1. general themes of transmembrane signaling - clustering and phosphorylation - receptor protein...
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1. General themes of transmembrane signaling- clustering and phosphorylation-receptor protein tyrosine kinases and receptor-assoicated protein tyrosine kinases
2. Signaling moleculesadaptor proteins, GEF and small G proteins, PLC-,
3. TCR and BCR structure
4. One step before activation
5. BCR signaling
6. TCR signaling
General Principles of Transmembrane Signaling
1. Binding of antigen leads to clustering of antigen receptors on lymphocytes
2. Clustering of antigen receptors leads to activation of intracellular signal molecules
3. Phosphorylation of receptor cytoplasmic tails by tyrosine kinases concentratesintracellular signaling molecules around the receptors
4. intracellular signaling components recruited to activated receptors transmitthe signal onward from the membrane and amplify it
Cross-Linking of antigen receptors is the first step in lymphocyte
activation
These enzyme domains are normally inactive, but when brought together by receptor clustering they are able to activate each other by trans-phosphorylation. Once activated, these tyrosine kinases can phosphorylate and activate other cytoplasmic signaling molecules.
Receptor Protein-Tyrosine Kinases Receptor with Intrinsic Tyros
ine Kinase Activity
insulin receptorEGF receptor
Receptor Protein-Tyrosine Kinases
Dimerization and Autophosphorylation of Receptor Protein-Tyrosine Kinases
ligand induced dimerizationautophosphorylation by cross-phosphorylation
Association of Downstream Signaling Molecules with Receptor Protein-
Tyrosine
Adaptor Proteins
Adaptor proteins are specialized signaling molecules that usually have no enzymatic activity themselves.
Adaptor Proteins
Adaptor Proteins
Small G proteins are switched from inactive to active states by Guanine-nucleotide Exchange Factors (SOS, Vav).
Most of the time, Ras is in the inactive state owing to its intrinsic GTPase activity
epinephrine = adrenaline
-adrenergic receptor
Gs: GTP-binding stimulatory G protein
Small G Proteins Are Different from GTP-binding StimulatoryG proteins
phospholipase C-
PLC- has two SH2 domains and phosphorylation of a tyrosine residuein PLC- activates it.
Ca2+-binding protein calmodulinNF-AT
(nuclear factor of activated T cells)
phosphorylation of the tyrosines in ITAMs serves as the first intracellular signal indicating that the lymphocyte has detected its specific antigen.
YXX[L/V]X6-9YXX[L/V]
Receptor without Intrinsic Tyrosine Kinase ActivityBCR TCR
JAKSrc
Cytokine Receptors
43 kDa
12 kDa
34 kDa29 kDa
co-stimulatory signal
TCR triggering and co-stimulatory signal must be delivered by the same APC.
CD4 binds MHC class II molecules at a site on the 2 domain through a region that lies mainly on a lateral face of the first domain (D1)
1. Cytoplasmic domain of the TCR heterodimer is 5-12 a.a. long2. The signal transduction function is carried out by a CD3 complex3. CD3 complex composed of CD3, CD3, CD3, and CD3
Receptor without Intrinsic Tyrosine Kinase Activity
receptor-associated tyrosine kinase:The antigen receptor of lymphocytes are associated with receptor-associated tyrosine kinase, mainly of the Src family, which bind to receptor tails via their SH2 domain
TCR BCR
Annu.Rev.Cell Dev.Biol.13:513.1997
為什麼 Signal Transduction 總是圍繞在 Phosphorylation?
Annu.Rev.Cell Dev.Biol.13:513.1997
Clinica Immunology and Immunopathology 83(3):205. 1997
Regulation of Src-Family Kinase Activity
SH2 SH2
Ig Ig
Blk, Fyn, or Lyn
Full phosphorylation of the ITAMs on clustered Ig or Ig chains
creates binding sites for Syk
Ig Ig
B-cell co-receptor is a complex of three proteins CD19, CD21, and CD81.
