Outline

1
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 a/b?
• Lipid rafts and T cell activation
• Microclusters vs. immune synapse or SMAC
• Partial activation and antagonism

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Proximal TCR signaling
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2
3
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The yin and yang of src kinase regulation
Partially Active Fully Active Transition
Inactive
394 in Lck
Csk
P
Y
505 in Lck
CD45
Y
Y Kinase
P
SH3
SH2
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Control of Lckby CD45 and Csk
Lipid Raft
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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

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CD45 isoform expression on B and T cells
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Model CD45 isoforms and control of lymphocyte
activation
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z clustering can be sufficient for T cell
activation
Initiation of Native TCR SignalingClustering
and/or Conformation?
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Models of TCR/CD3 Distribution - Resting State
from Alarcon et al., EMBO Reports 7490
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Pre-clustering ofTCR/CD3 increases sensitivity
to natural peptide/MHC ligands
note contribution of self peptides to activation
from Alarcon et al., EMBO Reports 7490
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What about a conformational change?
Evidence from GPCRs for conformational change
transmitted through transmembrane domains 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 CD3 and/or zeta
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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/ effectors

from Aivazian and Stern, Nat. Struct. Biol. 2000
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Nck
JNK (MAPK) Activation --gt AP-1 transcription
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2. Indirect Evidence for Conformational Change in
CD3? to allow binding of Nck

• Expt. setup GST fusion with Nck SH3 domain
• Pull-downs from lysates of T cells (unstim. or
  stimulated w/different abs - APA 1/1 or APA 1/2)
• Blot for CD3 e

--gt 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 109901
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Model from Gil et al. paper
Nck SH3 domain CD3 e proline-rich
from Gil et al., Cell 109901
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3. Crosslinking the TCR With a Modified ? Chain
Antigens for stimulation
NIP hapten
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
from Minguet et al., Immunity 2643
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Full T cell activation requires both the
conformational change and clustering
conform. change
Both
clustering (distant)
from Minguet et al., Immunity 2643
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New model conformation and clustering
pre-formed clusters
from Minguet et al., Immunity 2643
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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

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Lipid modifications of proteins (acylation)

• palmitoylation, myristoylation and prenylation
• double acylation (e.g. myristpalmit or
  palmitpalmit) leads to lipid raft localization
• some src family kinases myristoylated and
  palmitoylated
• LAT double palmitoylated
• Ras palmitylated and prenylated

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Lipid rafts and TCR signaling
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Isolation/visualization of lipid rafts
Gradient centrifugation
Fluorescent microscopy
Overlay
Lck-GFP
CT-B-Rhod.
CT-B Cholera Toxin B subunit
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Some Signaling Proteins are Recruited into Rafts
anti-CD3? Ab
large increase in tyrosine phosphorylation in
lipid rafts and recruitment of proteins to lipid
rafts
western blot for tyrosine phosphorylation
C cytoplasm M membrane D detergent-insoluble
(rafts)
from Xavier et al., Immunity 8723
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LAT palmitylation is required for TCR signaling
Experiment conducted in LAT-deficient T cell line
no lipid raft localization
from Lin et al., J Biol Chem 27428861
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Immune Synapse (or SMAC) Model of Monks and
Kupfer
SMAC supra-molecular activation
cluser
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Immunological Synapse and SMAC
Live T cells on lipid bi-layers
Fixed T cellAPC conjugates
LFA-1
PKC q
ICAM (LFA-1)
MHC/peptide (TCR)
from Monks et al., Nature 39582
from Grakoui et al., Science 285221
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ButSignaling can occur in the first few minutes
of T cellAPC contact (no mature synapse)
from Lee et al., Science 2951539
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Current Model Initial signaling occurs in
microclusters, which eventually fuse to form
the mature immune synapse
CD3???clusters
phospho-ZAP-70
CD3??? pZAP-70
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Possible functions for the immune synapse
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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?

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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?

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Immunogenic vs. altered peptides
Kd On-rate Off-rate Structural changes
CD3,zeta phosphor. Signaling pathways Transcriptio
n factors