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?
• Partial activation and antagonism
• Lipid rafts and T cell activation
• Immune synapse or SMAC

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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
4
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 may affect the degree of
  homo-dimerization
• Issue of potential ligand(s) is controversial

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CD45 isoforms
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CD45 isoform expression on B and T cells
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Summary CD45 isoforms and control of lymphocyte
activation
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Initiation of TCR SignalingClustering or
Conformation?
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Irving and Weiss expt. demonstrating that z
crosslinking can be sufficient for T cell
activation
CD8a ecto and t.m.
transfect into T cell line
X-link with anti-CD8 Ab
z intracellular
IL-2, etc.
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Models of TCR/CD3 Stoichiometry - Resting State
from Alarcon et al., EMBO Reports 7490
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TCR/CD3 Pre-clustering increases sensitivity to
natural peptide/MHC ligands
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
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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., whereupon it presumably
  interacts with effectors

from Aivazian and Stern, Nat. Struct. Biol. 2000
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2 - Indirect evidence for conformational change
in CD3?, leading to recruitment of adaptor
protein Nck
JNK (MAPK) Activation --gt AP-1 transcription
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Inducible binding of Nck to CD3 e

• 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 can crosslink TCR, so difference
may be in ability to induce a CD3
conformational change
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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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 (but w/out a change in TCR ???
conformation
from Minguet et al., Immunity 2643
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Butfull T cell activation requires both the
conformational change and clustering
conform. change
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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Altered peptide ligands (APL)

• analogs of antigenic peptides
• usually single amino acid change (TCR contact
  residue)
• antagonists can inhibit antigenic peptide when
  mixed
• partial agonists stimulate subset of T cell
  responses
• e.g. cytokine release but not proliferation
• what properties determine differences?

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Antigenic vs. altered peptides
Kd On-rate Off-rate Structural changes
CD3,zeta phosphor. Signaling pathways Transcriptio
n factors
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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 of lipid rafts
Lipid rafts
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Work of Seed and colleagues linking lipid rafts
to T cell activation distribution /- activation
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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Possible functions for the immune synapse
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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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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?