1. Overview Events controlled by signaling
Development, Activation, Homing, Death
Antigens, cytokines, chemokines, etc.
Antigen receptors:
Associated chains, ITAM’s, tyrosine kinases, adaptors, downstream effectors (more ubiquitous)
Spatial organization: lipid rafts, immunol. synapse (SMAC)
Other receptors: TNFr’s, chemokines, TLR’s

2. Signal Transduction Pathways Relay Information from the Cell Surface to the Nucleus

3. Major types of phosphorylation: Tyrosine and serine/threonine
Reversible Protein Phosphorylation is a general mechanism for transmitting signals from the cell membrane P
ATP
ADP
Kinase
Protein
Protein
Phosphatase Pi
Major types of phosphorylation:
Tyrosine and serine/threonine

4. Specialized domains mediate protein-protein interactions during signal transduction
SH2 P
Tyr
SH3
Poly-proline
sequence

5. Crosslinking and receptor tyrosine kinases

6. Surface Ig Crosslinking --> B Cell Activation
Similarly, TCR crosslinking activates T cells

7. TCR/CD3/ Complex and Surface Ig with Ig and Ig
These are relatively stable complexes, and can isolated intact
by immuno-precipitation, when mild detergents are used
Assembly of the entire complex is necessary
for efficient expression at the cell surface

8. Irving and Weiss expt. demonstrating that z crosslinking is sufficient for T cell activation
CD8a
ecto
+ t.m.
transfect into
CD8 neg. T cell line
X-link with
anti-CD8 Ab
z intracellular
IL-2
Similar results seen by others with chimeras containing
intracellular domain of CD3 or Ig or Ig

9. TCR-Responsive Promoters: Models for studying signaling pathways from the TCR to the nucleus
PKC
IKK/IkB
Ras/
MAP kinase
Calcium
IL-2
NFAT
CD28- RE
NF-B
AP-1
AP-1
NFAT

10. How do TCR and surface Ig access intracellular signaling pathways?
Unlike RTK’s, no intrinsic catalytic activity
Conserved motif with 2 tyrosines in , CD3 chains and Ig-a and Ig-b proteins
First recognized by Michael Reth
Now called an ITAM
Immuno-receptor Tyrosine-based Activation Motif
Also found in receptors on NK and monocytic cells

11. TCR- and Surface Ig-Associated Proteins
Immuno-receptor tyrosine-based activation motif (ITAM)
D/E X X Y X X L X7 Y X X L

12. Src vs. Syk Family Tyrosine Kinases
SH2
catalytic
Src
Yes
Fgr
Hck
Lyn
Blk
Fyn
Lck
Syk - B cells, early T cell
development, NK cells,
platelets, monocyte lineage
ZAP-70 - Throughout T
cell development, NK cells
B cells
T cells

13. Early events in signaling through the TCR
- Sequential involvement of src and Syk family
src family
tyrosine kinases
Syk/ZAP-70 family
tyrosine kinase

14. Sequential tyrosine kinase activation in B cells

15. Multi-domain adaptors nucleate signaling complexes
SH2 (src-homology 2) :: phospho-tyrosine
SH3 :: proline-rich sequences
Different SH2 and SH3 domains have different specificities

16. Cytoplasmic adaptors of lymphocytes

17. Transmembrane adaptors are also intermediates in non-receptor tyrosine kinase systems

18. Transmembrane lymphocyte adaptors
LAB/NTAL B cells

19. Nucleation of signaling complex at LAT
Formation of this
complex results in
stable membrane
association of PLC-1,
activation of which is
critical for generation
of second messengers

20. Second messengers relay signals from receptors at the plasma membrane
DAG and IP3 are second messengers

21. Small G protein activation is aided by Guanine nucleotide Exchange Factors

22. Ras activation is also aided by a guanine nucleotide releasing protein (GRP)
GEF
GRP

23. Small G proteins activate MAPK Cascades
MAPK = mitogen-activated protein kinase

24. Three major transcription factors downstream of the TCR
PIP2

25. 1.Tyrosine phosphorylation 2. “Second messengers”
3. Downstream kinases
4. Transcription factors 1 3 2
IP3
second
messengers
AP-1
activation
transcription
factors
NF-kB
activation

26. Ways to study the contributions of signaling proteins to T cell development and activation
Generate mouse knockout
Germ-line; tissue-specific; inducible
Generate mutant cell line
Chemical or radiation mutagenesis
RNAi - oligos or plasmid-based - variable
Each approach has advantages and drawbacks

27. Checkpoints in Thymic T Cell Development
(Pre-TCR)
+ pre-T alpha
very low/

28. Effect of SLP-76 deficiency on T cell development
LAT k.o. looks identical

29. Signaling defects in SLP-76-deficient Jurkat T cells
Ras/MAPK
IP3/Ca2+

30. Some Remaining Big-Picture Questions
Exactly how does crosslinking initiate the whole signaling cascade?
Is crosslinking/dimerization actually sufficient?
Any role for conformational change?
Maybe a little of both…
Changes in local concentration of ITAMs, sub-cellular localization (i.e. access to kinases and/or lack of access to phosphatases ?

31. Lipid rafts and Ig/TCR signaling
Distribution of lipids in p.m. not uniform
High concentration of sphingolipids and cholesterol in mobile ‘rafts’
Singer/Nicholson fluid mosaic model…
Proteins with certain lipid modifications partition preferentially to lipid rafts
including some signaling molecules (lck, LAT)
Chemical disruption of rafts prevents activation
May be important for initiation of signaling

32. MIRR = multi-chain immune recognition receptor

33. SMAC or Immunologic Synapse
SMAC = supra-molecular activation cluster
Kupfer - deconvolution IF microscopy of T cell/APC interactions - --> organized, bulls-eye type structure
c-SMAC
p-SMAC
LFA-1/Talin, CD45
c-SMAC
TCR/CD3, CD28, PKC q, lck
- Non-activating, altered, peptides do not support
the formation of these structures
- Precise functional role still controversial

34. SMAC or Immunologic Synapse
LFA-1
TCR
APC
T cell
LFA-1: an integrin; binds ICAM on APC

35. Negative regulation and down-regulation of TCR and BCR signaling
Internalization; trafficking to endosomes/lysosomes
role for the immune synapse in this process?
Ubiquitination --> proteosomal degradation of receptor
Cbl and other adaptors
Phosphatases - remove tyrosines, etc.
SHP-1 and others

36. Other types of receptors in lymphocyte biology
G-protein coupled receptors (GPCR)
e.g. chemokine receptors
Jak/Stat signaling
cytokine receptors
Toll-like receptors
TNF receptor family
TNFr, CD40, Fas (co-stimulation; death)

37. GPCR Signaling
DAG
PLCb
PI4,5P2
ATP
Ca2+
(CREB, etc.)

38. Some Toll-Like receptors and their ligands

39. Major Signaling Pathways Activated by TLR’s and IL-1R
MyD88-Independent Pathway
MAPKs

40. Cytokine Receptor Signaling
that
bind

41. Specificity in Cytokine
Receptor Signaling

42. TNFr family - death inducers

43. TNFr family - co-stimulators
ligands
co-stimulators

44. TNFr family - proximal signaling
Downstream pathways:
MAP Kinases + AP-1
IKK’s + NF-κB
anti-apoptotic
Caspases (apoptosis)