Signal transduction in cells of the immune system Dr. Stefanie Gnipp

Department of Experimental Pneumology

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What does it really mean………

..cells get activated… …T-cell polarization…

…cellular differentiation/determination…. …induces an inflammatory response…

… induces cell maturation… …cells are recruited to site of….

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How does it work?

General Introduction

Receptors

• Receptors that penetrate the plasma membrane and have intrinsic enzymatic activity • Receptors that penetrate the plasma membrane and have no intrinsic enzymatic activity • Receptors that are coupled, inside the cell, to GTP-binding and hydrolyzing proteins • Intrinsic receptors/Nuclear receptors

Pathways of Intracellular Signal Transduction

• The JAK/STAT Pathway • Ras, Raf, and the MAP Kinase Pathway • Phospholipids and Ca2+ • The cAMP Pathway: Second Messengers and Protein Phosphorylation • Cyclic GMP

Signal Transduction Pathways in Immunology

• TGFß • TLR • Cytokines • TCR und BCR

Termination of Signal Transduction

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SIGNAL

RECEPTION

TRANSDUCTION

RESPONSE(S)

AMPLIFICATION

Principles of Signal Transduction

An environmental signal is first received by interaction with a cellular component, most often a cell-surface receptor. The information that the signal has arrived is then converted into other chemical forms, or transduced. The signal is often amplified before evoking a response. Feedback pathways regulate the entire signaling process. Biochemistry. 5th edition.Berg JM, Tymoczko JL, Stryer L.New York: W H Freeman ;2002. 4

Signal-transduction cascades mediate the sensing and processing of stimuli. These molecular circuits detect, amplify, and integrate diverse external signals to generate responses such as changes in enzyme activity, gene expression, or ion-channel activity.

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Communication of cells by extracellular signals involves 6 steps: 1. Synthesis of signaling molecule (=ligand) (or ligand is an environmental stimulus!) 2. Release of signaling molecule 3. „Transport“ of the signal to the target cell 4. Detection of the signal by a specific receptor 5. Change in cellular metabolism/function/development triggered by Ligand-Receptor complex 6. Removal of signal (e.g.Ubiquitination (proteasomal degradation); Dephosphorylation)

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4 Types of signaling

Molecular Cell Biology, 4th edition; Harvey Lodish

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General Introduction

Receptors

• Receptors that penetrate the plasma membrane and have intrinsic enzymatic activity • Receptors that penetrate the plasma membrane and have no intrinsic enzymatic activity • Receptors that are coupled, inside the cell, to GTP-binding and hydrolyzing proteins • Intrinsic receptors/Nuclear receptors • Ion channel receptors

Pathways of Intracellular Signal Transduction

• The JAK/STAT Pathway • Ras, Raf, and the MAP Kinase Pathway • Phospholipids and Ca2+ • The cAMP Pathway: Second Messengers and Protein Phosphorylation • Cyclic GMP

Signal Transduction Pathways in Immunology

• TLR • TGFß • Cytokines • Apoptosis

Receptor Classes

1. Receptors that penetrate the plasma membrane and have intrinsic enzymatic activity

• tyrosine kinases (RTKs) : many growth factor receptors • serine/threonine kinases (RSKs) : e.g. TGF-β receptor(s) • tyrosine phosphatases (e.g. CD45 protein of T cells and macrophages) • guanylate cyclases (e.g. natriuretic peptide receptors)

Additionally, several families of receptors lack intrinsic enzyme activity, yet are coupled to intracellular tyrosine kinases by direct protein-protein interactions:

1a. PTKs: Non-Receptor Protein Tyrosine Kinases: • includes all of the cytokine receptors • as well as the CD4 and CD8 cell surface glycoproteins of T cells and the • T cell antigen receptor (TCR) Most of the proteins of non-receptor PTKs couple to cellular receptors that lack enzymatic activity

2. Receptors that are coupled, inside the cell, to GTP-binding and hydrolyzing proteins

• adrenergic receptors, odorant receptors, certain hormone receptors (e.g. glucagon, angiotensin, vasopressin and bradykinin).

