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