1. CD19 is expressed on all B cells from an early stage in their development,before CD21 and CD81 are expressed, and it appears to contribute to signaling through the B-cell receptor even in the absence of co-ligation through CD21.
2. CD19-/- mice
B cells from mice that lack CD19 fail to proliferate in response to B-cell receptor cross-linking and do not fully activate the intracellular signaling pathways normally generated when the B-cell receptor is cross-linked.
3. These experiments suggest that CD19 can associate with the B-cell receptor, either constitutively or after receptor activation, and contribute to signaling even when the co-receptor has not been engaged through CD21 binding to complement.
Vav
guanine-nucleotide exchange factor
Receptor crosslinking activates Blk, Fyn, and Lyn
activated Blk, Fyn, and Lyn phosphorylate ITAMs
Activated Syk phosphorylates CD19, BLNK, PLC-, and GEFs (Vav)
PLC- cleavages PIP2 to yield DAG and IP3
GEF activates small G proteins- Rac and Ras
Syk binds to phosphorylated ITAM and becomes activated
Small G proteins, Ras and Rac, activate MAP kinase cascades
The Ras-induced kinase cascade induces and activates Fos
DAG and Ca2+ activate PKC
IP3 increases intracelluloar Ca2+ concentration activating a phosphotase calcinerrin
PKC activates NF-B
The NF-B, NFAT, and AP-1 act to induce specific gene transcription
Calcineurin activates NFAT (nuclear factor activated T cells)
Antigen receptor signaling is enhanced by co-receptors that bind the same ligand
Clinica Immunology and Immunopathology 83(3):205. 1997
Binding of SH2 (Src homology 2 domain) to phosphotyrosines is a crucial mechanism for recruiting intracellular signaling molecules to an activated receptor
SH3: binds to proline rich region
Annu.Rev.Immunol. 17:89. 1999
A Second Method Controlling the Activity of Src-Family Kinases
Annu. Rev. Immunol. 17:89.1999
Adapter Proteins Grb2, Linker of Activation in T Cells (LAT), and SH2-Domain
Leukocyte Protein of 76 kDa (SLP-76) in
Mediating Positive T Cell Receptor Signals
Phosphorylated SLP-76 recruits Vav
Phosphorylated LAT recruits Grb2 and PLC to membrance
Overexpression of SLP-76 augment ERK activation and AP-1 promoter activity, suggesting that SLP-76 impacts the Ras/ERK signaling pathway
Following TCR ligation, ZAP-70 is activated and phosphorylates LAT and SLP-76.
LAT LAT
SLP-76
Grb2
LAT may link TCR-stimulated PTKs with the phosphatidylinositol second messenger and/or Ras pathways
Shc itself is tyrosine phosphorylated and may interact with the SH2 domain of Grb2 to allow its translocation to the cell surface
recruitment of Grb2 along with Sos, results in activation of Ras
In T cells, the interaction of Shc with the tyrosine phosphorylated ITAMs of the TCR has been suggested to mediate the translocation of Grb2 to the plasma membrane
Clinica Immunology and Immunopathology 83(3):205. 1997
Shc
Clinica Immunology and Immunopathology 83(3):205. 1997
LAT
Overexpression of SLP-76 augment ERK activation and AP-1 promoter activity, suggesting that SLP-76 impacts the Ras/ERK signaling pathway
Immuol.Today 20:431.1999
cysteine residues that are palmitoylated and thus become associated with membrane lipid rafts
SLP-65 or BLNK in B-cell
Syk phosphorylates BLNK BLNKp recruits Tec Tec was phosphorylated by Src Tec phosphorylates PLC-
In B: shc-Grb2-SOS-Ras-Raf
In T: LAT-GADS-SOS-Ras-Raf
CD19 Vav-Rac
The human immunodeficiency disease X-linked agammaglobulinemia (XLA), in which B cells fail to mature, results from a mutation of Btk, while the same gene mutated in mice leads to a similar immunodeficiency called xid.
