Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

T CELL RECEPTOR SIGNALING

Tímea Berki and Ferenc BoldizsárSignal transduction

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

TÁMOP-4.1.2-08/1/A-2009-0011

DN1

CD44+

CD25-

DN2

CD44+

CD25+

DN3

CD44-

CD25+

DN4

CD44-

CD25-

DP

CD4+

CD8+

HSC

CD4-

CD8+

SP

CD4+

CD8-

SP

Surface receptors

Transcription factors

HES-1, GATA-3 Sox4, HEB, NFATc

Ikaros E2A, STAT5 TCF-1-Lef-1, NF-B, p53

Frizzled receptor,Death receptor, pre-

TCR

Notch-1

c-Kit, IL-7R-c TCRβ

Commitment TCRβcheckpoint

Pre-TCRcheckpoint

β-selection

Role of transcription factors in thymocyte development

TÁMOP-4.1.2-08/1/A-2009-0011

CD4+

CD8-CD4+

CD8-

CD8+

CD4-

Positive selection

Lineage commitment

Post-commitment

TOX

RUNX

No TOX

ApoptosisNo GATA3

or MYB

GATA3

MYB

ThPOK

GATA3

CD4hi

CD8hiCD4mid

CD8midCD4hi

CD8mid

No ThPOK

No RUNX

Th - Tc cell decision

TÁMOP-4.1.2-08/1/A-2009-0011

CD43

CD44 CD45

TCR-CD3-peptide-MHCCD28-CD80/CD86CTLA4-CD80/CD86

AgrinPKC

CD4LCK

LFA1-ICAM1Talin

CD2-CD48/CD59

LCK

CD44

CD45

CD4/CD8

CD43

CD28 CD80/CD86

CD2 CD48/CD59

LFA1 ICAM1

CTLA4 CD80/CD86

TCR Peptide-MHC

CD3

APCT cell

PI3K

SHIP2

ZAP70

Molecules of the „immunological synapse”

TÁMOP-4.1.2-08/1/A-2009-0011

Co-stimulatory pathways regulate the TcR signal

TCR

b

d e e zz

CD28

Plasma membrane

Calcium flux,NFAT activation

MAPK activation,AP1 formation

GRB2

SOS

RAS

RASGRP

1

DAG

GTP

NFAT FOS JUN IL-12Nucleus

ZAP70

AP1

CD3

TÁMOP-4.1.2-08/1/A-2009-0011Binding of ZAP-70 kinase (Zeta-chain Associated Protein kinase 70 kDa)

APC

T cell

TCR

CD3

b z z dee

YY

YY

YY

YY

YY

YY

YY

YY

YY

YY

P

P

P

P

Peptide-MHCII

CD4

ZAP-70

Lck

TCR

CD3

b z z dee

YY

YY

YY

YY

YY

YY

YY

YY

YY

YY

P

P

P

P

P

Peptide-MHCII

CD4

ZAP-70

Lck

PPP

PPP

Downstream signalsGene transcription

T cell activation

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.

TÁMOP-4.1.2-08/1/A-2009-0011

Phosphorylation sites of the ZAP-70 kinase

0 100 200 300 400 500 600

N C

Y69 Y126 Y178 Y238

Y292

Y315/319 Y474 Y493

Y492

Y597/598

Function unclear

Activating phosphorylation sites Inhibitory phosphorylation sites

Y69 Y315/319 Y292Y126 Y474 Y492Y178 Y493 Y597/598Y238

TÁMOP-4.1.2-08/1/A-2009-0011

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 autophosphorylation haa also 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.

TÁMOP-4.1.2-08/1/A-2009-0011

T cell activation pathways

L

R

T

E

N/RE

Apoptosis

Apoptosis

FADDMort1

FasTNFR

FasLTNF

Caspases 1-14

Activation

Cytokine production

CD28

CD80CD86

PIP2 Ras

IP3DAG

Raf-1MEK

MAPK

CD3dez

TCRβ

CD4

Allopeptide

Ras

Raf-1MEK

MAPK

Lck Fyn ZAP70PIP2

PLC1

CaMPKCPKCβPKCCaN

IP3DAGCa2+

NFATc, NF-B, Jun/Fos, NFATn, c-Rel, IB, Oct-1

Cellular proliferation

IL-2RIL-2RβIL-2R

IL-2IL-2

Lyk SykFyn SHC

Raf-1MEK

MAPK

STAT3STAT5aSTAT5b

IP3DAG

Ras JAK1JAK3

PIP2

Bcl-2, Bcl-x, Kip1, cyclinE/Cdk2

TÁMOP-4.1.2-08/1/A-2009-0011

2 signals are required for T-cell activation

Signal 1: TCR-CD3 complexAntigen specific

Signal 2: co-stimulation signalCD28 - B7 interactionNOT antigen specific

T-cell differentiationand proliferation

Effector and memoryT-cells

TÁMOP-4.1.2-08/1/A-2009-0011

Initial steps of T cell activation 1 ITAM: Immunoreceptor Tyrosine-based Activation

