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Signal Transduction Transmission of external signals across the membrane and conversion of these signals into intracellular biochemical events Peptide:MHC TCR ? Changes in gene expression 1 TCR signaling: Big Picture RECEPTOR ? ? ? ? ? ? ? ? ? Alterations in 2 Gene expression General principle of signal transduction: Ligand induced dimerization is the initiating step in signal transduction (or clustering) 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 How does the TCR transduce signals with such a little tail? 4 TCR associates with the CD3 Complex Analogous to the Ig and Ig proteins that associate with BCR on B cells 5 ITAM 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 6 SH2 domains bind to specific phosphotyrosines within ITAMs 7 Protein Tyrosine Kinases in initial TCR signaling Lck and Fyn - Src family kinases Y SH3 SH2 Y Kinase Regulatory Tyrosines Zap-70 - Syk family kinase Y SH2 SH2 Kinase 8 How do the ITAMs on CD3 get phosphorylated? 9 Lck binds to the tails of CD4 and CD8 Lck 10 Initiation of TCR signaling 11 Initial events in TCR signaling 1. 2. 3. 4. 5. 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 Initial TCR signaling RECEPTOR Protein tyrosine kinases 13 Regulation of early TCR signaling Lck or Fyn Phosphorylation is a dynamic, reversible way of modulating protein function • 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 14 The phosphatase CD45 regulates PTK activity CD45 removes inhibitory phosphates from Lck and Fyn 15 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 16 Lipid rafts help regulate T cell activation If rafts are disrupted (by depleting cholesterol), T cell activation is blocked 17 Downstream TCR signaling RECEPTOR Protein tyrosine kinases ? 18 ZAP-70 phosphorylates SLP-76 and LAT which leads to activation of 3 downstream signaling pathways 1 2 3 19 TCR signaling: Big Picture RECEPTOR Protein tyrosine kinases Phospholipase C Intracellular Ca2+ 1 Protein kinase C 2 Transcription factors Ras (G-proteins) MAP kinases 3 Alterations in 20 Gene expression Zap-70 phosphorylates PLC, activating it to cleave PIP2 21 IP3 binds to Ca2+ channels and opens them 2nd messengers amplify initial signals 22 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 Immunosuppressive drugs block NF-AT activation FK506 and cyclosporin block NF-AT activation by inhibiting calcineurin Essential drugs to prevent transplant rejection 24 Cyclosporin Tolypocladium inflatum, the white mold from Norway 25 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 26 Gene expression IP3 binds to Ca2+ channels and opens them 2nd messengers amplify initial signals 27 Activating NF-kB Ca++ 28 TCR signaling: Big Picture RECEPTOR Protein tyrosine kinase Phospholipase C Intracellular Ca2+ 1 Ras (G-proteins) Protein kinase C 2 NFAT NF-kB MAP kinases 3 ? Alterations in 29 Gene expression ZAP-70 phosphorylates SLP-76 and LAT which leads to activation of 3 downstream signaling pathways 1 2 3 30 Ras is a small G-protein GTPase activating protein GEF Guanine nucleotide exchange factor 31 Ras activates the MAP kinase pathway TCR activation Raf (MAP kinase kinase kinase) MEK (MAP kinase kinase) MAP kinase AP-1 (Transcription factor) 32 RECEPTOR Protein tyrosine kinase Phospholipase C Intracellular Ca2+ NF-AT Ras (G-proteins) Protein kinase C NF-kB AP-1 MAP kinases The IL-2 gene promoter 33 Example of T cell activation TCR binds peptide:MHC Induces expression of genes T cells divides many times creating more T cells with the same TCR T cells differentiate and acquire new characteristics 34 IL-2 is one of hundreds of genes induced by TCR signaling 35 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? 36 Initial T cell/APC contact is mediated by adhesion molecules 37 Clustering leads to formation of an “immunological synapse” B-Cell (Peptide-pulsed) QuickTime™ and a Vi deo decompre ssor are n eede d to see thi s pi ctu re. T-Cell minutes in 20 seconds of video cluster at TCR, MHC & 8adhesion molecules interface between T cell and APC 38 Many additional receptor interactions contribute to T cell activation 39 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: PLC - Ca2+ - NFAT PLC - PKC - NFkB 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. 40