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How does receptor engagement lead to lymphocyte activation?. Y. ?. Basic signal transduction. -signals are transmitted through changes in specific activity or location of proteins

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How does receptor engagement lead to lymphocyte activation

Basic signal transduction

-signals are transmitted through changes in specific activity or location of proteins

-these changes in protein function are mediated by binding small molecules (e.g. Ca++) or by covalent modifications (e.g. phosphorylation), and also by protein-protein interactions


How does receptor engagement lead to lymphocyte activation

PO4-

Tyr

Pro

Pro

Pro

Interaction domains found in signaling proteins can modify function through changes in activity and/or localization.

Domain

Ligand

Phosphotyrosine

SH2

Proline-rich

sequences

SH3

Phosphatidylinositol

Triphosphate (PIP3)

PH


How does receptor engagement lead to lymphocyte activation

-P-P-P-

Three types of tyrosine kinases are involved in antigen receptor signaling. Each has a conserved catalytic domain and protein interaction domains

Src kinase family

(e.g. Lck, Fyn, Lyn, Blk)

Syk/ZAP-70 family

Tec kinase family

(e.g. Itk,Btk,Rlk)

SH3

SH2

Catalytic domain


How does receptor engagement lead to lymphocyte activation

Tyrosine kinase activity is regulated

by tyrosine phosphorylation and protein interaction domains

Lck

The Csk kinase phosphorylates

the inhibitory tyrosine and the

CD45 phosphatase can dephosphorylate this tyrosine.

Src kinases autophosphorylate the activating tyrosine.



How does receptor engagement lead to lymphocyte activation

How does a signal cross the cell membrane? structure of Src kinases

?


How does receptor engagement lead to lymphocyte activation

Receptor clustering can signal the presence of bound ligand by juxtaposition of the cytoplasmic domains of the receptor.

Association of kinase domains, present in many growth factor receptors, leads to kinase activation and the initiation of intracellular signaling.


Figure 6 3
Figure 6-3 by juxtaposition of the cytoplasmic domains of the receptor.

Aggregation of receptors may lead to their localization in membrane rafts where Src kinases can initiate signaling


How does receptor engagement lead to lymphocyte activation

In some cases, receptor activity directly modifies transcription factor function - cytokine receptor signaling to STAT transcription factors


How does receptor engagement lead to lymphocyte activation

MAP kinase signal transduction pathway: transcription factor function - cytokine receptor signaling to STAT transcription factors

Guanine nucleotide exchange factors (GEFs) activate small G-proteins to initiate a sequence of kinase phosphorylation events

which culminate in transcription factor activation


How does receptor engagement lead to lymphocyte activation

Phosphatidylinositol signaling pathway: Phospholipase C activity

generates second messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG) which increase levels of intracellular calcium and activate Protein kinase C (PKC).


How does receptor engagement lead to lymphocyte activation

Adaptor proteins have no catalytic activity, but can provide binding sites which bring together effector and target proteins to promote efficient coupling of receptors to intracellular signaling pathways



How does receptor engagement lead to lymphocyte activation

e receptors mediate signaling

e

g

d

z

z

Immunoreceptor Tyrosine-based activation motifs (ITAMs) are present in multiple copies in the cytoplasmic domains of antigen receptors

a b

hz1 Q L Y N E L N L G R R E E - Y D V L

hz2 G L Y N E L Q K D K M A E A Y S E I

hz3 G L Y Q G L S T A T K D T - Y D A L

hCD3g Q L Y Q P L K D R E D D Q - Y S H L

hCD3e P D Y E P I R K G Q R D L - Y S G L

hCD3d Q V Y Q P L R D R D D A Q - Y S H L

mIga N L Y E G L N L D D C S M - Y E D I

mIgb H T Y E G L N I D Q T A T - Y E D I


How does receptor engagement lead to lymphocyte activation

e receptors mediate signaling

e

g

d

z

z

z

z

ITAM motifs were shown to be sufficient for signaling when expressed as chimeras, independent of the receptor

CD8

a b


How does receptor engagement lead to lymphocyte activation

Receptor aggregation can initiate signaling receptors mediate signaling


How does receptor engagement lead to lymphocyte activation

Membrane mobility and cytoskeletal function allows TCR’s that bind MHC-peptide to cluster at the T cell-APC interface.


How does receptor engagement lead to lymphocyte activation

Src family kinases are responsible for the tyrosine phosphorylation of ITAM motifs in antigen receptors


How does receptor engagement lead to lymphocyte activation

-P phosphorylation of ITAM motifs in antigen receptors

e

e

e

e

d

d

g

g

z

z

z

z

P-

P-

P-

P-

Receptor phosphorylation leads to recruitment and activation of the ZAP-70 tyrosine kinase

a b

a b

Lck

ZAP-70


How does receptor engagement lead to lymphocyte activation

Y phosphorylation of ITAM motifs in antigen receptors

PTKs

Initiation of Antigen Receptor signaling depends upon non-receptor protein tyrosine kinases (PTKs)


How does receptor engagement lead to lymphocyte activation

Models of antigen receptor signal initiation phosphorylation of ITAM motifs in antigen receptors

Receptor-associated kinase aggregation leads to kinase activation and ITAM phosphorylation.

