Signaling Through Immune System Receptors (Ch. 7) 1. General principles of signal transduction and propagation. 2. Antigen receptor signaling and lymphocyte activation. 3. Other receptors and signaling pathways.
1. General principles of signal transduction Signal Transduction: The processes by which a cell transforms one type of extracellular signal into the different biochemical forms - The final destination of the signal transduction: Nucleus (or cytoplasm) - The primary cellular response: changes in gene expression (or cellular metabolism)
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IKK IKK IKK IKK Enzymatic activation (Kinase activation)
Protein Kinase O Protein OH + ATP Protein O P P i Protein Phosphatase H 2 O O O + ADP A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein. A protein phosphatase catalyzes removal of the P i by hydrolysis.
Many enzymes are regulated by covalent attachment of phosphate to the side-chain hydroxyl group of a particular amino acid residue (serine, threonine, or tyrosine). -Serine/Threonine Kinase: Major -Tyrosine Kinase: Minor (e.g.. BCR, TCR)
Phosphorylation: 1. directly alter activity of an enzyme (e.g., by promoting a conformational change). 2. alter signaling activity by binding another protein that specifically recognizes a phosphorylated domain. JPEG file adapted from
P - P - P- + + + - -- P - P - P -
A + + + - - - B - -- + + + P - P - P -
Scaffold or Adaptor proteins: - No enzymatic activity - Function to recruit other proteins to a signaling complex so that they can interact each other 1. Scaffold proteins: larger proteins, recruit many different proteins 2. Adaptor proteins: Smaller proteins, link two proteins together JPEG file adapted from
Receptors: 1. Contains an intrinsic kinase activity in the cytoplasmic portion of the receptor 2. Does not contain an intrinsic kinase activity, but non-covalently associates with a cytoplasmic tyrosine kinase JPEG file adapted from
IKK IKK IKK IKK
How to recruit intracellular signaling proteins to the plasmamembrane: JPEG file adapted from
1. Phosphorylation of the receptor and the subsequent recruitment of SH2-domaincontaining signaling proteins to the receptor: IKK IKK P P P P IKK IKK Domain Domain
2. The activation of membrane-associated small G proteins: Small G proteins (or small GTPase): e.g. the Ras family proteins - GTP- (active) or GDP- (inactive) bound form - Guanine nucleotide exchange factors (GEFs) mediates the exchange of the GDP for GTP - Each G protein has its own GEF - G proteins contain the intrinsic GTPase activity to convert itself into the inactive GDP-bound form. - GEFs are recruited to the site of receptor activation at the cell membrane by binding to adaptor proteins. - G proteins localize to the inner surface of the plasma membrane via fatty acids that are attached to the G protein post-translationally.
IKK IKK IKK IKK Domain GEF P P P P G G Domain GEF
3. The local production of modified membrane lipids: - Phosphorylation of the membrane phospholipid (phosphatidylinositol) by phosphatidylinositol kinases - Phosphatidylinositol 3,4,5-triphosphate (PIP 3 ) is generated from Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by phosphatidylinositol 3-kinase (PI3-kinase) -PIP 3 or PIP 2 is recognized by proteins containing a PH or a PX domain.
IKK IKK IKK IKK P P Domain PI3K P P P P PH/ PX Domain PI3K
The amplification of signaling: 1. Kinase cascade 2. Secondary messengers - low-molecular-weight intracellular biochemical mediators - e.g.. Ca 2+, diacylglycerol (DAG), phosphatidylinositol, camp, cgmp JPEG file adapted from
Lipid raft (detergent-insoluble glycolipid-rich domains (DIG) or glycolipidenriched microdomains (GEMs): - small cholesterol-rich areas in the cell membrane - Enriched in particular lipids, sphingolipids and cholesterol - Enriched in certain signaling proteins GPI: Glycosylphosphatidylinositol
The regulation of signaling: 1. Protein phosphatase 2. Ubiquitin-dependent protein degradation (ubiquitinated membrane proteins lysosome; other ubiquitinated proteins proteasome) JPEG file adapted from
JPEG file adapted from E1: Ubiquitin Activating Enzyme E2: Ubiquitin Conjugating Enzyme E3: Ubiquitin Ligase (determines substrate specificity)
2. Lymphocyte signaling pathways B cell receptor complex T cell receptor complex JPEG file adapted from ITAM: Immunoreceptor Tyrosine-based Activation Motif (Two YXXL/I motifs separated by 6-9 aa)
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Activation of lymphocyte receptors: 1. BCR: Cross-linking of receptors JPEG file adapted from
2. TCR: JPEG file adapted from i) Conformation change ii) Clustering iii) Immunological synapse formation SMAC: SupraMolecular Activation Complex
