Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

T CELL RECEPTOR SIGNALING

Tímea Berki and Ferenc Boldizsár Signal transduction

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: 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-g

TCRβ

Commitment TCRβ

checkpoint Pre-TCR

checkpoint β-selection

Role of transcription factors in

thymocyte development

CD4⁺ CD8^- CD4⁺

CD8^- CD8⁺ CD4^-

Positive selection

Lineage commitment

Post-commitment

TOX

RUNX No TOX

Apoptosis No GATA3

or MYB

GATA3

MYB ThPOK

GATA3

CD8^hi

CD4^mid CD8^mid

CD4^hi CD8^mid

No ThPOK No RUNX

Th - Tc cell decision

CD43

CD44 CD45

TCR-CD3-peptide-MHC CD28-CD80/CD86 CTLA4-CD80/CD86

Agrin PKC

CD4 LCK LFA1-ICAM1

Talin

CD2-CD48/CD59

LCK

CD44 CD45 CD4/CD8

CD43

CD28 CD80/CD86

CD2 CD48/CD59

LFA1 ICAM1

CTLA4 CD80/CD86

TCR Peptide-MHC

CD3

APC T cell

PI3K

SHIP2 ZAP70

Molecules of the „immunological

synapse”

Co-stimulatory pathways regulate the TcR signal

TCR

 b g

d e e z z

CD28

Plasma membrane

Calcium flux, NFAT activation

MAPK activation, AP1 formation GRB

2 SOS

RAS

RAS GRP

1 DAG

GTP

NFAT FOS JUN IL-12

Nucleus

ZAP70

AP1 CD3

Binding of ZAP-70 kinase (Zeta-chain Associated Protein kinase 70 kDa)

APC

T cell TCR

CD3 b

 z z g e e d

Y Y Y Y Y Y

Y Y Y Y Y Y

Y Y Y Y Y Y Y Y

P P P P

Peptide-MHCII CD4

ZAP-70 Lck

TCR

CD3 b

 z z g e e d

Y Y Y Y Y Y

Y Y Y Y Y Y

Y Y Y Y Y Y Y Y

P P P P P

Peptide-MHCII CD4

ZAP-70 Lck

P P P P

P P

Downstream signals Gene 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.

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 Y292

Y126 Y474 Y492

Y178 Y493 Y597/598

Y238

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.

T cell activation pathways

L

R T

E

N/RE

Apoptosis Apoptosis

FADD Mort1

TNFR Fas FasL TNF

Caspases 1-14

Activation

Cytokine production

CD28 CD80 CD86

PIP2 Ras IP3

DAG

Raf-1 MEK MAPK

CD3 gdezh TCR β

CD4

Allopeptide

Ras

Raf-1 MEK MAPK Lck Fyn ZAP70 PIP2

PLCg1

CaM PKC

PKCβ PKCf CaN

IP3 DAG Ca²⁺

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

Cellular proliferation IL-2R

IL-2Rβ IL-2Rg IL-2 IL-2

Lyk Syk Fyn SHC

Raf-1 MEK MAPK

STAT3 STAT5a STAT5b

IP3 DAG Ras JAK1

JAK3

PIP2

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

2 signals are required for T-cell activation

Signal 1: TCR-CD3 complex Antigen specific

Signal 2: co-stimulation signal CD28 - B7 interaction

NOT antigen specific

T-cell differentiation and proliferation

Effector and memory

T-cells

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

Th differentiation

Naive CD4

T cell

Treg

IL-2 FOXP3

RUNX1

Th2

Th2 cytokine locus IL-12Rβ2 GATA3

Th17

IL-17 RORgt

RUNX1

Th1

T-bet IFNg

RUNX3

Th2 cytokine locus

Anergy

TCR

 b g

d e e z z

CD28

Calcium flux, NFAT activation

GRB 2

SOS RAS

RAS GRP

1 DAG

GDP

NFAT IL-12

Nucleus

ZAP70 CD3

P DGK

a

Phosphatidic acid

Similarities in TcR and FceR signaling

Lyn Lck

g g 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 z z g

d e e

CD3

ITAM

Syk

ZAP-70 ZAP-70

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.

Non-receptor tyrosin kinases

Myr

Fes/Fer Syk/Zap70 Jak Tec Fak Ack Src Csk Srm Rak/Frk Brk/Sik

Abl

Cdc-42

Y Myr

PH

Y Y

Y

Y

Y

Y

Y Y

Y Y

Y

Y

Y

Y _SH2

PTK catalytic

SH3

Integrin-binding/JEF domain Kinase-like domain

Characteristics of Src family kinases

Src family member Pattern of expression Isoforms Oncogenic forms

Blk B cells

Fgr Myeloid cells, B cells Oncogenic 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, brain Overexpressed in T-cell acute lymphocytic

leukemias

Lyn Brain, B cells, myeloid

cells Two alternatively spliced forms

Src Ubiquitous Neuron-specific isoforms

Mutated 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

Overview of TcR/CD3 signaling pathway

Erk

PKC

GRP Ras

PIP3

lB

NF-B Calcineurin

MKK3/6

p38

MKK4/7

JNK GRB2 PLCg1 LAT

SOS

VAV GADS

SLP76 ADAP NCK

Akt

IKK

IKKg IKKβ

NFAT NFAT

NFAT Raf

MEK1/2 CaM

Ras

lBβ

Fos Jun Rel

NIK MEKKK Wasp

Calpain

Rac/

cdc42

MEKK Ca²⁺

Ca²⁺

Proteasomal degradation Proteasomal

degradation NF-B

Rel lBβ

 β g d e e z z Ca²⁺

Carma1 Bcl 10 Malt1

NF-B Rel lB

TCR CD4 CD45

PI3K CD28

Cytoplasm

Nucleus

IL-2 Gene

CRAC Channel

F-Actin Clustering Talin

LFA1

Actininin

ER

Intracellular Ca²⁺ store

Zap70 Lck

DAG PIP2

IP3