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  • Explain function of CD45 (dephosphorylation of C-terminal regulatory tyrosine of Src-kinases). Say that Src-kinases are important in initiating immunoreceptor signaling Stress that CD45 is thus positive regulator of immunoreceptor signaling Surprisingly, not much defect in B and M cells
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    1. 1. Microbiology 204 TCR Signal Transduction Art Weiss October 9, 2009
    2. 2. Naïve T cell Clonal T cell progeny with effector functions
    3. 3. Key Points: 1. T cell activation occurs during a complex cell-cell interaction 2. Stimulation of a G O T cell results in cell cycle progression as well as enormous metabolic and synthetic changes as well as cell enlargement 3. Activation is initiated by the TCR, but also requires multiple other receptors involved in intercellular interactions 4. Key event is the production of lymphokines and the cellular response to them 5. End result is the clonal expansion and acquisition of effector functions
    4. 4. Complexity of the T Cell : Antigen Presenting Cell Interaction
    5. 5. T Cell Target Cell MTOC Polymerized Actin T Cells Polarize During Antigen- Specific Recognition 2. MTOC and microtubules polarize 3. Actin cytoskeleton polymerizes and polarizes 4. Requires TCR signals: Rac/Cdc42 activation and Ca 2+ elevation (calcineurin- and CaMK- independent events) 5. Polarization of cytoskeletal elements contribute to polarized secretion of cytolytic granules or lymphokines 1. T cell polarizes, APC does not
    6. 6. Immunologic Synapse
    7. 7. SMAC (supramolecular activation complex) <ul><li>Kupfer - deconvolution IF microscopy of T cell/APC interactions - organized, bulls-eye type structure </li></ul>c-SMAC c-SMAC p-SMAC TCR/CD3, CD2, CD28, PKC  lck LFA-1, Talin, CD43, CD45, CD148 <ul><li>Non-activating, altered, peptides do not support the formation of these structures </li></ul>
    8. 8. Formation of an Immunological Synapse Involves Coordinated Spatial Organization Red= ICAM-1 Green=MHC/pep Grakoui et al. (1999) Science 285 :221.
    9. 9. Signaling Occurs in TCR Microclusters at the Periphery of the Synapse Yokosuka et al., Nature Immunol. 6, 1253, 2005
    10. 10. Function(s) of the Immunological Synapse? 1. Signal Initiation : probably not P-tyr and Calcium responses precede mature synapse formation 2. Signal enhancement : perhaps But no biochemical correlate and mature synapse does not appear to be required (CD2-AP KO signals better than wt but does not form synapse) Peripheral microclusters appear to be most active signaling complexes 3. Directed secretion : fits well Directed secretion by CTL and Th cells makes teological sense and has been well documented 4. Receptor downmodulation : likely Recent modeling and CD2-AP KO data support this New data on TCR microclusters also supportive
    11. 14. The TCR is an Oligomer and Utilizes CD3 and  Chain ITAMs to Initiate Signal Transduction TCR  and      
    12. 15. <ul><li>How do TCRs transmit their ligand occupancy state across </li></ul><ul><li>the plasma membrane? </li></ul><ul><li>2. Do TCRs undergo conformational changes when they bind ligand? </li></ul>
    13. 16. Mechanisms for Signaling Ligand Occupancy States Across Membranes Cochran et al, TIBS, 28:304, 2001 GPCRs RTKs Antigen Receptors? TCR/CD4 Cytokine Receptors? Bacterial Asp Receptor
    14. 17. <ul><li>Support for the Oligomerization Model </li></ul><ul><li>Bivalent (but usually not univalent Fab) mAbs can induce </li></ul><ul><ul><li>T cell responses </li></ul></ul><ul><li>Many different mAbs can induce T cell signals and responses </li></ul><ul><li>The more multivalent, the better (role of immobilization to surface </li></ul><ul><li>or to Fc receptor) </li></ul><ul><li>Chimeric receptors (ITAM containing chimeras) </li></ul><ul><li>Hetero-oligomerization with coreceptors (ie. CD4) increases </li></ul><ul><ul><ul><li>sensitivity to peptide/MHC molecule </li></ul></ul></ul>
    15. 18. Irving and Weiss, Cell, 1991 CD3 mAbs Against the TCR/CD3 Complex or Chimeric Receptors can Induce Similar Signals and Responses
    16. 19. A Hetero-Oligomerization Model The TCR and CD4 coreceptor both interact with peptide/MHC complex, bringing the Lck kinase into close proximity with TCR.