3. Receptors that are found intracellularly

• steroid and thyroid hormone receptors

http://themedicalbiochemistrypage.org/signal-transduction.php 8

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Protein phosphorylation

Protein Kinases

Tyrosine Serine/Threonine

RSKs RTKs/PTKs

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Protein Phosphatases

Protein dephosphorylation

PTPs+PSPs

Threonin

>> termination of signal

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Receptor Thyrosine Kinases and Thyrosine Kinase associated Receptors: Cross-phosphorylation of receptor-dimers upon ligand-binding

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GPCRs: G-protein coupled receptors

Activation of the G alpha subunit of a G-protein-coupled receptor In unstimulated cells, the state of G alpha (orange circles) is defined by its interaction with GDP, G beta-gamma (purple circles), and a G-protein-coupled receptor (GPCR; light green loops). Upon receptor stimulation by a ligand called an agonist, the state of the receptor changes. G alpha dissociates from the receptor and G beta-gamma, and GTP is exchanged for the bound GDP, which leads to G alpha activation. G alpha then goes on to activate other molecules in the cell. © 2002 Nature Publishing Group Li, J. et al. The Molecule Pages database. Nature 420, 716-717 (2002).

What Do GPCRs Do? As their name implies, GPCRs interact with G proteins in the plasma membrane. When an external signaling molecule binds to a GPCR, it causes a conformational change in the GPCR. This change then triggers the interaction between the GPCR and a nearby G protein

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The relationships of G proteins to the plasma membrane In this diagram of G-protein-coupled receptor activation, the alpha, beta, and gamma subunits are shown with distinct relationships to the plasma membrane. After exchange of GDP with GTP on the alpha subunit, both the alpha subunit and the beta-gamma complex may interact with other molecules to promote signaling cascades. Note that both the alpha subunit and the beta-gamma complex remain tethered to the plasma membrane while they are activated. These activated subunits can act on ion channels in the cell membrane, as well as cellular enzymes and second messenger molecules that travel around the cell. © 2010 Nature Education

GPCRs: G-protein coupled receptors

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Signaling cascades within a cell can interact to affect multiple molecules in the cell, leading to secretion of substances from the cell, ion channel opening, and transcription. Binding of an agonist to the seven-transmembrane G-protein-coupled receptor in the plasma membrane activates a pathway that involves G proteins as well as cAMP-related pathways that modulate cellular signaling. In this example, the activated G alpha (Gαi/0) proteins inhibit (-) adenylyl cyclase (AC, on the right), the enzyme that induces formation of cAMP, which in turn results in the activation of protein kinase A (PKA). This in turn activates a molecule called cAMP-responsive element-binding protein (CREB), which modulates gene transcription. The activated G alpha proteins can also have a variety of other effects, shown at the left. These effects include activating the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. Activation of the enzyme phospholipase A2 (PLA2) may also occur, which induces the release of arachidonic acid (AA), as well as inhibition of the Na+/H+ exchanger in the plasma membrane, and the lowering of intracellular Ca2+ levels (exact mechanism unknown, ?). Subsequent activation of the MAPK and PI3K pathways results in the phosphorylation of extracellular signal-regulated kinases (ERKs) and protein kinase B (PKB), respectively. Activated PKB will subsequently phosphorylate and thereby inhibit the action of glycogen synthase kinase 3beta (GSK3beta), a major kinase in the brain. © 2005 Nature Publishing Nature Reviews Drug Discovery 4, 107-120 (2005).

What Second Messengers Do GPCR Signals Trigger in Cells?