2. Receptor aggregation leads to co-localization with kinase and ITAM phosphorylation.


Potential mechanisms regulating the initiation of tcr signaling by src kinases

1. Receptor aggregation leads to co-localization of receptor and kinase.

2. Kinase phosphorylates receptor

1. Receptor aggregation leads to transphosphorylation and activation of receptor-associated kinase.

2. Kinase phosphorylates receptor

Potential mechanisms regulating the initiation of TCR signaling by Src kinases

Co-localization model

Activation model

-P

P-


How does receptor engagement lead to lymphocyte activation

CD4 and CD8 co-receptors bind to Lck and help initiate signaling by delivering the kinase to the cytoplasmic domains of the T cell receptor following binding to MHC-peptide


How does receptor engagement lead to lymphocyte activation

Y signaling by delivering the kinase to the cytoplasmic domains of the T cell receptor following binding to MHC-peptide

PTKs

?


How does receptor engagement lead to lymphocyte activation

Protein tyrosine kinases use adaptor proteins to link antigen receptors to intracellular phosphatidylinositol (PI) and MAP kinase pathways

Y

PTKs

adaptors

PI

pathway

MAP kinase

pathway


How does receptor engagement lead to lymphocyte activation
ZAP-70 phosphorylates two adaptor molecules: Linker for Activated T cells (LAT) and SH2 domain containing Leukocyte Protein (SLP-76)


How does receptor engagement lead to lymphocyte activation

R Activated T cells (LAT) and SH2 domain containing Leukocyte Protein (SLP-76)

a

s

Ras

G

T

P

GDP

Raf

Y-PO

4

SOS

MEK

ERK

The transmembrane adaptor LAT recruits SOS, a GTP exchange factor, to the membrane where it activates Ras and initiates the (ERK) MAP kinase pathway

GRB2

LAT

MEK


How does receptor engagement lead to lymphocyte activation

~P Activated T cells (LAT) and SH2 domain containing Leukocyte Protein (SLP-76)

~P

~P

The LAT and SLP-76 adaptors co-localize PLCg and the Tec family kinase ITK. Activation of PLCg by ITK induces cleavage of PIP2 to DAG and IP3.

DAG

PLCg1

IP3

ITK

LAT

P~

SLP-76


How does receptor engagement lead to lymphocyte activation

P~ Activated T cells (LAT) and SH2 domain containing Leukocyte Protein (SLP-76)

~P

P~

~P

LAT

LAT

SOS

SOS

GRB2

GRB2

GRB2

GRB2

Each SOS exchange factor can bind two Grb2 adaptor molecules

Houtman, et al. Nature Structural Biology 13: 798-805, 2006


How does receptor engagement lead to lymphocyte activation

Mutation of the tyrosine phosphorylation sites in the carboxy-terminus of LAT that mediate Grb2 binding prevents LAT clustering following TCR stimulation



How does receptor engagement lead to lymphocyte activation

Regulation of gene expression through increased transcription factor activity

NFAT: nuclear localization regulated by calcium-activated phosphatase

AP-1: expression and activity regulated by MAPK pathways

NFkB: nuclear localization regulated by degradation


How does receptor engagement lead to lymphocyte activation

The NFAT transcription factor is normally phosphorylated and sequestered in the cytosol. The PI pathway induces calcium influx which activates Calcineurin phosphatase activity and promotes nuclear translocation of the NFAT transcription factor.



How does receptor engagement lead to lymphocyte activation

MAP kinase pathways increase AP-1 dependent transcription by increasing expression of the Fos subunit, and by increasing activity of the Jun subunit

Fos

-P

-P


How does receptor engagement lead to lymphocyte activation

Stimulated antigen receptors utilize tyrosine kinases to induce intracellular signaling pathways leading to transcription factor activation and the induction of cell growth and differentiation.

Y

PTKs

adaptors

PI

pathway

MAP kinase

pathways

Interleukin-2, and

effector functions


How does receptor engagement lead to lymphocyte activation

The NF-kB transcription factor is sequestered in the cytosol by IkB which is phosphorylated and degraded in response to receptor signals

nucleus


How does receptor engagement lead to lymphocyte activation

Rawlings by IkB which is phosphorylated and degraded in response to receptor signalset al.Nature Reviews Immunology6, 799–812. 2006)

Antigen receptor activation of NF-kB depends upon assembly of a CARMA - BCL10 - MALT - TRAF complex that leads to the ubiquitylationof IKKassociation of TAK, and IKK phosphorylation