Fyn or Lck phosphorylate tyrosine residues on the CD3and , ITAMS, allowing ZAP-70 to bind
Fyn or Lck phosphorylate tyrosine residues onThe CD3and , ITAMS, allowing ZAP-70 to bind
Fyn or Lck protein tyrosine kiniase clustering activates kinase activity
Lck activated ZAP-70, which in turn phosphorylates LAT and SLP-76. SLP-76 binds and activates PLC-, GEFs, and Tec kinases
PLC- cleaves PIP2 to yield DAG and IP3
GEFs activate Ras which in turn activates a MAP kinase cascade
The Ras-induced kinase cascade induces and activates Fos, a component of the AP-1 transcription factor
IP3 increases intracellular Ca2+ concentration, activating a phosphotase, calcineurin
Calcineurin activates a transcription factor NFAT
DAG and Ca2+ activate PKC
PKC activates a transcription factor NFB
The transcription factors, NF-kB, NFAT, AP-1 act to induce specific gene transcription, leading to cell proliferation and differentiation.
Src Was Associated with Focal Adhesion
MAPK: mitogen-activated protein kinase
Raf (a serine/threonine kinase)
MEK Erk (extracellular-signal regulated kinase(phosphorylated at TEY)
MAP kinase cascades activate transcription factors
Initiation of MAP Kinase Cascadesin both Antigen Receptor and Co-stimulatory Signaling
Jnk: Jun N-terminal kinase
J. Immunol. 160:4182. 1998
Elkc-Jun
a serine/threonine protein phosphatase
cyclosporin A and FK506 Jun N-termianl kinase
Other receptors that pair with ITAM-containing chains can deliver activating
signals
KAR: killer activatory receptor
these receptors can activate NK cells to kill infected or abnormal target cells. The KARsignal through their associated ITAM-containing homodimer for the release of the cytotoxic granules by which NK cells kill their targets.
one ITAMYxx[L/V]xx6-9Yxx[L/V]
Some lymphocyte cell-surface receptors contain motifs
involved in downregulating activation.
ITIM: immunoreceptor tyrosine based inhibitory motif [I/V]xYxxL
1. It functions by recruiting one or other of the inhibitory phosphatases SHP-1, SHP-2 and SHIP.
2. SHIP is an inositol phosphatase and removes the 5’ phsophate from PI-3,4,5-P. it is thought to inhibit the activation of PLC- by inhibiting the recruitment of the Tec family of kinases, including Btk and Itk, and thus the production of DAG and IP3 and the associated mobilization of calcium
SHP-2
= IK+IK
SIIK (serine/threonine innate immunity kinase)= IRAK (IL-1R associated kinase)
The pahway that NF-B is activated by signals from TLR
Also activated is the gene for IB itself, which is rapidly synthesized and inactivates the NFB signal
1. fMLP receptor2. photoreceptor bacteriorhodopsin (the only solved structure of seven-transmembrance protein)3. receptor for anaphylotoxins4. chemokine receptors
large G protein = heterotrimeric G proteinsmall G protein
Seven-Transmembrane Receptor
Important targets for the activated G protein subunits are adenylate cyclase and phospholipase C, whose activation gives rise to the second messengers cyclic AMP, IP3 and Ca2+.
Subfamilies of Class I Cytokine Receptors Have Signaling Subunits in Common
Cytokine Receptors
JAK = Janus kinaseSTAT = signal transducers and activators of transcription
Signalling by the JAK/STAT pathway for a typical type I cytokine
Pathways for the induction of expression of IFN-
SOCS proteins are negative-feedback inhibitors of cytokine signal transduction
The cross-talk between cytokine-signalling pathways
Schematic representation of signalling pathways activated by class II cytokine receptor
s
Early death-inducing events after trophic-factor withdrawal