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Intrinsic receptors/Nuclear receptors Steroid signaling

Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com)

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General Introduction

Receptors

• Receptors that penetrate the plasma membrane and have intrinsic enzymatic activity • Receptors that penetrate the plasma membrane and have no intrinsic enzymatic activity • Receptors that are coupled, inside the cell, to GTP-binding and hydrolyzing proteins • Intrinsic receptors/Nuclear receptors

Pathways of Intracellular Signal Transduction

• The JAK/STAT Pathway • Ras, Raf, and the MAP Kinase Pathway • Phospholipids and Ca2+ • The cAMP Pathway: Second Messengers and Protein Phosphorylation • Cyclic GMP

Termination of Signal Transduction

Signal Transduction Pathways in Immunology

• TLR • TGFß • Cytokines • Apoptosis

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One Signal – diverse directions Main Intracellular Signaling Cascades downstream of RTK/PTK Activation

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1. JAK-STAT signaling

Nature Reviews Immunology 3, 900-911 (November 2003)

JAK: Janus Kinase

→PTKs →2 tandem-like kinase domains → JAK1,2,3 and Tyk2

STAT: signal transducers and activators of transcription

→transcription factor 1. JAK cross-phosphorylation = activation upon (dimeric) ligandbinding 2. JAK phosphorylates tyrosinresidue of receptor =activation 3. Binding of STATs with SH2-domain to receptor 4. Phosphorylation of STATs by JAKs 5. Dimerization of STATs and translocation to nucleus 6. activation of transcription

(dimerization)

(dimeric)

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2. Ras, Raf, and the MAP Kinase Pathway

One of the most common intracellular signaling pathways triggered by RTKs is known as the mitogen-activated protein (MAP) kinase cascade, because it involves three serine-threonine kinases. The pathway starts with the activation of Ras, a small G protein anchored to the plasma membrane. In its inactive state, Ras is bound to GDP. However, when SH2-containing proteins join with activated RTKs, they cause Ras to bind GTP in place of GDP and become active. Next, the GTP-bound Ras (which is not itself a kinase) activates the first serine-threonine kinase (e.g. Raf) in the MAP kinase cascade. Each of the three kinases in this cascade then activates the next by phosphorylating it. Because all three kinases in this pathway phosphorylate multiple substrates, the initial signal is amplified at each step. Then, the final enzyme in the pathway phosphorylates transcription regulators, leading to a change in gene transcription

Raf activation (= 1. PSK= MAPKKK/MAP3K)

Gbr2 and Sos

Adaptor with SH2-domains binds RTK Two SH3 domains bind Sos

Guanine nucleotide exchange factor (GEF)

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MAPK-signaling

The MAPK signaling cascade. The simplified MAPK pathway (left): complex parallel and crossover signaling cascades link the three main MAPK families, ERK, JNK, and p38, which are activated by cytokines, stress, and growth factors (right). The top level shows the MAP3Ks, the second tier shows the MKKs, and the third tier comprises the MAPKs (ERK, JNK, p38) that regulate various genes through phosphorylation of transcription factors (e.g. c-Jun, ATF2) and other kinases (MAPKAPK2/MK2). The primary, but overlapping, responses include cell growth and differentiation (ERK), matrix regulation (JNK), and inflammatory cytokine production (p38). Abbreviations: ATF2, activating transcription factor 2; ERK, extracellular signal-related kinases; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAPK, MAPK-activated protein kinase; MAP3K, MKK kinase; MEK, MAPK and ERK kinase; MK2, MAPKAPK2; MKK, MAPK kinase.

Nature Clinical Practice Rheumatology (2007) 3, 651-660

Common second messengers

Second messengers are intracellular molecules that change in concentration in response to environmental signals. That change in concentration conveys information inside the cell.

