in out Lipid bilayer of plasma membrane Antigen recognition Signal transduction (contains one or more ITAMs) + - Src-family tyrosine kinase reversibly associated with receptor

Consensus ITAM (amino acid) ….---.asp tyr leu tyr leu.---…. glu ile ile The one letter code is usually used: xx(D/E)xxYxx(L/I)xxxxxxxYxx(L/I) These tyrosines are phosphorylated by various src family kinases upon recognition receptor ligation

Src-family tyrosine kinases Unique region reversibly associates with certain membrane receptors. Differs among family members. SH3 motif involved in binding to many proteins that carry a proline rich motif (e.g. P-X-X-P). SH2 motif involved in binding to phospho-tyrosines in certain sequence contexts, including phosphorylated ITAMs. Kinase domain motif catalyzes tyrosine phosphorylation. Myristylation/palmitylation sites present in many family members, promotes association with membranes and membrane “rafts”. (e.g. CH 3 (CH) 12 CONH-Glycine..)

CD45 (plasma membrane tyrosine phosphatase) csk (cytoplasmic tyrosine kinase)

Adapted from Molecular Cell Biology 3rd Ed., p894

Table 1. Expression of Src family kinases Src Ubiquitous; two neuron-specific isoforms Fyn Ubiquitous; T cell-specific isoform (Fyn T) Yes Ubiquitous Lyn Brain, B-cells, myeloid cells; two alternatively spliced forms Hck Myeloid cells (two different translational starts) Fgr Myeloid cells, B-cells Blk B-cells Lck T-cells, NK cells, brain Frk subfamilyPrimarily epithelial cells

+ - in out Antigen recognition Signal transduction (with ITAMs) T Cell Receptor  CD3  - CD3   - + CD3 functions both as signal transducers and in transport of antigen receptors to the plasma membrane

in out Antigen recognition Signal transduction (with ITAMs) B Cell Receptor Ig  Lyn Blk Hck Fyn Lck syk ACTIVATION!

Antigen receptor signaling may be similar to that of other surface receptors such as receptor tyrosine kinases like epidermal growth factor in which receptor crosslinking leads to initial trans-phosphorylation.

+ - in out Signal transduction (with ITAMs)  - + CD3  Fyn T Cell Receptor And coreceptor CD3  -  Lck CD4 Cholesterol and sphingolipid rich microdomain

+ -  - + CD3  T Cell Receptor CD3  -  ZAP70 ACTIVATION! Lck CD4

TABLE 2. Protein with Src homology-2 domains that may be associated with T cell-activation (partial list) Protein tyrosine kinases Fyn Lck Syk ZAP-70 Csk ltk Protein tyrosine phosphatases SHP-1 SHP-2 Proteins with enzymatic functions Phospholipase C  1 (PLC  1) Phosphatidylinositol 3”-kinase (P1 3-kinase), p85 subunit Adaptors and Regulators GTPase acttivating protein (GAP) SOS Vav Slp-76 Shc Nck Grb2 Crk From Fundamental Immunology 4th Ed, Paul WE Ed., pp 425

TABLE 3. Some of the proteins that are tyrosine phosphorylated following TCR stimulation. TCR subunits CD3 , , ,  Protein tyrosine kinases ltk Lyn Lck MAPKs Pyk2 Syk ZAP-70 Proteins with enzymatic function Phospholipase C  1 (PLC  1) Others Cbl CD5 CD6 Ezrin LAT Shc Slap-130 Slp-76 Shc  Tubulin Valosin containing protein Vav From Fundamental Immunology 4th Ed, Paul WE Ed., pp 427

+ -  - + CD3  T Cell Receptor activation (cont) CD3  -  ACTIVATION! ZAP70 Lck CD4 LAT Plc  PIP2 IP 3 DAG Raf-1 MEK MAPK Immediate early genes Jun/Fos Ca ++ Endoplasmic reticulum IP 3 receptor [Ca ++ ] i Ca ++ CRAC Ca ++ calcineurin PO 4 NFAT IL-2 gene NFAT Fos/Jun nucleus PKC RasGRP RAS GTP GDP

From Fundamental Immunology 4th Ed, Paul WE Ed., pp 428

+ -  - + CD3  T Cell Receptor activation (cont) CD3  -  ACTIVATION! ZAP70 Lck CD4 LAT nucleus Gads Slp-76 SLAP 130 vav rho/rac Nck Pak WASP Actin/cytoskeletal reorganization Adhesion migration Grb2 sos Ras GDP GAPs Ras GTP Ras effectors

+ -  - + CD3  T Cell Receptor activation (cont) CD3  -  ACTIVATION! Lck CD4 LAT nucleus CD28 “Costimulatory” B7 molecules on antigen presenting cells B7.1 or B7.2 ? PI3K p85 PIP 3 PI3K PIP 2 Itk ACTIVATION! Antigen presenting cell PKB

P PI3K Removed by SHIP--1

Where does cell type specificity come from? Preexisting substrate differences. Specificity of src and ZAP70/syk kinases PKC isoform expression PLC  isoform expression NFAT isoform expression Transcription factor isoform expression Inhibitory receptor expression etc….

1,2 Ig-like extracellular domains NKp46 NKp30 FcRIII + - in out “Antigen” recognition Signal transduction (with ITAMs) Activating Receptors (on Monocytes, Macrophages, Neutrophils, Mast cells, and NK cells) 2-4 Ig-like extracellular domains Fc  RI Fc  RIIa * Fc  RIII Fc  RI ILT1 ILT7 ILT8 LIR6a Fc common  chain + - CD3  Fc  chain  1,2-? Ig-like extracellular domains Fc  RIII Fc  RI 1-3 Ig-like extracellular domains NK activatory receptors Fc  RI DAP12 Type 2 membrane protein NKp80

in out Activating receptor + - Activating ligand Inhibitory ligand ITIMs Motif: I/VxYxxL/V Src-family tyrosine kinase reversibly associated with receptor ITAMs Tyrosine phosphatase e.g., SHP-1 Tyrosine kinase e.g., ZAP-70, syk SHIP lipid phosphatase

+ - + Weak signal no response nucleus Normal cell Defective cell “Natural killer” cell recognition “Missing self” Initiation of killing program MHC class I

CD28 nucleus APC + T cell down- regulation late in response CTLA4 signaling turns response off TCR CD28 MHC/peptide + + Ag signal + costimulation T cell activation CTLA4 sequestered - CTLA4 on surface B7 Late in the response... B7 APC

in out “Antigen” recognition Signal transduction (with ITIMs) Inhibitory Receptors (on Monocytes, Macrophages, Neutrophils, Mast cells, and NK cells) 1-6 Ig-like extracellular domains ILT5 LIR8 ILT4 Siglec 6 CD33 CD22 FcgRIIb PIRB SIRPs LAIR1 ILT3 LIR5 PD-1 KIR2DL KIR3DL CTLA4 Type 2 membrane proteins Ly49 CD94/ NKG2A CD72

Immunoprecipitation Detergent Anti-Y-PO 4 precipitate antibodies with S. aureus protein A-coupled resin, wash, elute Fractionate by electrophoresis on denaturing polyacrilamide gel - + sample Mobility inversely proportional to the Log of molecular weight markerscontrol

Generation of gene knockouts in mice: an important technique in establishing the role of signaling molecules in normal cells. Adapted from Molecular Cell Biology 3rd Ed., p294-5

Adapted from Molecular Cell Biology 3rd Ed., p296

Yeast two hybrid assay is one way that protein: protein interactions can be measure and is an important way that new molecules are discovered. Adapted from Molecular Cell Biology 3rd Ed., p896