in

out

Lipid bilayerof plasma membrane

Antigen recognition

Signal transduction(contains one or more ITAMs)

+ -

Src-family tyrosine kinase reversibly associated with receptor

Consensus ITAM (amino acid) ….---.asp.---.---.tyr.---.---.leu.---.---.---.---.---.---.---.tyr.---.---.leu.---…. glu ile ile The one letter code is usually used:

xx(D/E)xxYxx(L/I)xxxxxxxYxx(L/I)

These tyrosines are phosphorylated by various src family kinases upon recognition receptor ligation

Src-family tyrosine kinases

Unique region reversibly associates with certain membrane receptors. Differs among family members.

SH3 motif involved in binding to many proteins that carry a proline rich motif (e.g. P-X-X-P).

SH2 motif involved in binding to phospho-tyrosines in certain sequence contexts, including phosphorylated ITAMs.

Kinase domain motif catalyzes tyrosine phosphorylation.

Myristylation/palmitylation sites present in many family members, promotes association with membranes and membrane “rafts”. (e.g. CH3(CH)12CONH-Glycine..)

CD45 (plasma membrane tyrosine phosphatase)

csk (cytoplasmic tyrosine kinase)

Adapted from Molecular Cell Biology 3rd Ed., p894

Table 1. Expression of Src family kinases

Src Ubiquitous; two neuron-specific isoformsFyn Ubiquitous; T cell-specific isoform (Fyn T)Yes UbiquitousLyn Brain, B-cells, myeloid cells; two alternatively spliced formsHck Myeloid cells (two different translational starts)Fgr Myeloid cells, B-cellsBlk B-cellsLck T-cells, NK cells, brain

Frk subfamily Primarily epithelial cells

+ -

in

out

Antigen recognition

Signal transduction(with ITAMs)

T Cell Receptor

CD3

-CD3

- +

CD3 functions both as signal transducers and in transport of antigen receptors to the plasma membrane

in

out

Antigen recognition

Signal transduction(with ITAMs)

B Cell Receptor

Ig

LynBlkHckFynLck

+ + - - + + - -

syk

ACTIVATION!

Antigen receptor signaling may be similar to that of other surface receptors such as receptor tyrosine kinases like epidermal growth factor in which receptor crosslinking

leads to initial trans-phosphorylation.

+ -

in

out

Signal transduction(with ITAMs)

- +

CD3

Fyn

T Cell ReceptorAnd coreceptor

CD3

-

Lck

CD4

Cholesterol and sphingolipid rich microdomain

+ -

- +

CD3

T Cell Receptor

CD3

-

ZAP70

ACTIVATION!Lck

CD4

TABLE 2. Protein with Src homology-2 domains that may be associated with T cell-activation (partial list)

Protein tyrosine kinases Fyn Lck Syk ZAP-70 Csk ltkProtein tyrosine phosphatases SHP-1 SHP-2Proteins with enzymatic functions Phospholipase C 1 (PLC 1) Phosphatidylinositol 3”-kinase (P1 3-kinase), p85 subunitAdaptors and Regulators GTPase acttivating protein (GAP) SOS Vav Slp-76 Shc Nck Grb2 Crk

From Fundamental Immunology 4th Ed, Paul WE Ed., pp 425

TABLE 3. Some of the proteins that are tyrosine phosphorylated following TCR stimulation.

TCR subunits CD3 , , ,

Protein tyrosine kinases ltk Lyn Lck MAPKs Pyk2 Syk ZAP-70Proteins with enzymatic function Phospholipase C 1 (PLC 1)Others Cbl CD5 CD6 Ezrin LAT Shc Slap-130 Slp-76 Shc Tubulin Valosin containing protein Vav

From Fundamental Immunology 4th Ed, Paul WE Ed., pp 427

+ -

- +

CD3

T Cell Receptoractivation (cont)

CD3

-

ACTIVATION!

