR CD28 4) TCR und Apoptose signal 1 signal 2 Possible consequences: - NF-"B activation - clonal expansion, IL-2 production - differentiation - immune response Signal transduction induced by T cell receptor:CD3 complex T cell receptor engagement • T cell receptor – # and $ chain heterodimer – antigen recognition MHC T-Cell TCR/CD3 • CD3 AntigenPresenting Cell – transmembrane proteins with extracellular domains and cytoplasmic tails - two % chains - one & chain - one ! chain – transmembrane/cytoplasmic ' homodimers Co-Receptors of the TCR -CD4 and CD8 recognize invariant amino acids on MHCII and MHCI, respectively -CD4 and CD8 are constitutively associated with Lck -CD4/CD8 concentrate Lck at the site of TCR-MHC interaction -this enhances signaling / activation Lipid Rafts • Membrane compartments enriched with cholesterol, glycosphingolipids and sphingomyelin • Contain lipid modified signal proteins – Src kinases (Lck) – Glycosylphophatidylinositol (GPI)- linked proteins (Ras proteins, G-proteins) – Adaptor proteins (LAT) -function of co-receptors is to increase efficiency of lymphocyte activation Lipid Rafts Accumulate in cSMAC of Mature IS Burack et al., 2002 • Selectively concentrate membrane proteins with lipid anchor of saturated acyl chains • Defined experimentally by detergent solubility, cholera toxin B binding and expression of GM1 The Immunologic Synapse of the T cell Lee et al, 2002 The mature IS on the side of the Antigen-Presenting-Cell Immunologic Synapse (IS) Bromley et al., 2001 Induction of a wave of tyrosine phosphorylation - + Delon and Germain, 2000 Induction of tyrosine phosphorylation TCR associated non-receptor tyrosine kinases MG (kDa) SH3 200 66 CD4 M SH3 CD4 45 ' ' & % ' ' & % CD3/' ' SH2 ZAP70 P P p59fyn Csk PH 21 T-Cell anti-pTyr WB (4G10) Kinase SH2 Kinase Syk-Kinases: ZAP-70, Syk Tec p56lck SH2 Src-Kinases: Fyn, Lck, Yes TZR % ! CD3/' ' T-Cell Kinase Csk TCR % ! SH2 116 97 anti-TCR anti-CD3% TH SH3 SH2 Kinase Tec-Kinases: Tec, Itk, Rlk, Mlk Induction of tyrosine phosphorylation Induction of tyrosine phosphorylation Non-receptor tyrosine kinases of the Src family T-Cell Antigen Receptor p56lck : Y394 p59fyn(T) : Y420 M U SH3 SH2 Pro SH1 ATP R Y CD4/CD8 # Binding of Proline-rich Motifs PxxP PxxPP(P/L)xP B-cell Antigen Receptor $ Y % ! COOH ' ' & % Mastcell-Fc%R1 # mIg TCR Fc!R CD64 CD16 CD3 CD3/' ' P P P P Receptor binding p56lck : CD4/CD8 p59fyn(T): TZR' CD3%, !, & = Immune receptor-Tyrosine-Activation Motifs Kinase Domain Autophosphorylation Receptorphosphorylation Membrane binding NH2 ITAM Binding to Phosphotyrosine-Motifs (pYEEI) regulatory Residues p56lck : Y505 p59fyn(T): Y531 Lck P P $ P ITAMs Fyn ! & $ % % P $ # # ! ! ITAM: ' ' '' oder !! DXXYXXLXXXXXXXY DXL Active 12 Aminoacids Src-Kinases phosphorylate TCR'- Components I/L in Position +3 Reth M. Antigen receptor tail clue. Nature 1989 Induction of tyrosine phosphorylation Induction of tyrosine phosphorylation Kinase Domain Autophosphorylation Receptorphosphorylation Membrane binding p56lck : Y394 p59fyn(T) : Y420 M ZAP-70 Zeta-associated U NH2 SH3 SH2 Pro SH1 ATP Y P R Y protein of 70 kDa COOH Binding of Proline-rich Motifs PxxP PxxPP(P/L)xP Binding to Phosphotyrosine-Motifs (pYEEI) katalytic Region ATP-Binding Phosphorylation of diverse Substrates (u.a. SLP-76, LAT, PLC!1, Cbl) P ATP NH2 Receptor binding p56lck : CD4/CD8 p59fyn(T): TZR' CD3%, !, & SH2 SH2 Kinase P COOH regulatory Residues p56lck : Y505 p59fyn(T): Y531 Tandem-Binding to ITAMs Active JB Bolen & JS Brugge, Annu Rev Immunol, 1997 Src-Kinases recruit and activate ZAP kinases (ZAP kinase: