outline control of src kinase activity by csk, cd45

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 ?

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

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 may affect the degree of homo-dimerization

Issue of potential ligand(s) is controversial

CD45 isoforms

CD45 isoform expression on B and T cells

Summary: CD45 isoforms and control of lymphocyte activation

Initiation of TCR Signaling:

Clustering or Conformation?

Irving and Weiss expt. demonstrating that crosslinking can be sufficient for T cell activation

CD8 ectoand t.m.

intracellular

transfect intoT cell line

X-link withanti-CD8 Ab

IL-2, etc.

Models of TCR/CD3 Stoichiometry - Resting State

from: Alarcon et al., EMBO Reports 7:490

TCR/CD3 Pre-clustering increases

sensitivity to natural

peptide/MHC ligands

from: Alarcon et al., EMBO Reports 7:490

contribution of selfpeptides 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

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

2 - Indirect evidence for conformational change in CD3, leading to recruitment of adaptor protein Nck

JNK (MAPK) Activation --> AP-1 transcription

Inducible binding of Nck to CD3

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 can crosslink TCR, so difference may be in ability to induce a CD3 conformational change

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

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

(but w/out a change in TCR

conformation

Antigens for stimulation NIP: hapten

But…full T cell activation requires both the conformational change and clustering

from: Minguet et al., Immunity 26:43

conform.change

clustering(distant)

New model: conformation and clustering

from: Minguet et al., Immunity 26:43

pre-formed clusters

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?

Antigenic vs. altered peptides

Kd

On-rateOff-rateStructural changes

CD3,zeta phosphor.Signaling pathwaysTranscription factors

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 of lipid rafts

Lipid rafts

Work of Seed and colleagues linking lipid rafts to T cell activation: distribution +/- activation

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

Possible functions for the immune synapse

But…Signaling can occur in the first few minutes of T cell:APC contact (no mature synapse)

from: Lee et al., Science 295:1539

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?