Signaling Through Immune System Receptors (Ch. 7)

Transcription

1 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.

2 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)

3 JPEG file adapted from

4 JPEG file adapted from

5 IKK IKK IKK IKK Enzymatic activation (Kinase activation)

6 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.

7 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)

8 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

9 P - P - P P - P - P -

10 A B P - P - P -

11 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

12 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

13 IKK IKK IKK IKK

14 How to recruit intracellular signaling proteins to the plasmamembrane: JPEG file adapted from

15 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

16 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.

17 IKK IKK IKK IKK Domain GEF P P P P G G Domain GEF

18 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.

19 IKK IKK IKK IKK P P Domain PI3K P P P P PH/ PX Domain PI3K

20 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

21 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

22 The regulation of signaling: 1. Protein phosphatase 2. Ubiquitin-dependent protein degradation (ubiquitinated membrane proteins lysosome; other ubiquitinated proteins proteasome) JPEG file adapted from

23 JPEG file adapted from E1: Ubiquitin Activating Enzyme E2: Ubiquitin Conjugating Enzyme E3: Ubiquitin Ligase (determines substrate specificity)

24 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)

25 JPEG file adapted from

26 Activation of lymphocyte receptors: 1. BCR: Cross-linking of receptors JPEG file adapted from

27 2. TCR: JPEG file adapted from i) Conformation change ii) Clustering iii) Immunological synapse formation SMAC: SupraMolecular Activation Complex

28 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

29 1) T cell receptor (TCR) signaling pathways: JPEG file adapted from

30 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

31 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

32 - 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)

33 PLC-γ -Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) -Inositol 1,4,5-triphosphate (IP 3 ) CRAC (calcium release-activated calcium) channels

34 JPEG file adapted from PLC-γ: PIP 2 IP 3 and DAG

35 Ca 2+ : Casein Kinase 2 (CK2) and Glycogen Synthase Kinase 3 (GSK3) JPEG file adapted from

36 DAG: JPEG file adapted from

37 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))

38 ERK phosphorylates Elk-1 to transactivate FOS JNK phosphorylates Jun AP1 = Fos + Jun SRF: Serum Response Factor SRE: Serum Response Element

39 DAG recruits PKC-θ to the membrane (Ca2+ is required for PKC-θ activity) JPEG file adapted from

40 Nuclear Factor kappa B (NF-κB) JPEG file adapted from

41 B. Signaling from the co-stimulatory receptor (CD28) JPEG file adapted from CD80 (= B7.1), CD86 (= B7.2) only on APCs

42 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

43 JPEG file adapted from

44 JPEG file adapted from CD80 (= B7.1), CD86 (= B7.2)

45 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

46 JPEG file adapted from

47 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.

48 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

49 3. Other signaling pathways - Cytokine receptors - Chemokine receptors - Death receptors

50 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)

51 Regulation of cytokine receptor signaling: 1. Phosphatase (SHP, CD45, etc.) 2. Cytokine induced inhibitors: promotes ubiquitination and degradation of receptors and pathway components.

52 Chemokine receptors: JPEG file adapted from

53 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

54 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..

55 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.

56 TNRF-1: JPEG file adapted from RIP: NF-κB TRAF2: MAPK

57 Intrinsic or mitochondrial pathway of apoptosis: JPEG file adapted from CAD: Caspase activated DNase

58 Bcl-2 family of proteins regulate Cytochrome C release from Mitochondria and apoptosis. JPEG file adapted from