The key receptor associated kinases in lymphocyte signaling: Tyrosine Kinases of the Src family (phosphorylates ITAM) The Src family kinases localize to the membrane either by myristate attachment or by palmitate modification CD45: The tyrosine phosphatase CSK: C-terminal Src kinase
1) T cell receptor (TCR) signaling pathways: JPEG file adapted from
A. Signaling from the TCR complex and co-receptor (CD4 or CD8) Src family kinases for TCR: Lck and Fyn Lck: associated with CD4 and CD8 Fyn: associated with CD3 and ζ JPEG file adapted from
JPEG file adapted from Src family kinases for TCR: Lck and Fyn (less important) Lck: associated with CD4 and CD8 Fyn: associated with CD3 and ζ ZAP-70: ζ-chain-associated tyrosine kinase, contains SH2 domain
- LAT (Linker of Activated T cells): transmembrane protein, posttranslationally modified by palmitate - LAT, SLP-76: scaffold protein, GADS: Adaptor protein - Tec family of cytoplasmic tyrosine kinase: Tec, Itk (T cells), Btk (B cells) Phosphorylates PLC- γ - PLC-γ (Phospholipase C-γ): breakdown PIP 2 to IP 3 (Inositol triphosphate) and Diacylglycerol (DAG)
PLC-γ -Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) -Inositol 1,4,5-triphosphate (IP 3 ) CRAC (calcium release-activated calcium) channels
JPEG file adapted from PLC-γ: PIP 2 IP 3 and DAG
Ca 2+ : Casein Kinase 2 (CK2) and Glycogen Synthase Kinase 3 (GSK3) JPEG file adapted from
DAG: JPEG file adapted from
DAG recruits RasGRP (GEF for the small G protein, Ras) to the membrane JPEG file adapted from MAPKKK: Raf (phosphorylates MAPKK (MEK)) MAPKK: MEK (phosphorylates Threonine and Tyrosine in ERK (MAPK))
ERK phosphorylates Elk-1 to transactivate FOS JNK phosphorylates Jun AP1 = Fos + Jun SRF: Serum Response Factor SRE: Serum Response Element
DAG recruits PKC-θ to the membrane (Ca2+ is required for PKC-θ activity) JPEG file adapted from
Nuclear Factor kappa B (NF-κB) JPEG file adapted from
B. Signaling from the co-stimulatory receptor (CD28) JPEG file adapted from CD80 (= B7.1), CD86 (= B7.2) only on APCs
CD28:Contains Non ITAM motif (YXXM, YXN) and Proline-rich motif (PXXP) Y X X M Y X N P P SH2 SH2 PI3K Grb2 SH3 SH3 PXXP PXXP SOS (GEF for Ras) PIP 2 PIP 3 Akt through PH domain - Akt promotes cell survival and stimulates cellular metabolism P X X P SH3 Lck or Itk
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JPEG file adapted from CD80 (= B7.1), CD86 (= B7.2)
IL-2: cytokine important for T cell proliferation, differentiation into effector cells JPEG file adapted from Oct1 is constitutively expressed and bound to IL-2 promoter
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2) B cell receptor (BCR) signaling pathways: Src family kinases for BCR: Fyn, Blk and Lyn Syk: ZAP-70 homologue in B cells Syk phosphorylates BLNK (= SLP-65). BLNK is a SH2 containing scaffold protein which recruits PLCγ. In B cells, Btk phosphorylates and activates PLCγ.
3) Inhibitory Receptors: Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM): (I/V)XYXX(L/I) Phosphorylated Tyrosine in ITIM recruits phosphatases. SHP (SH2-containing phosphatase) SHIP (SH2-containing inositol phosphatase): PIP 3 PIP 2
3. Other signaling pathways - Cytokine receptors - Chemokine receptors - Death receptors
Cytokine Receptors: JPEG file adapted from - JAK family: Jak1, Jak2, Jak3 and Tyk2 - STAT (Signal Transducers and Activators of Transcription): STAT1-5, 6a, 6b - Homo- or Hetero-dimers of STATs can transactivate genes for lymphocyte growth and differentiation (e.g.. STAT4 for TH1 development, STAT6 for TH2 development)
Regulation of cytokine receptor signaling: 1. Phosphatase (SHP, CD45, etc.) 2. Cytokine induced inhibitors: promotes ubiquitination and degradation of receptors and pathway components.
Chemokine receptors: JPEG file adapted from
Death receptors: Induce programmed cell death (or apoptosis) to eliminate unnecessary immune cells. Apoptosis is mediated by proteases called Caspases. Caspase Procaspase - Initiator caspases: cleave and activate other caspases - Effector caspases: initiate the cellular changes by cleaving and degrading nuclear structural proteins and activating endonucleases
Apoptosis: 1. Extrinsic pathway of apoptosis: mediated by the activation of death receptors by extracellular ligands. 2. Intrinsic or mitochondrial pathway of apoptosis: mediated by intracellular stimuli such as UV, starvation, etc..
Extrinsic pathway of apoptosis: Death Receptors: Fas (CD95) and Tumor Necrosis Factor Receptor 1 (TNRF-1) FAS: JPEG file adapted from The high local concentration of caspases allows it to cleave itself.
TNRF-1: JPEG file adapted from RIP: NF-κB TRAF2: MAPK
Intrinsic or mitochondrial pathway of apoptosis: JPEG file adapted from CAD: Caspase activated DNase
Bcl-2 family of proteins regulate Cytochrome C release from Mitochondria and apoptosis. JPEG file adapted from