zeta-associated protein of 70kDa) Latour and Veillette, 2001 ZAP-Kinase Phosphorylation by Src-Kinases phosphorylates docking sites for SH2-Proteins Time Course of pTyr Migration in T cell:APC Conjugates Composition of a large signaling complex via tyrosine phosphorylation and SH2/SH3 interactions Lee et al, 2002 TCR induced cytosolic calcium influx Clinical Application: Immunosuppression by FK506 and Cyclosporin A Composition of a large signaling complex via Composition of a large signaling complex via tyrosine phosphorylation and SH2/SH3 interactions tyrosine phosphorylation and SH2/SH3 interactions PKC( IKK Members of the NF-kB signaling cascade induced by T cell costimulation CD28 CD4 TCR CD3 PTKs lipid raft LAT P P ZAP-70 P PIP3 ? P SLP-76 Vav1 ITK RAC/ RHO DAG PLC! 1 P PKC) P CARMA1/ CARD11 1. Activation of a specific receptor/co-receptor PI3-K AKT ? P Bcl10 MALT1 P 2. Recruitment to lipid rafts PDK1 3. Receptor-modification (Tyrosine phosphorylation) COT ? MLK3 General steps of signaling via the TCR IKK! NIK P IKK$ P IKK# P p50 I"B P ? P p65 NF-"B P p50 p65 CaMKII RIP2 ? 4. Protein-protein-interactions 5. Composition of a large signaling complex 6. Cytosceletal reorganisation and induced gene expression Die Rolle der CD8+ T Zellen: Apoptoseinduktion durch CTL 1) Prinzipien und Komponenten intrazellularer Signaltransduktion 2) Das Immunsystem und T-Zellen 3) Signaltransduktion des TCR 4) TCR und Apoptose Zytotoxische CD8+ T Zellen fungieren als ‚Serienkiller‘. Sie können mehr als eine Zielzelle durch Induktion von Apoptose (programmierter Zelltod) eliminieren Two ways of killing for cytotoxic T cells CD95/CD95Lmediated Perforinmediated CTL Target cell CD95 Caspase-8 Caspase-8 GrB GrB GrB Ag TCR Bid MHC Apoptosis MHC GrB Ag Caspase-3, Caspase-3, -10 cell number CTL GrB GrB CD95L Die Immunantwort durch T Zellen TCR time antigen challenge clonal expansion and effector phase down phase memory phase resistant sensitive resistant apoptosis phenotype activation-induced cell death (AICD) activated T cell Paul (ed.) „Fundamental Immunology“, 2003, 5th Edition, Lippincott Williams & Wilkins autocrine suicide 1 Literatur Janeway et al., „Immunobiology“, 2004, 6th Edition, Garland. CD95L MHC + AG Abul K. Abbas, Andrew H. Lichtman „Cellular and Molecular Immunology“, 2003, MP TCR CD95 CD95L paracrine activated T cell 5th Edition, W.B. Saunders Company Horejsi V, Zhang W, Schraven B. „Transmembrane adaptor proteins: organizers of immunoreceptor signalling“, Nat Rev Immunol. 2004 Aug;4(8):603-16. activated T cell 2 fratricide Leo A, Wienands J, Baier G, Horejsi V, Schraven B. „Adapters in lymphocyte signaling“, J Clin Invest. 2002 Feb;109(3):301-9. Praktikum im Institut für Molekulare Medizin (14.3. - 24.3.05 oder 29.3. - 8.4.05) NF"B Klausur Dienstag 8.3.2004 18-19 Uhr Zellzahlbestimmung, Zellstimulation und Zellyse Nachweis der IKK Aktivität im Immunkomplexkinaseassay Nachweis der Phosphorylierung von IkB und des NF-kB Faktors p65 durch phosphospezifische Antikörper im Western-blot Nachweis der Degradation von IkBalpha (Western Blot) Nachweis der transkriptionellen Aktivtät von NFkB im Reportergenassay Nachweis der Kernwanderung von p65 mit Hilfe der Immunfluoreszenzmikroskopie Apoptose Zellzahlbestimmung, Zellstimulation und Zellyse Lichtmikroskopische Analyse der Apoptose Nachweis der CD95 Expression durch FACSAnalyse Nachweis der DNA-Degradation während der Apoptose (FACS) Co-Immunpräzipitation des CD95 death-inducing signaling complex (DISC) und Analyse per Western Blot Nachweis der Translokation von GFP-Bax mit Hilfe der Immunfluoreszenzmikroskopie