21 Biochemistry, 5th edition Jeremy M Berg, John L Tymoczko, and Lubert Stryer.

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One of the most widespread pathways of intracellular signaling is based on the use of second messengers derived from the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is a minor component of the plasma membrane, localized to the inner leaflet of the phospholipid bilayer. A variety of hormones and growth factors stimulate the hydrolysis of PIP2 by phospholipase C—a reaction that produces two distinct second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP3). Diacylglycerol and IP3 stimulate distinct downstream signaling pathways (protein kinase C and Ca2+ mobilization, respectively), so PIP2 hydrolysis triggers a two-armed cascade of intracellular signaling.

3. Phospholipids and Ca2+: PLC-PKC signaling

Hydrolysis of PIP2 is activated downstream of both G protein-coupled receptors and protein-tyrosine kinases. This occurs because one form of phospholipase C (PLC-β) is stimulated by G proteins, whereas a second (PLC-γ) contains SH2 domains that mediate its association with activated receptor protein-tyrosine kinases. This interaction localizes PLC-γ to the plasma membrane as well as leading to its tyrosine phosphorylation, which increases its catalytic activity.

The Cell, 2nd edition A Molecular Approach Geoffrey M Cooper. Boston University Sunderland (MA): Sinauer Associates; 2000.

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Ca2+ is pumped from the cytosol into the endoplasmic reticulum, which therefore serves as an intracellular Ca2+ store. IP3 binds to receptors that are ligand-gated Ca2+ channels in the endoplasmic reticulum membrane, thereby allowing the efflux of Ca2+ to the cytosol.

Phospholipids and Ca2+

IP3 triggers Ca2+ mobilization

The Cell, 2nd edition A Molecular Approach Geoffrey M Cooper. Boston University Sunderland (MA): Sinauer Associates; 2000.

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Function of calmodulin Calmodulin is a dumbbell-shaped protein with four Ca2+-binding sites. The active Ca2+/calmodulin complex binds to a variety of target proteins, including Ca2+/calmodulin-dependent protein kinases.

Phospholipids and Ca2+

Ca2+ activates intracellular Kinases

Many of the effects of Ca2+ are mediated by the Ca2+-binding protein calmodulin, which is activated by Ca2+ binding when the concentration of cytosolic Ca2+ increases to about 0.5 μM. Ca2+/calmodulin then binds to a variety of target proteins, including protein kinases. One example of such a Ca2+/calmodulin-dependent protein kinase is myosin light-chain kinase, which signals actin-myosin contraction by phosphorylating one of the myosin light chains. Other protein kinases that are activated by Ca2+/calmodulin include members of the CaM kinase family, which phosphorylate a number of different proteins, including metabolic enzymes, ion channels, and transcription factors.

e.g. activation of transcription factor NFAT (nuclear factor of activated T-Cells)

Biochemistry, 5th edition Jeremy M Berg, John L Tymoczko, and Lubert Stryer.

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Phospholipids and Ca2+

Activation of the transcription factor NFAT via Ca2+ dependent Proteins

Activation of Protein Kinase C (PKC): one Kinase with multiple targets

wikipedia

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The cAMP Pathway: Second Messengers and Protein Phosphorylation

In many animal cells, increases in cAMP activate the transcription of specific target genes that contain a regulatory sequence called the cAMP response element, or CRE. In this case, the signal is carried from the cytoplasm to the nucleus by the catalytic subunit of protein kinase A, which is able to enter the nucleus following its release from the regulatory subunit. Within the nucleus, protein kinase A phosphorylates a transcription factor called CREB (for CRE-binding protein), leading to the activation of cAMP-inducible genes. Such regulation of gene expression by cAMP plays important roles in controlling the proliferation, survival, and differentiation of a wide variety of animal cells.

Biochemistry, 5th edition Jeremy M Berg, John L Tymoczko, and Lubert Stryer.

