Overview

1
Overview

• Events controlled by signaling
• development, activation, homing, death
• Antigens, cytokines, chemokines, etc.
• Antigen receptors
• associated chains, ITAMs, tyrosine kinases,
  adaptors, downstream effectors (more ubiquitous)
• Spatial organization lipid rafts, immunol.
  synapse (SMAC)
• Other receptors TNFrs, chemokines, TLRs

2
Signal Transduction Pathways Relay Information
from the Cell Surface to the Nucleus
3
Reversible Protein Phosphorylation is a general
mechanism for transmitting signals from the cell
membrane
P
ATP
ADP
Kinase
Protein
Phosphatase
Pi
Major types of phosphorylation Tyrosine and
serine/threonine
4
Specialized domains mediate protein-protein
interactions during signal transduction
SH2
P
Tyr
SH3
Poly-proline sequence
5
Crosslinking and receptor tyrosine kinases
6
Surface Ig Crosslinking --gt B Cell Activation
Similarly, TCR crosslinking activates T cells
7
TCR/CD3/? Complex and Surface Ig with Ig? and
Ig?
These are relatively stable complexes, and can
isolated intact by immuno-precipitation, when
mild detergents are used
Assembly of the entire complex is necessary for
efficient expression at the cell surface
8
Irving and Weiss expt. demonstrating that z
crosslinking is sufficient for T cell activation
Cell (1991) 64 891.
CD8a ecto t.m.
transfect into CD8 neg. T cell line
X-link with anti-CD8 Ab
z intracellular
IL-2
Similar results seen by others with chimeras
containing intracellular domain of CD3? or Ig?
or Ig?
9
TCR-Responsive Promoters Models for studying
signaling pathways from the TCR to the nucleus
Ras/ MAP kinase
PKC IKK/IkB
Calcium
IL-2
CD28- RE
AP-1
NFAT
AP-1
NFAT
NF-?B
10
How do TCR and surface Ig access intracellular
signaling pathways?

• Unlike RTKs, no intrinsic catalytic activity
• Conserved motif with 2 tyrosines in ?, CD3 chains
  and Ig-a and Ig-b proteins
• First recognized by Michael Reth
• Now called an ITAM
• immuno-receptor tyrosine-based activation motif

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TCR- and Surface Ig-Associated Proteins
Immuno-receptor tyrosine-based activation motif
(ITAM) D/E X X Y X X L X7
Y X X L
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Src vs. Syk Family Tyrosine Kinases
Src Yes Fgr Hck Lyn Blk Fyn Lck
Syk - B cells, early T cell development, NK
cells, platelets, monocyte lineage ZAP-70 -
Throughout T cell development, NK cells
B cells
T cells
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Early events in signaling through the TCR
- Sequential involvement of src and Syk family
src family tyrosine kinases
Syk/ZAP-70 family tyrosine kinase
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Sequential tyrosine kinase activation in B cells
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Multi-domain adaptors nucleate signaling complexes
SH2 (src-homology 2) phospho-tyrosine SH3
proline-rich sequences Different SH2 and SH3
domains have different specificities
16
Cytoplasmic adaptors of lymphocytes
17
Transmembrane adaptors are also intermediates in
non-receptor tyrosine kinase systems
18
Transmembrane lymphocyte adaptors
LAB/NTAL B cells
19
Nucleation of signaling complex at LAT
Formation of this complex results in stable
membrane association of PLC-?1, activation of
which is critical for generation of second
messengers
20
Second messengers relay signals from receptors at
the plasma membrane
DAG and IP3 are second messengers
21
Small G protein activation is aided by Guanine
nucleotide Exchange Factors
22
Ras activation is also aided by aguanine
nucleotide releasing protein (GRP)
GRP
GEF
23
Small G proteins activate MAPK Cascades
MAPK mitogen-activated protein kinase
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Three major transcription factors downstream of
the TCR
PIP2
25
1.Tyrosine phosphorylation 2. Second
messengers 3. Downstream kinases 4.
Transcription factors
1
3
2
IP3
second messengers
AP-1 activation
transcription factors
NF-kB activation
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Ways to study the contributions of signaling
proteins to T cell development and activation

• Generate mouse knockout
• Germ-line tissue-specific inducible
• Generate mutant cell line
• Chemical or radiation mutagenesis
• RNAi - oligos or plasmid-based - variable
• Each approach has advantages and drawbacks

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Checkpoints in Thymic T Cell Development
(Pre-TCR)
pre-T alpha
very low/
28
Effect of SLP-76 deficiency on T cell development
LAT k.o. looks identical
29
Signaling defects in SLP-76-deficient Jurkat T
cells
Ras/MAPK IP3/Ca2
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Some Remaining Big-Picture Questions

• Exactly how does crosslinking initiate the whole
  signaling cascade?
• Is crosslinking/dimerization actually sufficient?
• Any role for conformational change?
• Maybe a little of both
• Changes in local concentration of ITAMs,
  sub-cellular localization (i.e. access to kinases
  and/or lack of access to phosphatases ?

31
Lipid rafts and Ig/TCR signaling

• Distribution of lipids in p.m. not uniform
• High concentration of sphingolipids and
  cholesterol in mobile rafts
• Singer/Nicholson fluid mosaic model
• Proteins with certain lipid modifications
  partition preferentially to lipid rafts
• including some signaling molecules (lck, LAT)
• Chemical disruption of rafts prevents activation
• May be important for initiation of signaling

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MIRR multi-chain immune recognition receptor
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SMAC or Immunologic Synapse
SMAC supra-molecular activation cluster

• Kupfer - deconvolution IF microscopy of T
  cell/APC interactions - --gt organized,
  bulls-eye type structure

c-SMAC
p-SMAC
LFA-1/Talin, CD45
c-SMAC
TCR/CD3, CD28, PKC q, lck
- Non-activating, altered, peptides do not
support the formation of these structures -
Precise functional role still controversial
34
SMAC or Immunologic Synapse
LFA-1
TCR
APC
T cell
LFA-1 an integrin binds ICAM on APC
35
Negative regulation and down-regulation of TCR
and BCR signaling

• Internalization trafficking to
  endosomes/lysosomes
• role for the immune synapse in this process?
• Ubiquitination --gt proteosomal degradation of
  receptor
• Cbl and other adaptors
• Phosphatases - remove tyrosines, etc.
• SHP-1 and others

36
Other types of receptors in lymphocyte biology

• G-protein coupled receptors (GPCR)
• e.g. chemokine receptors
• Jak/Stat signaling
• cytokine receptors
• Toll-like receptors
• TNF receptor family
• TNFr, CD40, Fas (co-stimulation death)

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GPCR Signaling
DAG
PI4,5P2
PLCb
ATP
Ca2
(CREB, etc.)
38
Some Toll-Like receptors and their ligands
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Major Signaling PathwaysActivated by TLRs and
IL-1R
MyD88-Independent Pathway
MAPKs
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Cytokine Receptor Signaling
which
bind
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Specificity in Cytokine Receptor Signaling
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TNFr family - death inducers
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TNFr family - co-stimulators
ligands
co-stimulators
44
TNFr family - proximal signaling

• Downstream pathways
• MAP Kinases AP-1
• IKKs NF-?B
• anti-apoptotic
• Caspases (apoptosis)