Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Tímea Berki and Ferenc Boldizsár Signaltransduction T cell receptor signaling
Role of transcriptionfactorsinthymocytedevelopment HES-1, GATA-3 Sox4, HEB, NFATc Transcriptionfactors Ikaros E2A, STAT5 TCF-1-Lef-1, NF-B, p53 Notch-1 c-Kit, IL-7R-c TCRβ Surface receptors Frizzled receptor, Death receptor, pre-TCR CD4+ CD8+ CD4+ CD8- CD44- CD25+ CD44- CD25- CD44+ CD25+ CD44+ CD25- CD4- CD8+ SP HSC DN1 DN2 DN3 DN4 DP β-selection SP Pre-TCR checkpoint Commitment TCRβ checkpoint
Th-Tc cell decision Lineagecommitment Positiveselection Post-commitment No TOX RUNX No RUNX TOX CD4+ CD8- CD8+ CD4- CD4+ CD8- CD4hi CD8hi CD4mid CD8mid CD4hi CD8mid No GATA3 or MYB No ThPOK Apoptosis ThPOK GATA3 GATA3 MYB
Molecules of the „immunologicalsynapse” T cell APC CD44 CD43 CD45 CD4 CD4/CD8 LFA1-ICAM1 LCK CD3 Talin Peptide-MHC TCR LCK TCR-CD3-peptide-MHC CD28-CD80/CD86 CTLA4-CD80/CD86 Agrin PKC ZAP70 CD28 CD80/CD86 PI3K CTLA4 CD80/CD86 SHIP2 CD44 CD45 CD2 CD48/CD59 CD2-CD48/CD59 ICAM1 LFA1 CD43
Co-stimulatorypathwaysregulatetheTcRsignal a b z z d e e g TCR CD28 CD3 Plasmamembrane DAG GRB2 ZAP70 SOS RAS GTP RAS GRP1 FOS JUN Calcium flux, NFAT activation MAPK activation, AP1 formation AP1 Nucleus IL-12 NFAT
Binding of ZAP-70 kinase(Zeta-chainAssociatedProtein kinase 70 kDa) b a b a APC Peptide-MHCII Peptide-MHCII CD4 CD4 TCR TCR CD3 CD3 z z g e e d z z g e e d T cell Lck Lck P P P P P P Y Y Y Y Y Y Y Y P Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Downstreamsignals Genetranscription T cellactivation Y Y Y Y Y Y Y Y Y Y Y Y ZAP-70 P P P P ZAP-70 P P P P Activation through the T cell receptor (TcR) initiates various signaling pathways. The T and NK cell specific protein tyrosine-kinase ZAP-70 plays a crucial role in early TcR signalling. Phosphorylation on ZAP-70 leads to downstream signaling events, including phosphorylaton of the MAPK cascade and the increase of intracellular calcium level.
Phosphorylation sites of the ZAP-70 kinase N C 0 100 200 300 400 500 600 Y69 Y126 Y178 Y238 Y315/319 Y474 Y493 Y597/598 Y292 Y492
The role of ZAP-70 • ZAP-70 consists of two tandem SH2 domains, and a C-terminal kinase domain separated by interdomains A and B. It contains a total of 30 tyrosine (Tyr) residues, 11 of them have been identified as phosphorylation sites, some of them still with unknown function (Tyr069, Tyr126, Tyr178 and Tyr238).Interdomain B plays an important role in regulating ZAP-70 conformation. ZAP-70 is phosphorylated by p56Lck but autophosphorylationhaaalso been described on some of the Tyr residues. • ZAP-70 is not only indispensible in T cell activation, but also an important prognostic marker in B-CLL. Moreover, in mice, spontaneous or induced ZAP-70 mutations lead to the development of autoimmunity.