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Cyclic GMP

Cyclic GMP (cGMP) is also an important second messenger in animal cells, although its roles are not as clearly understood as those of cAMP. Cyclic GMP is formed from GTP by guanylyl cyclases and degraded to GMP by a phosphodiesterase (=counteraction/termination of signal). Different types of guanylyl cyclases are activated by both nitric oxide (NO-GCs/soluble GCs) and peptide ligands (particulate GCs). Stimulation of these guanylyl cyclases leads to elevated levels of cGMP, which then mediate biological responses, such as blood vessel dilation. The action of cGMP is frequently mediated by activation of a cGMP-dependent protein kinase (PKG), although cGMP can also act to regulate other targets, including ion channels.

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General Introduction

Receptors

• Receptors that penetrate the plasma membrane and have intrinsic enzymatic activity • Receptors that penetrate the plasma membrane and have no intrinsic enzymatic activity • Receptors that are coupled, inside the cell, to GTP-binding and hydrolyzing proteins • Intrinsic receptors/Nuclear receptors

Pathways of Intracellular Signal Transduction

• The JAK/STAT Pathway • Ras, Raf, and the MAP Kinase Pathway • Phospholipids and Ca2+ • The cAMP Pathway: Second Messengers and Protein Phosphorylation • Cyclic GMP

Signal Transduction Pathways in Immunology

• TGFß • TLR • Cytokines • TCR/BCR

Fig. 1 Smad-dependent and Smad-independent pathways in TGF-β signaling. TGF-β binds a complex of transmembrane receptor serine/threonine kinases (types I and II) inducing transphosphorylation of the GS segments in the type I receptor by the type II receptor kinases. The activated type I receptors phosphorylate selected Smads. Receptor-activated Smads (R-Smads) form a complex with the common Smad4. Subsequently the Smad complexes translocate into the nucleus, where they regulate transcription of target genes. The structurally divergent inhibitory Smads (Smad6 and Smad7), negatively regulate TGF-β signaling. In addition, TGF-β signals through Smad-independent cascades (green) activating Erk, JNK, p38MAPK, Protein Phosphatase 2A (PP2A) and RhoA pathways.

Cardiovascular Research 74 (2007) 184–195

The TGFß pathway (Transforming growth factor beta)

Example for Serine/Threonine Kinase-Signaling

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PAMP: pathogen associated molecular pattern PRR: pattern recognition receptor

The Toll like receptor (= TLR) pathway(s)

Nature Reviews Immunology 13, 453–460 (2013) 31

The Toll like receptor (= TLR) pathway(s)

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Activation of NFκB

6.36 Die MyD88-unabhängigen Signale der TLR werden durch TRIF vermittelt. TLR-4 sendet auch Signale über den MyD88-unabhängigen Weg. In diesem Signalweg wird anstelle von MyD88 das Adaptorprotein TRIF, das eine TIR-Domäne enthält, zum Rezeptor mobilisiert. TRIF kann direkt an TRAF-6 binden und stimuliert dadurch die Aktivierung von NFκB. TRIF kann auch die beiden Serin/Threonin-Kinasen IκKε und TBK1 aktivieren. Die Aktivierung dieser Kinasen stimuliert den interferonregulierenden Faktor (IRF), einen Transkriptionsfaktor, der die Transkription des Gens für das Interferon IFN-β stimuliert.

6.35 Toll-ähnliche Rezeptoren aktivieren NFκB. Die Toll-ähnlichen Rezeptoren (TLR) aktivieren NFκB über einen Signalweg, dessen erste Stufen sich von dem Weg unterscheiden, der von den Antigenrezeptoren oder TNF-Rezeptoren ausgeht. Die TLR senden ihre Signale über die TIR-Domäne in ihren cytoplasmatischen Schwänzen. Die TIR-Domäne mobilisiert eine Familie von Adaptorproteinen, die ebenfalls eine TIR-Domäne enthalten. MyD88 ist von diesen Adaptoren am besten bekannt. MyD88 enthält neben seiner TIR-Domäne auch eine Todesdomäne (DD), durch die die Serin/Threonin-Kinase IRAK aktiviert und mobilisiert wird. Die aktivierte IRAK-Kinase mobilisiert das Adaptorprotein TRAF-6, das die Aktivierung von TAK1 stimuliert, eine MAPKKK. TAK1 stimuliert die Aktivierung von IKK, wodurch IκB zerstört und NFκB aktiviert wird. TAK1 stimuliert auch die Aktivierung der MAP-Kinasen JNK und p38.

