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Signal Transduction

Transmission of external signals across themembrane and conversion of these signals intointracellular biochemical events

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TCR

Peptide:MHC

Changes in gene expression2

RECEPTOR

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Alterations inGene expression

TCR signaling: Big Picture

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General principle of signal transduction:Ligand induced dimerization is the initiating step in signal transduction

Example: Kit signaling

General principle:Phosphorylation is adynamic, reversible way ofmodulating protein function

(or clustering)

A kinase is an enzyme thatattaches phosphate groupsto specific amino acids ofproteins (Tyr, Ser, Thr)

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How does the TCR transduce signals with such a little tail?

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TCR associates with the CD3 Complex

Analogous to the Igαand Igβ proteins thatassociate with BCR onB cells

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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, itcreates a binding site for other signaling molecules

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SH2 domains bind to specific phosphotyrosines within ITAMs

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Protein Tyrosine Kinases in initial TCR signaling

Lck and Fyn - Src family kinasesY Y

YZap-70 - Syk family kinase

SH3 SH2

SH2 SH2

Kinase

Kinase

Regulatory Tyrosines

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How do the ITAMs on CD3 get phosphorylated?

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Lck binds to the tails of CD4 and CD8

Lck

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Initiation of TCR signaling

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Initial events in TCR signaling

1. TCR + CD4 or CD8 bind peptide:MHC (clustering)2. Lck and Fyn phosphorylate ITAMs on CD3 complex3. ZAP-70 binds to P-ITAMs on CD3zeta chains4. Lck and Fyn phosphorylate and activate ZAP-705. ZAP-70 initiates downstream signaling events…

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Initial TCR signaling

RECEPTOR

Protein tyrosine kinases

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Regulation of early TCR signaling

• Activating and inhibitory Tyr-phosphorylation sites

• CD45 (B220) is a phosphatasewhich activates PTKs bydephosphorylating inhibitory P-Tyr.

• Csk is a kinase which inhibits PTKsby phosphorylating inhibitory Tyr

Phosphorylation is adynamic, reversible way ofmodulating protein function

Lck or Fyn

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The phosphatase CD45 regulates PTK activityCD45 removes inhibitory phosphates from Lck and Fyn

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• 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

Regulation of early TCR signaling

Fyn

TCR

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Lipid rafts help regulate T cell activation

If rafts are disrupted (by depleting cholesterol), T cell activation is blocked

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Downstream TCR signaling

RECEPTOR

Protein tyrosine kinases

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ZAP-70 phosphorylates SLP-76 and LAT which leads toactivation of 3 downstream signaling pathways

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Phospholipase Cγ Ras (G-proteins)

Transcription factors Alterations inGene expression

RECEPTOR

Protein tyrosine kinases

TCR signaling: Big Picture

Protein kinase CIntracellular Ca2+ MAP kinases1 2 3

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Zap-70 phosphorylates PLCγ,activating it to cleave PIP2

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IP3 binds to Ca2+ channels and opens them2nd messengers amplify initial signals

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RECEPTOR

Protein tyrosine kinase

Phospholipase C Ras (G-proteins)

Protein kinase CIntracellular Ca2+ MAP kinases

Alterations inGene expression

TCR signaling: Big Picture

1 2 3

NFAT ?? 24

Immunosuppressive drugs block NF-AT activation

FK506 and cyclosporin blockNF-AT activation byinhibiting calcineurin

Essential drugs to preventtransplant rejection

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Tolypocladium inflatum, the white mold from Norway

Cyclosporin

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RECEPTOR

Protein tyrosine kinase

Phospholipase C Ras (G-proteins)

Protein kinase CIntracellular Ca2+ MAP kinases

Alterations inGene expression

TCR signaling: Big Picture

1 2 3

NFAT ??

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IP3 binds to Ca2+ channels and opens them2nd messengers amplify initial signals

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Activating NF-κB

activates kinase

Ca++

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RECEPTOR

Protein tyrosine kinase

Phospholipase C Ras (G-proteins)

Protein kinase CIntracellular Ca2+ MAP kinases

Alterations inGene expression

TCR signaling: Big Picture

1 2 3

NFAT NF-κB ? 30

ZAP-70 phosphorylates SLP-76 and LAT which leads toactivation of 3 downstream signaling pathways

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Guanine nucleotide exchange factorGEF

GTPaseactivatingprotein

Ras is a small G-protein

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Ras activates the MAP kinase pathway

Raf (MAP kinase kinase kinase)

MEK (MAP kinase kinase)

MAP kinase

AP-1 (Transcription factor)

TCR activation

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RECEPTOR

Protein tyrosine kinase

Phospholipase C

Protein kinase CIntracellular Ca2+

Ras (G-proteins)

MAP kinases

The IL-2 genepromoterNF-κB AP-1NF-AT

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Example of T cell activation

TCR binds peptide:MHC

Induces expression of genes

T cells divides many timescreating more T cells withthe same TCR

T cells differentiateand acquire newcharacteristics

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IL-2 is one of hundreds of genes induced by TCR signaling

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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:MHCcomplexes ever recognized?

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Initial T cell/APC contact is mediated by adhesion molecules

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T-Cell

B-Cell(Peptide-pulsed)

8 minutes in 20 seconds of video

Clustering leads to formation of an “immunological synapse”

TCR, MHC & adhesion molecules cluster atinterface between T cell and APC

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Many additional receptor interactions contributeto T cell activation

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Summary of TCR signaling

• TCR signaling is initiated by TCR binding peptide:MHC andclustering 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 activationallows the TCR to enter lipid rafts and associate with PTKs.

• T cell signaling activates 3 general downstream pathways:PLCγ - Ca2+ - NFATPLCγ - PKC - NFκBRas - MAPKs - AP-1

• Coreceptors and adhesion molecules contribute to T cell:APCinteractions. These molecules are organized into an immunologicalsynapse which facilitates prolonged T cell signaling.