Signal Transduction Transmission of external signals across the

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 CD 3 Complex Analogous to the Ig and Ig proteins that associate with BCR on B cells 5

ITAMs are the essential basis of TCR signal transduction ITAM: Immune Tyrosine Activation Motif ITAM structure is Y- X - I/L - X 6 -8 - Y - X -I/L When a tyrosine within an ITAM is phosphorylated, it creates a binding site for other signaling molecules 6

SH 2 domains bind to specific phosphotyrosines within ITAMs 7

Protein Tyrosine Kinases in initial TCR signaling Lck and Fyn - Src family kinases Y SH 3 SH 2 Y Kinase Regulatory Tyrosines Zap-70 - Syk family kinase SH 2 Y Kinase 8

How do the ITAMs on CD 3 get phosphorylated? 9

Lck binds to the tails of CD 4 and CD 8 Lck 10

Initiation of TCR signaling 11

Initial events in TCR signaling 1. 2. 3. 4. 5. TCR + CD 4 or CD 8 bind peptide: MHC (clustering) Lck and Fyn phosphorylate ITAMs on CD 3 complex ZAP-70 binds to P-ITAMs on CD 3 zeta chains Lck and Fyn phosphorylate and activate 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 • CD 45 (B 220) 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 CD 45 regulates PTK activity CD 45 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 Ca 2+ 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 PIP 2 21

IP 3 binds to Ca 2+ channels and opens them 2 nd messengers amplify initial signals 22

TCR signaling: Big Picture RECEPTOR Protein tyrosine kinase Phospholipase C Intracellular Ca 2+ 1 Ras (G-proteins) Protein kinase C 2 NFAT ? MAP kinases 3 ? Alterations in 23 Gene expression

Immunosuppressive drugs block NF-AT activation FK 506 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 Ca 2+ 1 Ras (G-proteins) Protein kinase C 2 NFAT ? MAP kinases 3 ? Alterations in 26 Gene expression

IP 3 binds to Ca 2+ channels and opens them 2 nd messengers amplify initial signals 27

Activating NF- B activ ates kina se Ca++ 28

TCR signaling: Big Picture RECEPTOR Protein tyrosine kinase Phospholipase C Intracellular Ca 2+ 1 Ras (G-proteins) Protein kinase C 2 NFAT NF- B 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) MEK (MAP kinase) MAP kinase AP-1 (Transcription factor) 32

RECEPTOR Protein tyrosine kinase Phospholipase C Intracellular Ca 2+ NF-AT Ras (G-proteins) Protein kinase C NF-k. B 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) T-Cell minutes in 20 seconds of video cluster at TCR, MHC & 8 adhesion 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 CD 4/CD 8 and other accessory molecules. • Clustering leads to activation of Src family protein tyrosine kinases (Lck and Fyn) that phosphorylate ITAMs of the CD 3 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 - Ca 2+ - NFAT PLC - PKC - NF B 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