T cellactivationpathways IL-2 Activation Apoptosis CD4 TCR β CD3 Fas TNFR FasL TNF CD28 IL-2 L IL-2R IL-2Rβ IL-2R R PIP2 Lck Fyn ZAP70 T PLC1 CD80 CD86 PIP2 Ras Allopeptide JAK1 JAK3 Ras PIP2 IP3 DAG Ca2+ E Ras Raf-1 MEK MAPK IP3 DAG FADD Mort1 CaM PKC PKCβ PKC Raf-1 MEK MAPK Raf-1 MEK MAPK IP3 DAG STAT3 STAT5a STAT5b Lyk Syk CaN Fyn SHC N/RE NFATc, NF-B, Jun/Fos, NFATn, c-Rel, IB, Oct-1 Caspases 1-14 Bcl-2, Bcl-x, Kip1, cyclinE/Cdk2 Cytokineproduction Cellularproliferation Apoptosis
2 signalsarerequiredforT-cellactivation Signal 1: TCR-CD3 complex Antigenspecific Signal 2: co-stimulationsignal CD28 - B7 interaction NOT antigenspecific T-celldifferentiation and proliferation Effector and memory T-cells
Initialsteps of T cellactivation • 1 ITAM:ImmunoreceptorTyrosine-based ActivationMotif • 2 TCR crosslinking results inthe association of co-receptors (CD4, CD3, CD45) closer to TcR • 3 Protein tyrosine kinase (PTK) Lck and Fyn activation: CD45 phosphatase removes an inhibitory phosphate (Pi) + addition of a phosphate (Pa) • 4 Fyn and Lckphosphorylate ITAMs of CD3 complex • 5 Docking of ZAP-70 PTK to z-chain ITAMs and its phosphorylation
Th differentiation Naive CD4+ T cell Th1 Th17 T-bet IFN RORgt RUNX3 RUNX1 IL-17 Th2 cytokine locus Treg Th2 IL-12Rβ2 FOXP3 GATA3 RUNX1 IL-2 Th2 cytokine locus
Anergy a b z z d e e g TCR CD28 CD3 DAG Phosphatidic acid P DGKa ZAP70 RAS GDP RAS GRP1 GRB2 Calcium flux, NFAT activation SOS Nucleus IL-12 NFAT
Similarities in TcR and FceR signaling a b Antigen-specific receptor TCR IgE d e e g z g z g a CD3 CD3 FcRI b Lck Lyn ZAP-70 ZAP-70 ITAM ITAM Syk Src-familykinase Syk-family kinase ZAP-70 expression is restrictedto T cells, NK cells and a subpopulation of CLL Syk is presentin most hematopoeticcelltypes
Characteristics of Src and Sykfamilykinases • Srckinasesconsist of eight non-receptor tyrosinekinases (Src, Fyn, Yes, Lck, Lyn, Hck, Fgr and Blk) thatinteractwiththeintracellulardomains of growthfactor/cytokinereceptors, GPCRs and integrins. Members of theSrckinasefamilyhave a verysimilardomainstructure. • ZAP-70 is an abbreviationforZeta-chain-associated protein kinase 70 (70 is themolecularweightinkDa). The protein is a memberintheSykprotein-tyrosinekinasefamily. ZAP-70 is normallyexpressedin T cells and naturalkillercellsand has a criticalroleintheinitiation of T-cellsignaling. • ZAP-70 in B cells is usedas a prognostic marker inidentifyingdifferentforms of chroniclymphocyticleukemia (CLL). • ZAP70 deficiencyresultsin a form of immunedeficiency.
Non-receptor tyrosinkinases DNA Actin Y Myr SH3 binding region Abl Y Fes/Fer Y Syk/Zap70 Y Jak PH Y Tec Y Fak Cdc-42 Y Ack Y Y Myr Src Y PTK catalytic Csk Kinase-like domain Y SH2 Srm SH3 Y Y Integrin-binding/JEF domain Rak/Frk Y Y Brk/Sik
Overview of TcR/CD3 signalingpathway β CRAC Channel LFA1 TCR CD4 CD45 CD28 Ca2+ GADS LAT Clustering PLC1 VAV SLP76 GRB2 Actininin Talin Lck PI3K NCK Zap70 SOS ADAP Ca2+ Rel PIP2 PKC lBβ CaM NF-B PIP3 Calpain DAG Carma1 Bcl 10 Malt1 Calcineurin F-Actin Akt Wasp Rac/ cdc42 Ras GRP Ca2+ MEKKK IKK IP3 IKK IKKβ Ras NIK Intracellular Ca2+store MEKK Rel ER Raf lB NFAT NF-B MKK3/6 MKK4/7 Proteasomal degradation lBβ MEK1/2 NFAT p38 JNK Erk Proteasomal degradation lB Cytoplasm Nuclear membrane NFAT Fos Jun NF-B Rel Nucleus IL-2 Gene