Outline
Control of src kinase activity by Csk, CD45
Control of CD45 activity by dimerization Evidence for TCR/CD3 conformational change zeta, CD3 epsilon; TCR ?
Lipid rafts and T cell activation Microclusters vs. immune synapse or SMAC Partial activation and antagonism
Proximal TCR signaling
1
2
3 3
The yin and yang of src kinase regulation
SH3 SH2 Y Kinase
P
Y
SH3 SH2 Y Kinase
Csk
Partially Active
Fully Active
Transition
Inactive
SH3 SH2 Y Kinase
P
Y
SH3 SH2
P
Y
Y Kina
seCD45
P
Y
394 in Lck
505 in Lck
Control of Lckby CD45 and Csk
Lipid Raft
CD45 isoforms and phosphatase activity CD45 is a transmembrane
phosphatase
Dimerization appears to inhibit activity
There are different size isoforms, created by alternative splicing of exons in the ecto domain
These isoforms have different quantities of glycosylation, which appears to affect the degree of homo-dimerization
Issue of potential ligand(s) is controversial
CD45 isoform expression on B and T cells
Model: CD45 isoforms and control of lymphocyte activation
clustering can be sufficient for T cell activation
Initiation of Native TCR Signaling:Clustering and/or Conformation?
CD8 ecto+ t.m.
intracellular
transfect intoCD8 neg. T cell line
X-link withanti-CD8 Ab
IL-2
Models of TCR/CD3 Distribution - Resting State
from: Alarcon et al., EMBO Reports 7:490
Pre-clustering of
TCR/CD3 increases
sensitivity to natural
peptide/MHC ligands
from: Alarcon et al., EMBO Reports 7:490
note: contribution of self
peptides to activation
What about a conformational change?
Evidence from GPCR’sfor conformationalchange transmitted
through transmembranedomains to cytoplasm
Structural evidence so far has not demonstrated such
a change in TCR itself
But, several indirect lines of evidence for
inducible change in CD3and/or zeta
1. Evidence for zeta conform. change
zeta assumes folded conformation in the presence of acidic phospholipids at P.M.
phosphorylation frees zeta to assume less-structured conform., which may facilitate its interaction w/ effectorsfrom: Aivazian and Stern, Nat. Struct. Biol. 2000
Nck
JNK (MAPK) Activation --> AP-1 transcription
2. Indirect Evidence for Conformational Change in CD3 to allow binding of Nck
from: Gil et al., Cell 109:901
Expt. setup: GST fusion with Nck SH3 domain
Pull-downs from lysates of T cells (unstim. or stimulated w/different ab’s - APA 1/1 or APA 1/2)
Blot for CD3
--> Either Ab 1/1 or 1/2 can crosslink TCR, so difference may be in ability to induce a CD3 conformational change, since a Fab fragment of APA 1/2 can also induce Nck assocation
from: Gil et al., Cell 109:901
Model from Gil et al. paper
Nck SH3 domain::CD3 proline-rich
from: Minguet et al., Immunity 26:43
3. Crosslinking the TCR With a Modified Chain
Thus: the conformational
change in CD3 that allows Nck binding can be induced by clustering TCR/CD3 complexes that are
close to one another
(apparently w/out a change in TCR
conformation
Antigens for stimulation NIP: hapten
…Full T cell activation requires both the conformational change and clustering
from: Minguet et al., Immunity 26:43
conform.change
clustering(distant)
Both
New model: conformation and clustering
from: Minguet et al., Immunity 26:43
pre-formed clusters
Lipid rafts
Distribution of lipids in p.m. not uniform
High concentration of sphingolipids and cholesterol in ‘rafts’ - regions of reduced mobility
Proteins with certain lipid modifications partition preferentially to lipid rafts
Lipid modifications of proteins (acylation)
palmitoylation, myristoylation and prenylation
double acylation (e.g. myrist+palmit or palmit+palmit) leads to lipid raft localization
some src family kinases: myristoylated and palmitoylated
LAT: double palmitoylated
Ras: palmitylated and prenylated
Lipid rafts and TCR signaling
Isolation/visualization of lipid rafts
Lipid rafts
Lck-GFP
Gradient centrifugation
Fluorescent microscopy
CT-B-Rhod. Overlay
CT-B = CholeraToxin B subunit
Some Signaling Proteins are Recruited into Rafts
C: cytoplasmM: membraneD: detergent-insoluble (rafts)
from: Xavier et al., Immunity 8:723
western blot for tyrosine phosphorylation
anti-CD3 Ab
large increase in tyrosinephosphorylation in lipid raftsand recruitment of proteins
to lipid rafts
LAT palmitylation is required for TCR signaling
from: Lin et al., J Biol Chem 274:28861
Experiment conducted inLAT-deficient T cell line
no lipid raft localization
Immune Synapse (or SMAC) Model
of Monks and Kupfer
SMAC = supra-molecular activation cluser
Immunological Synapse and SMAC
ICAM (LFA-1)
MHC/peptide (TCR)
LFA-1 PKC
Live T cells on lipid bi-layersFixed T cell:APC conjugates
from: Grakoui et al., Science 285:221 from: Monks et al., Nature 395:82
But…Signaling can occur in the first few minutes of T cell:APC contact (no mature synapse)
from: Lee et al., Science 295:1539
CD3clustersphospho-ZAP-70CD3pZAP-70
Current Model:
Initial signaling occurs in ‘microclusters’, which eventually fuse to form the mature immune synapse
Possible functions for the immune synapse
Questions about the immunological synapse/SMAC
Is it actually required for early signaling events or more important for later activation? What kind of structures are associated with early signaling?
Is it required for down-regulation of signaling? Internalization of TCR
Recruitment of phosphatases
What cell biological and signaling processes control its formation?
Altered peptide ligands (APL)
Analogs of antigenic peptides
Usually single amino acid change (TCR contact residue)
Antagonists: can inhibit the effects of antigenic peptide when mixed
Partial agonists: stimulate subset of T cell responses e.g. cytokine release but not proliferation
What properties determine differences?
How does the TCR discriminate between different ligands?
Immunogenic vs. altered peptides
Kd
On-rateOff-rateStructural changes
CD3,zeta phosphor.Signaling pathwaysTranscription factors