ZAP70

Lck

CD4

LAT

PlcPIP2

IP3

DAG

Raf-1

MEK

MAPKImmediate early genes

Jun/Fos

Ca++

Endoplasmic reticulum

IP3 receptor

[Ca++]i

Ca++

CRAC

Ca++

Ca++

Ca++

Ca++

Ca++

calcineurin

PO4

NFAT NFAT

IL-2 gene

NFAT Fos/Jun

nucleus

PKC

RasGRP RAS

GTP GDP

From Fundamental Immunology 4th Ed, Paul WE Ed., pp 428

+ -

- +

CD3

T Cell Receptoractivation (cont)

CD3

-

ACTIVATION!

ZAP70

Lck

CD4

LAT

nucleus

Gads

Slp-76

SLAP130

vavrho/racNck

PakWASP

Actin/cytoskeletalreorganizationAdhesion

migration

Grb2sos

RasGDP

GAPs

RasGTP

Ras effectors

+ -

- +

CD3

T Cell Receptoractivation (cont)

CD3

-

ACTIVATION!

Lck

CD4

LAT

nucleus

CD28

“Costimulatory” B7 molecules

on antigen presenting cells

B7.1or B7.2

?PI3K p85

PIP3

PI3K

PIP2

Itk

ACTIVATION!

Antigen presenting cell

PKB

P

PI3KRemoved by SHIP--1

Where does cell type specificity come from?

• Preexisting substrate differences.•Specificity of src and ZAP70/syk kinases•PKC isoform expression•PLC isoform expression•NFAT isoform expression•Transcription factor isoform expression•Inhibitory receptor expression•etc….

1,2Ig-like extracellular domains

NKp46NKp30FcRIII

+ -

in

out

“Antigen” recognition

Signal transduction(with ITAMs)

Activating Receptors (on Monocytes, Macrophages, Neutrophils, Mast cells, and NK cells)

2-4Ig-like

extracellular domains

FcRIFcRIIa*

FcRIIIFcRIILT1ILT7ILT8

LIR6a

Fc commonchain

+ -

CD3 Fc

chain

1,2-?Ig-like

extracellular domains

FcRIIIFcRI

1-3Ig-like

extracellular domains

NK activatory receptors

FcRI

DAP12

Type 2 membrane

proteinNKp80

in

out

Activating receptor

+ -

Activatingligand

Inhibitoryligand

ITIMs

Motif: I/VxYxxL/V

Src-family tyrosine kinase reversibly associated with receptor

ITAMs

Tyrosinephosphatasee.g., SHP-1

Tyrosinekinase

e.g., ZAP-70,syk

SHIPlipid phosphatase

+ -+

Weak signal no response

nucleus nucleus

Normal cell Defective cell

“Natural killer”cell recognition

“Missingself”

Initiation of killing program

MHCclass I

CD28

nucleus nucleus

APC

+

T celldown-

regulationlate in

response

CTLA4 signalingturns response off

TCR CD28

MHC/peptide

++

Ag signal +costimulation

T cell activationCTLA4

sequestered

- CTLA4on surface

B7

Late in the response...

B7

APC

in

out

“Antigen” recognition

Signal transduction(with ITIMs)

Inhibitory Receptors (on Monocytes, Macrophages, Neutrophils, Mast cells, and NK cells)

1-6Ig-like extracellular domains

ILT5LIR8ILT4

Siglec 6CD33CD22

FcgRIIbPIRBSIRPsLAIR1ILT3LIR5PD-1

KIR2DLKIR3DLCTLA4

Type 2 membrane proteins

Ly49CD94/NKG2ACD72

Immunoprecipitation

Detergent Anti-Y-PO4

precipitate antibodies with

S. aureus protein A-coupled resin,

wash, elute

Fractionate by electrophoresis on

denaturing polyacrilamide gel

-

+

sample

Mobilityinversely

proportional to the Log of

molecular weight

markerscontrol

Generation of gene knockouts in mice:an important technique in establishingthe role of signaling molecules in normalcells.

Adapted from Molecular Cell Biology 3rd Ed., p294-5

Adapted from Molecular Cell Biology 3rd Ed., p296

Yeast two hybrid assay is one way

that protein: protein interactions can be measure and is an important way that new molecules are

discovered.

Adapted from Molecular Cell Biology 3rd Ed., p896