Motif 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 Lck phosphorylate ITAMs of CD3 complex 5 Docking of ZAP-70 PTK to z-chain ITAMs and its

phosphorylation

TÁMOP-4.1.2-08/1/A-2009-0011

Th differentiation

NaiveCD4+ T cell

Treg

IL-2

FOXP3

RUNX1

Th2

Th2 cytokinelocus

IL-12Rβ2

GATA3

Th17

IL-17

RORt

RUNX1

Th1

IFNT-bet

RUNX3

Th2 cytokinelocus

TÁMOP-4.1.2-08/1/A-2009-0011

Anergy

TCR

b

d e e zz

CD28

Calcium flux,NFAT activation

GRB2

SOS

RAS

RASGRP

1

DAG

GDP

NFAT IL-12Nucleus

ZAP70

CD3

PDGK

a

Phosphatidicacid

TÁMOP-4.1.2-08/1/A-2009-0011

Similarities in TcR and FceR signaling

LynLck

b

IgE

ITAM

FceRI

Antigen-specific receptor

Src-family kinase

Syk-family kinase

ZAP-70 expression is restricted to T cells, NK cells and a subpopulation of CLL

Syk is present in most hematopoetic cell types

TCR

CD3 b

zzd e e

CD3

ITAM

SykZAP-70 ZAP-70

TÁMOP-4.1.2-08/1/A-2009-0011

Characteristics of Src and Syk family kinases• Src kinases consist of eight non-receptor tyrosine kinases (Src,

Fyn, Yes, Lck, Lyn, Hck, Fgr and Blk) that interact with the intracellular domains of growth factor/cytokine receptors, GPCRs and integrins. Members of the Src kinase family have a very similar domain structure.

• ZAP-70 is an abbreviation for Zeta-chain-associated protein kinase 70 (70 is the molecular weight in kDa). The protein is a member in the Syk protein-tyrosine kinase family. ZAP-70 is normally expressed in T cells and natural killer cells and has a critical role in the initiation of T-cell signaling.

• ZAP-70 in B cells is used as a prognostic marker in identifying different forms of chronic lymphocytic leukemia (CLL).

• ZAP70 deficiency results in a form of immune deficiency.

TÁMOP-4.1.2-08/1/A-2009-0011

Non-receptor tyrosin kinasesMyr

Fes/Fer

Syk/Zap70

Jak

Tec

Fak

Ack

Src

Csk

Srm

Rak/Frk

Brk/Sik

Abl SH3 binding region DNA Actin

Cdc-42

Y

Myr

PH

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y SH2

PTK catalytic

SH3Integrin-binding/JEF domain

Kinase-like domain

TÁMOP-4.1.2-08/1/A-2009-0011

Characteristics of Src family kinases

Src family member Pattern of expression Isoforms Oncogenic forms

Blk B cells

Fgr Myeloid cells, B cellsOncogenic fusion with gag sequences in feline sarcoma virus; overexpressed in some leukemias and lymphomas

Fyn Ubiquitous T-cell-specific isoform (Fyn T)

Hck Myeloid cells Two different translational starts

Lck T cells, NK cells, brainOverexpressed in T-cell acute lymphocytic leukemias

LynBrain, B cells, myeloid cells

Two alternatively spliced forms

Src Ubiquitous Neuron-specific isoformsMutated and truncated in retroviruses; truncated in colon cancer; overexpressed in mammary, pancreatic and other cancers

Yes Ubiquitous

Oncogenic fusion with gag sequences in avian sarcoma viruses; highly expressed in colon, malignant melanoma and other cancers

Yrk Ubiquitous

TÁMOP-4.1.2-08/1/A-2009-0011

Overview of TcR/CD3 signaling pathway

Erk

PKC

RasGRP

PIP3

lB

NF-B

Calcineurin

MKK3/6

p38

MKK4/7

JNK

GRB2LAT

PLC1

SOS

VAVGADS

SLP76

ADAPNCK

Akt

IKK

IKK

IKKβ

NFAT

NFAT

NFAT Raf

MEK1/2

CaM

Ras

lBβ

Fos Jun Rel

NIK

MEKKK

Wasp

Calpain

Rac/cdc42

MEKK

Ca2+

Ca2+

Proteasomaldegradation

Proteasomaldegradation

NF-BRel

lBβ

β

d e e zz

Ca2+

Carma1 Malt1Bcl 10

NF-BRel

lB

TCR CD4 CD45

PI3K

CD28

Cytoplasm

Nucleus

Nuclear membrane

IL-2 Gene

CRACChannel

F-Actin

Clustering

Talin

LFA1

Actininin

ER

IntracellularCa2+ store

Zap70Lck

DAG

PIP2

IP3