Transcript Lecture 6 - The Scripps Research Institute

Antigen
recognition
out
+ in
Lipid bilayer
of plasma membrane
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
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.
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
Fyn
Yes
Lyn
Hck
Fgr
Blk
Lck
Ubiquitous; two neuron-specific isoforms
Ubiquitous; T cell-specific isoform (Fyn T)
Ubiquitous
Brain, B-cells, myeloid cells; two alternatively spliced forms
Myeloid cells (two different translational starts)
Myeloid cells, B-cells
B-cells
T-cells, NK cells, brain
Frk subfamily
Primarily epithelial cells
T Cell Receptor
a
b
CD3
Antigen
d e ge
recognition
out
-+ +in
CD3 z
Signal
transduction
(with ITAMs)
-
CD3 functions both as signal
transducers and in transport
of antigen receptors to the
plasma membrane
B Cell
Receptor
Ig
a b
Antigen
recognition
out
+
in
+
Signal
transduction
(with ITAMs)
-
-
+
+
-
-
syk
Lyn
Blk
Hck
Fyn
Lck
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.
T Cell Receptor
And coreceptor
a
b
CD4
CD3
d e ge
Cholesterol and sphingolipid
rich microdomain
out
- -+ +-
in
Signal
transduction
(with ITAMs)
Fyn
Lck
CD3 z
T Cell Receptor
a
b
CD4
CD3
d e ge
- -+ +ZAP70
ACTIVATION!
CD3 z
Lck
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 g1 (PLC g1)
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
TABLE 3. Some of the proteins that are tyrosine phosphorylated
following TCR stimulation.
TCR subunits
CD3 d, e, g , z
Protein tyrosine kinases
ltk
Lyn
Lck
MAPKs
Pyk2
Syk
ZAP-70
Proteins with enzymatic function
Phospholipase C g1 (PLC g1)
Others
Cbl
CD5
CD6
Ezrin
LAT
Shc
Slap-130
Slp-76
Shc
a Tubulin
Valosin containing protein
Vav
From Fundamental Immunology 4th Ed, Paul WE Ed., pp 427
T Cell Receptor
activation (cont)
a
b
CD4
CD3
ge
d e
Ca++
LAT
CRAC
-
-+
+-
DAG RasGRP RAS
PIP2
ZAP70
Plcg
PKC
GTP
Lck
Ca++
Ca
Ca++
Ca++
Ca++
++
CD3 z
GDP
IP3
ACTIVATION!
Raf-1
[Ca++]i
calcineurin
IP3
receptor
Ca++
NFAT
NFAT
MEK
Endoplasmic reticulum
PO4
nucleus
NFAT
Fos/Jun
IL-2 gene
MAPK
Immediate early genes
Jun/Fos
From Fundamental Immunology 4th Ed, Paul WE Ed., pp 428
T Cell Receptor
activation (cont)
a
b
CD4
CD3
ge
d e
LAT
-
-+
+-
Grb2
ZAP70
sos
Lck
CD3 z
ACTIVATION!
GAPs
Gads
Slp-76
SLAP
130
vav
Nck rho/rac
Pak
WASP
Adhesion
migration
nucleus
Ras
GTP
Ras
GDP
Ras effectors
Actin/cytoskeletal
reorganization
Antigen presenting cell
B7.1
or B7.2
T Cell Receptor
activation (cont)
a
b
CD4
CD3
ge
d e
“Costimulatory” B7
molecules
on antigen presenting
cells
CD28
LAT
-
-+
+-
PI3K
Itk
PKB
Lck
ACTIVATION!
CD3 z
ACTIVATION!
nucleus
p85
?
PIP2
PIP3
PI3K
PI3K
Removed by SHIP--1
P
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….
Activating Receptors (on Monocytes, Macrophages, Neutrophils,
Mast cells, and NK cells)
2-4
Ig-like
extracellular
domains
“Antigen”
recognition
1,2
Ig-like
extracellular
domains
FcgRI
FcgRIIa*
FcgRIII
FcaRI
ILT1
ILT7
ILT8
LIR6a
NKp46
NKp30
FcRIII
1,2-?
Ig-like
extracellular
domains
FcgRIII
FceRI
1-3
Ig-like
extracellular
domains
NK activatory
receptors
FcaRI
Type 2
membrane
protein
NKp80
out
+-
+in
Signal
transduction
(with ITAMs)
Fc common
g chain
CD3
z
DAP12
b
Fc
g chain
Activating
ligand
Inhibitory
ligand
Activating
receptor
Motif: I/VxYxxL/V
out
+ in
ITAMs
SHIP
lipid phosphatase
Src-family tyrosine
Tyrosine
kinase reversibly
kinase
associated with
e.g., ZAP-70,
receptor
syk
ITIMs
Tyrosine
phosphatase
e.g., SHP-1
Normal cell
Defective cell
MHC
class I
“Missing
self”
“Natural killer”
cell recognition
+
-
+
Weak signal
no response
Initiation of killing program
nucleus
nucleus
APC
APC
Late in the response...
B7
MHC/peptide
CD28
TCR
+
+
B7
CD28
+
Ag signal +
costimulation
CTLA4
sequestered
T cell
downregulation
late in
response
T cell activation
nucleus
-
CTLA4
on surface
CTLA4 signaling
turns response off
nucleus
Inhibitory Receptors (on Monocytes, Macrophages, Neutrophils, Mast
cells, and NK cells)
1-6
Ig-like extracellular domains
ILT5
LIR8
ILT4
Siglec 6
“Antigen”
CD33
recognition
CD22
FcgRIIb
PIRB
SIRPs
LAIR1
ILT3
LIR5
PD-1
out
KIR2DL
KIR3DL
CTLA4
in
Signal
transduction
(with ITIMs)
Type 2
membrane
proteins
Ly49
CD94/
NKG2A
CD72
Immunoprecipitation
Detergent
Anti-Y-PO4
precipitate
antibodies with
S. aureus protein
A-coupled resin,
wash, elute
sample control markers
Mobility
inversely
proportional
to the Log of
molecular
weight
+
Fractionate by
electrophoresis on
denaturing
polyacrilamide gel
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
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