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1. General themes of transmembrane signaling - clustering and phosphorylation -  receptor protein tyrosine kinases  and  receptor-assoicated protein.

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Presentation on theme: "1. General themes of transmembrane signaling - clustering and phosphorylation -  receptor protein tyrosine kinases  and  receptor-assoicated protein."— Presentation transcript:

1 1. General themes of transmembrane signaling - clustering and phosphorylation -  receptor protein tyrosine kinases  and  receptor-assoicated protein tyrosine kinases  2. Signaling molecules adaptor proteins, GEF and small G proteins, PLC- , 3. TCR and BCR structure 4. One step before activation 5. BCR signaling 6. TCR signaling

2 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 concentrates intracellular signaling molecules around the receptors 4. intracellular signaling components recruited to activated receptors transmit the signal onward from the membrane and amplify it

3 Cross-Linking of antigen receptors is the first step in lymphocyte activation

4 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 Tyrosine Kinase Activity

5 insulin receptorEGF receptor Receptor Protein-Tyrosine Kinases

6 Dimerization and Autophosphorylation of Receptor Protein-Tyrosine Kinases ligand induced dimerization autophosphorylation by cross- phosphorylation

7 Association of Downstream Signaling Molecules with Receptor Protein-Tyrosine

8 Adaptor Proteins

9 Adaptor proteins are specialized signaling molecules that usually have no enzymatic activity themselves. Adaptor Proteins

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11 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

12 epinephrine = adrenaline  - adrenergic receptor Gs: GTP-binding stimulatory G protein Small G Proteins Are Different from GTP-binding Stimulatory G proteins

13 phospholipase C-  PLC-  has two SH2 domains and phosphorylation of a tyrosine residue in PLC-  activates it.

14 Ca 2+ -binding protein calmodulin  NF-AT (nuclear factor of activated T cells)

15 phosphorylation of the tyrosines in ITAMs serves as the first intracellular signal indicating that the lymphocyte has detected its specific antigen. YXX[L/V]X 6-9 YXX[L/V] Receptor without Intrinsic Tyrosine Kinase Activity BCR TCR

16 JAK Src Cytokine Receptors

17 43 kDa 12 kDa 34 kDa29 kDa

18 co-stimulatory signal TCR triggering and co- stimulatory signal must be delivered by the same APC.

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20 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)

21 1.Cytoplasmic domain of the TCR heterodimer is 5-12 a.a. long 2.The signal transduction function is carried out by a CD3 complex 3.CD3 complex composed of CD3 , CD3 , CD3 , and CD3 

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23 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

24 Annu.Rev.Cell Dev.Biol.13: 為什麼 Signal Transduction 總是圍繞在 Phosphorylation?

25 Annu.Rev.Cell Dev.Biol.13:

26 Clinica Immunology and Immunopathology 83(3): Regulation of Src-Family Kinase Activity

27 SH2 Ig  Ig  Blk, Fyn, or Lyn

28 Full phosphorylation of the ITAMs on clustered Ig  or Ig  chains creates binding sites for Syk Ig  Ig 

29 B-cell co-receptor is a complex of three proteins CD19, CD21, and CD 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 CD 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

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31 guanine- nucleotide exchange factor

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34 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 PIP 2 to yield DAG and IP 3 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 Ca 2+ activate PKC IP 3 increases intracelluloar Ca 2+ 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)

35 Antigen receptor signaling is enhanced by co- receptors that bind the same ligand

36 Clinica Immunology and Immunopathology 83(3): 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

37 Annu.Rev.Immunol. 17: A Second Method Controlling the Activity of Src-Family Kinases

38 Annu. Rev. Immunol. 17: 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 SLP-76 Grb2

39 LAT may link TCR-stimulated PTKs with the phosphatidylinositol second messenger and/or Ras pathways

40  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): Shc

41 Clinica Immunology and Immunopathology 83(3): LAT Overexpression of SLP-76 augment ERK activation and AP-1 promoter activity, suggesting that SLP-76 impacts the Ras/ERK signaling pathway

42 Immuol.Today 20: cysteine residues that are palmitoylated and thus become associated with membrane lipid rafts  SLP-65 or BLNK in B-cell

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44 Syk phosphorylates BLNK  BLNK p 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.

45 Fyn or Lck phosphorylate tyrosine residues on the CD3  and , ITAMS, allowing ZAP-70 to bind

46 Fyn or Lck phosphorylate tyrosine residues onThe CD3  and , 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 PIP 2 to yield DAG and IP 3 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 IP 3 increases intracellular Ca 2+ concentration, activating a phosphotase, calcineurin Calcineurin activates a transcription factor NFAT DAG and Ca 2+ activate PKC PKC activates a transcription factor NF  B The transcription factors, NF-kB, NFAT, AP-1 act to induce specific gene transcription, leading to cell proliferation and differentiation.

47 Src Was Associated with Focal Adhesion

48 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

49 Initiation of MAP Kinase Cascades in both Antigen Receptor and Co-stimulatory Signaling Jnk: Jun N-terminal kinase

50 J. Immunol. 160: Elk c-Jun

51 a serine/threonine protein phosphatase cyclosporin A and FK506 Jun N-termianl kinase

52 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 KAR signal through their associated ITAM-containing homodimer for the release of the cytotoxic granules by which NK cells kill their targets. one ITAM Yxx[L/V]xx 6-9 Yxx[L/V]

53 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

54 = I  K  +I  K  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 I  B itself, which is rapidly synthesized and inactivates the NF  B signal

55 1. fMLP receptor 2. photoreceptor bacteriorhodopsin (the only solved structure of seven-transmembrance protein) 3. receptor for anaphylotoxins 4. chemokine receptors large G protein = heterotrimeric G protein small 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 Ca 2+.

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62 Subfamilies of Class I Cytokine Receptors Have Signaling Subunits in Common

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66 Cytokine Receptors JAK = Janus kinase STAT = signal transducers and activators of transcription

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68 Signalling by the JAK/STAT pathway for a typical type I cytokine

69 Pathways for the induction of expression of IFN- 

70 SOCS proteins are negative-feedback inhibitors of cytokine signal transduction

71 The cross-talk between cytokine-signalling pathways

72 Schematic representation of signalling pathways activated by class II cytokine receptors

73 Early death-inducing events after trophic-factor withdrawal

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