Consensus ITAM amino acid

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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
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Src-family tyrosine kinases
Myristylation/palmitylation sites present in many
family members, promotes association with
membranes and membrane rafts. (e.g.
CH3(CH)12CONH-Glycine..)
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.
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CD45 (plasma membrane tyrosine phosphatase)
csk (cytoplasmic tyrosine kinase)
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Adapted from Molecular Cell Biology 3rd Ed., p894
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Table 1. Expression of Src family kinases Src
Ubiquitous two neuron-specific isoforms Fyn
Ubiquitous T cell-specific isoform (Fyn T) Yes
Ubiquitous Lyn Brain, B-cells, myeloid cells
two alternatively spliced forms Hck Myeloid
cells (two different translational starts) Fgr
Myeloid cells, B-cells Blk B-cells Lck
T-cells, NK cells, brain Frk subfamily Primaril
y epithelial cells
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T Cell Receptor a b
CD3 functions both as signal transducers and in
transport of antigen receptors to the plasma
membrane
CD3 d e
Antigen recognition
g e
out
-
-
-
CD3 z
in
Signal transduction (with ITAMs)
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B Cell Receptor
Ig a b
Antigen recognition
out
Signal transduction (with ITAMs)
in
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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.
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T Cell Receptor And coreceptor
a b
out
-
-
in
Signal transduction (with ITAMs)
Fyn
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T Cell Receptor
a b
-
-
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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 ltk Protein tyrosine
phosphatases SHP-1 SHP-2 Proteins with
enzymatic functions Phospholipase C ?1 (PLC
?1) Phosphatidylinositol 3-kinase (P1
3-kinase), p85 subunit Adaptors and Regulators
GTPase acttivating protein (GAP) SOS Vav
Slp-76 Shc Nck Grb2 Crk
From Fundamental Immunology 4th Ed, Paul WE Ed.,
pp 425
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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-70 Proteins 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
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T Cell Receptor activation (cont)
a b
Ca
-
-
PIP2
RasGRP
Plcg
nucleus
IL-2 gene
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From Fundamental Immunology 4th Ed, Paul WE Ed.,
pp 428
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T Cell Receptor activation (cont)
a b
-
-
nucleus
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Antigen presenting cell
T Cell Receptor activation (cont)
a b
CD4
-
-
Lck
nucleus
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• Where does cell type specificity come from?
• Preexisting substrate differences.
• Specificity of src and ZAP70/syk kinases
• PKC isoform expression
• PLCg isoform expression
• NFAT isoform expression
• Transcription factor isoform expression
• Inhibitory receptor expression
• etc.

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Inhibitory ligand
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Normal cell
Defective cell
Missing self
Natural killer cell recognition

Weak signal no response
Initiation of killing program
nucleus
nucleus
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APC
MHC/peptide
CD28
TCR
CTLA4 signaling turns response off
CTLA4 sequestered
nucleus
nucleus
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Inhibitory Receptors (on Monocytes,
Macrophages, Neutrophils, Mast cells, and NK
cells)
1-6 Ig-like extracellular domains ILT5 LIR8 ILT4 S
iglec 6 CD33 CD22 FcgRIIb PIRB SIRPs LAIR1 ILT3 LI
R5 PD-1 KIR2DL KIR3DL CTLA4
Antigen recognition
out
in
Signal transduction (with ITIMs)
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Immunoprecipitation
Detergent
Anti-Y-PO4
precipitate antibodies with S. aureus protein
A-coupled resin, wash, elute
sample
markers
control
-
Fractionate by electrophoresis on denaturing
polyacrilamide gel
Mobility inversely proportional to the Log of
molecular weight

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Generation of gene knockouts in mice an
important technique in establishing the role of
signaling molecules in normal cells.
Adapted from Molecular Cell Biology 3rd Ed.,
p294-5
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Adapted from Molecular Cell Biology 3rd Ed., p296
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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