Degradation of inhibitory complex allows NFκB to enter the nucleus

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Cytokine signaling

Dimeric ligands/ receptors Trimeric lignands/ receptors

Cytokines of hemopoetin family Cytokines of TNF/lymphotoxin family

Nature Reviews Cancer 10, 561-574 (August 2010)

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TCR signaling cascades

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BCR signaling cascades

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Termination of signal transduction

One method of terminating or stopping a specific signal is to degrade or remove the ligand so that it can no longer access its receptor. Further: deactivation of receptor, degradation/recycling of second messengers….and active inhibitory signaling! After a signaling process has been initiated and the information has been transduced to affect other cellular processes, the signaling processes must be terminated. Without such termination, cells lose their responsiveness to new signals. Moreover, signaling processes that fail to be terminated properly may lead to uncontrolled cell growth and the possibility of cancer.

Dephosphorylation (Protein phosphatases: turnover of IP3>>Inositol) Protein degradation (Ubiquitin ligation: e.g. by Cbl) cAMP is degraded into AMP by phosphodiesterase, and the release of calcium stores is reversed by the Ca2+ pumps that are located in

the external and internal membranes of the cell.

Attenuation of immune receptor signaling: Lymphocyte activation needs to be tightly controlled

How to limit the immune response to foreign antigens? How to prevent reactions against self antigens?

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Summary

Signal transduction: cell communication : 6 steps! 4 types of signaling: • endocrine, paracrine, autocrine, plasma-membrane attached

Receptors: • penetrate the plasma membrane: extracellular domain; intracellular domain • With intrinsic enzymatic activity or without ( couple to PTKs like JAK) • Kinases: activation via phosphorylation of proteins (serin, threonin, tyrosin residue) • GPCRs: 7 transmenbrane protein, interacts with G-Proteins: Gα,β,γ, GDP-GTP exchange • hormone-receptors: ligand passes the membrane, binds receptor, complex enters nucleus and activates transcription of target genes

Main intracellular signal transduction downstram of RTK/PTK: • JAK-STAT: Janus kinase activates transcriptionfactor STAT, dimerization of STAT • MAPK signaling: 3 consecutive serin-threonin-kinases: MAP3K>MAPKK>MAPK TRKs/PTKs first activate Ras (G-Protein!!) which then activates the Kinase-cascade • PLC-PKC : triggered by GPCRs (PLC β) and RTKs/PTKs (PLC γ) activation of PLC>hydrolysis of PIP2 > 2 second messengers: IP3 and DAG two-armed signaling cascade! 1)IP3: Ca2+ efflux from ER> activation of Ca2+ -dependend enzymes 2)DAG (associated with membrane): activates Protein Kinase C (PKC)

Principles of termination of signal transduction

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Summary

Signal transduction pathways in immunology • TGFß: upon ligand binding to type I and II receptors (RSKs)>multimeric receptor-complex Type II activates Type I> activates R-SMADs + co-SMADs > translocation to nucleus> transcription • TLR pathway(s): 10 receptors human/11 mouse , activated by microorgnisms and their products (PAMPS) they activate different intracellular cascades via TIR-domain NFκB: inhibitor (IκB) is degraded by IKK and allows NFκB to enter nucleus • Cytokine signaling: dimeric/trimeric; associated with PTKs • TCR and BCR: large receptor-complexes which trigger mainly 2 intracellular signaling cascades: PLC-PKC and MAPK leading to transcriptional activation by 3TFs/TF-complexes: NFκB, NFAT, AP-1 !

Please mind the complexity of the latter ones……!!

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Thank You