    17. 20. Some recent data support a conformational change
    18. 21. Conformational Change in the TCR  Constant Region A-B Loop in the LC13 TCR CD3  thought to bind here Ligated - red Unligated - cyan Kjer-Nielsen, et al., Immunity, 2003
    19. 22. A CD3  Proline Motif May Become Accessible During a Putative TCR Conformational Change: Models (Alarcon, et al, Cell , 2002; Gil, et al, J. Exp. Med ., 2005; Levin and Weiss, J. Exp. Med ., 2005) SH3 SH3 SH3 SH2 N c k actin cytoskeleton Pak kinase ????
    20. 23. The TCR is an Oligomer and Utilizes ITAMs to Initiate Signal Transduction TCR  and      
    21. 26. Lipid rafts, GEMs, DIGs <ul><ul><ul><li>Cholesterol-rich microdomains in the plasma membrane </li></ul></ul></ul><ul><ul><ul><li>(25 to 70 nm in diameter) </li></ul></ul></ul><ul><ul><ul><li>Enriched in GPI-linked receptors and glycosphingolipids </li></ul></ul></ul><ul><ul><ul><li>Enriched in many signaling molecules, including: </li></ul></ul></ul><ul><ul><ul><li>PIP2 and PIP3, several GTPases including Ras, </li></ul></ul></ul><ul><ul><ul><li>Src kinases, some heterotrimeric G-proteins </li></ul></ul></ul><ul><ul><ul><li>Operationally defined based on detergent solubility </li></ul></ul></ul><ul><ul><ul><li>Some of Lck (50% or less) partitions into lipid rafts </li></ul></ul></ul><ul><ul><ul><li>(reversibility and dynamic changes?) </li></ul></ul></ul><ul><ul><ul><li>May be a focal point of signal initiation or sequestration </li></ul></ul></ul><ul><ul><ul><li>- A topic of considerable controversy! </li></ul></ul></ul>
    22. 29. CD45 <ul><li>Receptor-like protein tyrosine phosphatase expressed on all nucleated hematopoietic cells </li></ul><ul><li>Distinct isoforms expressed in a cell-type and developmental-stage specific manner </li></ul><ul><li>Required for T cell and B cell antigen receptor signaling and for normal development - dephosphorylates negative regulatory tyrosine in Src kinases. </li></ul><ul><li>Mutations associated with SCID and </li></ul><ul><li>autoimmunity in humans and mice </li></ul><ul><li>Contains tandemly duplicated phosphatase domains, but only domain 1 is functionally active. </li></ul>
    23. 30. PRIMED Dynamic Lck Regulation by Activating and Inhibitory Tyrosine Phosphorylation CD45 Csk Receptor Stimulation CD45 or PTPN22 Y Y INACTIVE P Y ACTIVE P Y
    24. 31. ZAP-70 in TCR Signaling <ul><ul><li>Expressed only in T cells and NK cells </li></ul></ul><ul><ul><li>Unique N-terminal SH2 domain (incomplete; unlike Syk) </li></ul></ul><ul><ul><li>Cooperative binding to doubly phosphorylated ITAMs </li></ul></ul><ul><ul><li>Catalytic activity induced by phosphorylation of activation loop </li></ul></ul><ul><ul><li>Positive and negative sites of phosphorylation </li></ul></ul><ul><ul><li>Negative sites Positive sites (? Allosteric) </li></ul></ul><ul><ul><li>Y292 - c-Cbl (Ub system) Y315 - Vav </li></ul></ul><ul><ul><li>Y492 - ? Allosteric Y319 - Lck, PLC gamma 1, Crk </li></ul></ul><ul><ul><li>Potential Substrates: LAT, Slp-76 </li></ul></ul><ul><ul><li>ZAP-70 mutations: Cell line - loss of TCR signaling </li></ul></ul><ul><ul><li> Mouse KO - developmental arrest (DP -> SP) </li></ul></ul><ul><ul><li>Human SCID - No CD8 T cells </li></ul></ul><ul><ul><li> CD4 T cells with TCR signaling defect </li></ul></ul><ul><ul><li>SKG mutant - C-terminal SH2 mutant - Balb/c - RA-like disease </li></ul></ul>
    25. 32. <ul><li>Key Features of the Full-Length ZAP-70 Structure </li></ul><ul><li>Y315F and Y319F are sequestered </li></ul><ul><li>by hydrophobic interactions involving </li></ul><ul><li>SH2 linker region and C-terminal </li></ul><ul><li>kinase domain - representing an auto- </li></ul><ul><li>inhibitory mechanism </li></ul><ul><li>The kinase domain is in an inactive </li></ul><ul><li>conformation due to interactions stabilizing </li></ul><ul><li>the hinge region </li></ul><ul><li>Alignment of SH2 domains is </li></ul><ul><li>not consistent with ITAM </li></ul><ul><li>binding </li></ul><ul><li>A conformational change likely </li></ul><ul><li>occurs after ITAM binding </li></ul>Deindl, et al., Cell, 2007
