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Signal Transduction TCR signaling: Big Picture Transmission of external signals across the membrane and conversion of these signals into intracellular biochemical events RECEPTOR ? ? ? Peptide:MHC TCR ? ? ? ? Changes in gene expression 1 ? ? ? Alterations in 2 Gene expression General principle of signal transduction: Ligand induced dimerization is the initiating step in signal transduction (or clustering) How does the TCR transduce signals with such a little tail? Example: Kit signaling A kinase is an enzyme that attaches phosphate groups to specific amino acids of proteins (Tyr, Ser, Thr) General principle: Phosphorylation is a dynamic, reversible way of modulating protein function 3 TCR associates with the CD3 Complex 4 ITAM Analogous to the Igα and Igβ proteins that associate with BCR on B cells ITAMs are the essential basis of TCR signal transduction ITAM: Immune Tyrosine Activation Motif ITAM structure is Y- X - X - I/L - X 6-8 - Y - X - X -I/L When a tyrosine within an ITAM is phosphorylated, it creates a binding site for other signaling molecules 5 6 1 Protein Tyrosine Kinases in initial TCR signaling SH2 domains bind to specific phosphotyrosines within ITAMs Lck and Fyn - Src family kinases Y SH3 SH2 Y Kinase Regulatory Tyrosines Zap-70 - Syk family kinase SH2 Y Kinase SH2 7 How do the ITAMs on CD3 get phosphorylated? 8 Lck binds to the tails of CD4 and CD8 Lck 9 Initiation of TCR signaling 10 Initial events in TCR signaling 1. 2. 3. 4. 5. 11 TCR + CD4 or CD8 bind peptide:MHC (clustering) Lck and Fyn phosphorylate ITAMs on CD3 complex ZAP-70 binds to P-ITAMs on CD3zeta chains Lck and Fyn phosphorylate and activate ZAP-70 ZAP-70 initiates downstream signaling events… 12 2 Regulation of early TCR signaling Initial TCR signaling Lck or Fyn Phosphorylation is a dynamic, reversible way of modulating protein function RECEPTOR Protein tyrosine kinases • Activating and inhibitory Tyrphosphorylation sites • CD45 (B220) is a phosphatase which activates PTKs by dephosphorylating inhibitory P-Tyr. • Csk is a kinase which inhibits PTKs by phosphorylating inhibitory Tyr 13 The phosphatase CD45 regulates PTK activity CD45 removes inhibitory phosphates from Lck and Fyn 14 Regulation of early TCR signaling • The plasma membrane is not homogeneous • Lipid rafts are enriched for saturated lipids and cholesterol making them more rigid • This composition regulates which types of proteins can localize to these rafts -most transmembrane proteins (e.g. TCR) are excluded -Lck and Fyn are enriched in lipid rafts TCR Fyn 15 Lipid rafts help regulate T cell activation 16 Downstream TCR signaling If rafts are disrupted (by depleting cholesterol), T cell activation is blocked RECEPTOR Protein tyrosine kinases ? 17 18 3 ZAP-70 phosphorylates SLP-76 and LAT which leads to activation of 3 downstream signaling pathways TCR signaling: Big Picture RECEPTOR 1 2 3 Protein tyrosine kinases Phospholipase Cγ Intracellular Ca2+ 1 19 Zap-70 phosphorylates PLCγ, activating it to cleave PIP2 21 Ras (G-proteins) Protein kinase C 2 MAP kinases 3 Transcription factors Alterations in 20 Gene expression IP3 binds to Ca2+ channels and opens them 2nd messengers amplify initial signals 22 Immunosuppressive drugs block NF-AT activation TCR signaling: Big Picture RECEPTOR Protein tyrosine kinase Phospholipase C Intracellular Ca2+ 1 Ras (G-proteins) Protein kinase C 2 NFAT ? MAP kinases 3 ? Alterations in 23 Gene expression FK506 and cyclosporin block NF-AT activation by inhibiting calcineurin Essential drugs to prevent transplant rejection 24 4 Cyclosporin TCR signaling: Big Picture RECEPTOR Protein tyrosine kinase Phospholipase C Intracellular Ca2+ 1 Ras (G-proteins) Protein kinase C 2 MAP kinases 3 Tolypocladium inflatum, the white mold from Norway 25 IP3 binds to Ca2+ channels and opens them NFAT ? Alterations in 26 Gene expression ? Activating NF-κB 2nd messengers amplify initial signals activ ates kina se Ca++ 27 TCR signaling: Big Picture 28 ZAP-70 phosphorylates SLP-76 and LAT which leads to activation of 3 downstream signaling pathways RECEPTOR 1 2 Protein tyrosine kinase Phospholipase C Intracellular Ca2+ 1 Protein kinase C 2 NFAT NF-κB 3 Ras (G-proteins) MAP kinases 3 ? Alterations in 29 Gene expression 30 5 Ras activates the MAP kinase pathway Ras is a small G-protein TCR activation GTPase activating protein GEF Raf (MAP kinase kinase kinase) Guanine nucleotide exchange factor MEK (MAP kinase kinase) MAP kinase AP-1 (Transcription factor) 31 RECEPTOR Example of T cell activation Protein tyrosine kinase TCR binds peptide:MHC Induces expression of genes Phospholipase C Intracellular Ca2+ NF-AT Ras (G-proteins) Protein kinase C NF-κB 32 AP-1 T cells divides many times creating more T cells with the same TCR MAP kinases The IL-2 gene promoter T cells differentiate and acquire new characteristics 33 IL-2 is one of hundreds of genes induced by TCR signaling 34 Mysteries in T cell antigen recognition • Affinity of TCR for peptide:MHC is quite low • How is T cell-APC interaction maintained? • How are low frequency peptide:MHC complexes ever recognized? 35 36 6 Initial T cell/APC contact is mediated by adhesion molecules Clustering leads to formation of an “immunological synapse” B-Cell (Peptide-pulsed) T-Cell minutes in 20 seconds of video cluster at TCR, MHC & 8adhesion molecules interface between T cell and APC 37 Many additional receptor interactions contribute to T cell activation 38 Summary of TCR signaling • TCR signaling is initiated by TCR binding peptide:MHC and clustering of TCR with CD4/CD8 and other accessory molecules. • Clustering leads to activation of Src family protein tyrosine kinases (Lck and Fyn) that phosphorylate ITAMs of the CD3 complex • In naïve T cells, the TCR is excluded from lipid rafts. TCR activation allows the TCR to enter lipid rafts and associate with PTKs. • T cell signaling activates 3 general downstream pathways: PLCγ - Ca2+ - NFAT PLCγ - PKC - NFκB Ras - MAPKs - AP-1 • Coreceptors and adhesion molecules contribute to T cell:APC interactions. These molecules are organized into an immunological synapse which facilitates prolonged T cell signaling. 39 40 7