    26. 34. Hypothetical Conformational Change in ZAP-70 Upon ITAM Binding Contributes to Its Activation
    27. 35. New Model: Intramolecular Regulatory Switch in ZAP-70: Initiation of Activation by Lck-Mediated Phosphorylation of a Negative Regulatory Switch Brdicka, et al., Mol. Cell Biol. June 2005
    28. 37. A Hetero-Oligomerization Model:
    29. 39. 2 Adapters Are Phosphorylated by ZAP-70: Required for PLC and Ras Activation in T Cells LAT SLP-76 LAT function: Lipid raft recruitment of effectors SLP-76 function: Organization of signaling complex - Y D S T - Y P R G - Y P P V - Y E N E - Y H N P - Y L V V - Y V N V - Y V N V - Y E N L SH2 . . . Y E S P . . . . . Y E S P . . . . . Y E P P . . . . . Pro-rich
    30. 41. Assembly of LAT and SLP-76 into a Signalosome in Lipid Rafts Is required for Multiple Downstream TCR Signaling Pathways Events requiring LAT & SLP-76 PLC  1 activation Ras activation Rac activation HPK1 activation
    31. 42. I t k / T e c / R l k K i n a s e S H 3 S H 2 PH T H Tec Kinases in T cells <ul><li>Itk </li></ul><ul><li>Expressed predominantly in T cells </li></ul><ul><li>PH domain binds to PIP3 which recruits Itk to membrane </li></ul><ul><li>Kinase domain is phosphorylated and activated by Lck </li></ul><ul><li>TH and SH3 domains interact in basal state to inhibit kinase </li></ul><ul><li>Autophosphorylation of Y180 adjacent to SH3 domain allows Itk to unfold, </li></ul><ul><li>rendering SH2 and SH3 domains accessible </li></ul><ul><li>Likely to phosphorylate and activate PLC  1 </li></ul><ul><li>KO in mice results in mild defect in PLC activation and Calcium flux </li></ul><ul><ul><li>(redundant roles of other family members, Rlk and Tec, in T cells) </li></ul></ul>Y Y
    32. 44. Sequential Events in TCR Signal Transduction
    33. 45. PLC Activation Leads to Second Messenger Generation and Mobilization of Calcium as well as PKC and RasGRP Activation
    34. 46. Evidence for the Importance of the Phosphatidylinositol Pathway in T cells 1. Activating and synergistic effects of calcium ionophores and phorbol esters 2. Inhibitory effects of calcium chelators and PKC inhibitors 3. Positive effects of activating alleles of calcineurin and PKC or Ras) 4. Ability of heterologous G-protein coupled receptors that activate PLC beta but not Src or Syk PTKs to induce IL-2 gene activation 5. T cell activation defects in PKC theta mice
    35. 47. The Phosphatidylinositol Pathway: Calcium Mobilization after TCR Stimulation + EGTA Initial release from Intracellular stores (results from IP3 interacting with receptors) Depletion of intracellular stores results in a transmembrane flux of calcium, Icrac Downstream Consequences: Activation of Calcineurin (Ca/CaM-dependent serine/threonine phosphatase), the target of Cyclosporin A and FK506 Activation of Ca/CaM kinase Time (min) 1 Lewis Annu. Rev. Immuno. 2001
    36. 48. Marchant, Current Biology, 2005 STIM is the ER Calcium Sensor and Orai is a Component of SOC Orai Prakriya, et al.,, Nature, 2006 Depletion of intracellular stores results in altered function and distribution of ER resident protein STIM. STIM migrates in “puncta” towards plasma membrane where it regulates the recently identified Icrac channel, Orai, in the plasma membrane to allow a transmembrane flux of calcium
    37. 49. The Phosphatidylinositol Pathway: PKC activation after TCR Stimulation PKC - a large family of serine/threonine kinases Multiple isozymes in T cells DAG-regulated, phorbol ester responsive Some are calcium responsive PKC theta localizes to cSMAC, and KO mice have selective defect in NF-kB activation in T cells Function: TCR downregulation NFkB activation Activation of RasGRP
    38. 50. TCR Stimulation Activates Ras R a s ( G D P ) i n a c t i v e R a s ( G T P ) a c t i v e P O 4 R a s G A P N e u r o f i b r o m i n G r b 2 / S O S RasGRP G T P G D P R a s G E F s R a s G A P s R a s E f f e c t o r s : R a f - 1 K i n a s e P I - 3 K i n a s e G A P R h o G T P a s e s
    39. 51. The TCR can Activate Ras via at Least 2 Mechanisms Roose and Weiss, Nat. Immunol., 2000

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