Trends Biochem. Sci.

Immunological Synapse Formation between T and APC

Science 2002 295:1539

Immunological Synapse

???TCR

IL-2

APC

Signal Transduction Determines Cell Response

1. Role of cytokines in immune system and hematopoiesis

2. Basic concepts of signal transduction

3. JAK-STAT pathway in cytokine signaling

4. Regulation of cytokine response

5. Negative regulators for cytokine signal transduction, SOCS and PIAS families

OUTLINE

General Properties of Cytokines

1. Polypeptides

2. Produced in response to microbes and antigens

3. Mediate and regulate immune and inflammatory responses

4. Pleiotropic and redundant

5. Regulate the synthesis and actions of other cytokines

6. Actions are local or systemic (autocrine, paracrine, or endocrine)

TNF, IL-1, Chemokines

IFN

IL-12

Modified from Cell. Mol. Immunol. Abbas & Lichtman 5th Ed. 2003

NK

DC

Neutrophils

Macrophage

Blood Vessel

TLR

NK

R

TLR

Cytokines/Chemokines Involved in Innate Immunity

Cytokines/Chemokines Involved in Innate Immunity

TNF

IL-1

IL-12

IFN

IFN

Chemokine

Cytokine Producers Targets:Effects

MØ, DC, T cells Neutrophils: activation Endothelial cells: activation (inflammation) Hypothalamus: Fever Many cells: apoptosis

MØ, endothelial Ditto

Modified from Cell. Mol. Immunol. Abbas & Lichtman 5th Ed. 2003

MØ, DC T cells & NK: IFN synthesis and CTL activity T cells: Th1 differentiation

IFN: MØ All cells: antiviral state, increase MHC I IFN: fibroblast NK cells: activation

NK, NKT MØ: activation (increased microbicidal)T cells, CD8 B cells: isotype switching to IgG2A

Many cells: Increase MHC I & MHC II

MØ, endothelial Leukocytes: chemotaxis, activation, Fibroblast, T cells migration to tissues

IL-2

CD4+ T Cell

IL2, IFNIL4, IL5

Plasma Cell Ab production

B Cell

BC

R

How Adaptive Immunity Works

T CellTCR

MHC:peptide

DC

Macrophage

Modified from Cell. Mol. Immunol. Abbas & Lichtman 5th Ed.

CD8+ CTLGranzyme,Perforin

IL2,

IFN

CD8+

Cytokines Involved in Adaptive Immunity

IL-2

IL-4

IL-5

Lymphotoxin

IFN

Cytokine Producers Targets:Effects

T cells T cells: proliferation, cytokine production B cells: proliferation, antibody production NK: proliferation and acitvation

Th2 B cells: isotype switching to IgE T cells: Th2 differentiation, proliferation

Modified from Cell. Mol. Immunol. Abbas & Lichtman 5th Ed. 2003

Th2 Eosinophils: activation, increase prod B cells: proliferation, IgA production

T cells Recruitment and activation of neutrophils

Th1, CD8 MØ: activation (increased microbicidal) NK, NKT B cells: isotype switching to IgG2A

Many cells: Increase MHC I & MHC II

Roles of Cytokines in Hematopoiesis

Cell. Mol. Immunol. 2003 Abbas & Lichtman 5th ed.

What Happens When Ligands Bind to Receptors

Conformational change of the receptors: Opens ion channel Facilitates binding of intracellular signaling proteins s

uch as chemokine receptor

Dimerization (Oligomerization) of the receptors: Bring signaling molecules into juxtaposition

-induces post-translational modification such as phosphorylation in the receptors or signal meidators

-activates downstream mediators

Phosphorylation of Proteins as a Controling Mechanism for Signal Transduction

Advantages

Rapid: does not require new protein synthesis or protein degradation

Reversible : easily reversed by action of protein phosphatases

Easy to relay signals: phosphorylation on Tyr, Thr, or Ser creating binding sites for other proteins

Tyrosine Phosphorylation Initiates Signaling(In general)

PTK : Protein Tyrosine KinasePTP : Protein Tyrosine Phosphatase

PTP

Active Form

P

Note: Src-family kinase

PTK Inactive Form

Signal mediator

Regulation of Activity of Src Family Kinase

Src family:

B cells: Lyn, Fyn, BlkT cells: Lck, Fyn,

Immunobiology 6th ed. 2005, Janeway et al.

Major Events in Signal Transduction Mediated by Receptor Tyrosine Kinase (RTK) or non-RTK

Ligand-induced receptor dimerization

Activation of kinases

Activation of signal mediators

Activation of transcription factors

Translocation of transcription factors into

nucleus and transactivation

Cytokine Receptor-mediated Signaling Pathways

PP

STAT2

JAK1 TYK2

IFNIFNRR

STAT1

PP

STAT3

PPCytosol

Nucleus

ISREISRE

STAT2STAT2

p48p48

PP

PP

STAT1STAT1STAT3STAT3

STAT1STAT1GASGAS GASGAS

STAT3STAT3PP

STAT1STAT1

STAT1STAT1GASGAS

STAT3STAT3PP

PP

PP

PP

PP

Interferon- Receptor Signaling Pathway

Kinase

Signal Transducer

Activator ofTranscription

Chris Schindler

Cloning STAT1

Xin-Yuan Fu

STAT2

David Levy

STAT3

Jim Darnell

Rockefeller U

Biochemical Approach

George Stark

Cleveland Clinic Fund

Ian Kerr

ICRF UK

Genetic Approach

Sandra Pellegrini

Biochemical Approach for Elucidating IFN Signaling Pathway

HeLa

IFN or IFN

Analysis promoter of IFN-inducible genes

Consensus binding sequence

GAS (gamma activated site): TTN 4-6 AAISRE (IFN-stimulated response element):AGTTN3TTC

Purification of ISGF3 or GAF Using GAS or ISRE-column

Microsequencing and cDNA cloning

ICR191 (frame shift mutagen)

Mol. Cell. Biol. 5th ed. 2004 Lodish et al.

Genetic Approach for Elucidating IFN Signaling Pathway

Complementationgroup

Response to Ligands

IFN IFNComplementing

Protein

U1

U2U3U4U5

U6

12

- + TYK2

IRF9(P48)STAT1JAK1IFNAR2

STAT2

JAK2

IFNGR2

--

±----

-++

++

±-

Complementation Groups for IFN (U1-U6) or IFN ( and ) responses

± :indicated that some genes do not respond, whereas others do respond well

Q: Why there is no STAT3 mutation in the complementation group ?

Modified from Science 1994 264:1415

Animation for JAK-STAT signaling

Structural and Functional Domains of JAK Family

JAK family : JAK1, JAK2, JAK3 and TYK2

Nat. Rev. Mol. Cell Biol. 2002 3:651

Nat. Rev. Mol. Cell Biol. 2002 3:651

Structural and Functional Domains of STATs

STAT family: STAT1, STAT2, STAT3, STAT4,

STAT5A, STAT5B, and STAT6

Activation of JAKs and STATs by Cytokines

Modified from Gene 2002 285:1

Ligand Jak kinases STATs

IFN familyType I IFN- IFN or IFN Tyk2, Jak1 STAT1, STAT2Type II IFN-IFN Jak1, Jak2 STAT1

C familyIL-2 Jak1, Jak3 STAT5IL-4 Jak1, Jak3 STAT5IL-7 Jak1, Jak3 STAT5

gp130 familyIL-6 Jak2 STAT3IL-11 Jak2 STAT3

Cytokine Receptor Families by Their Structures

(wsxws motif for -helical cytokine)

(serpentine)

Molecular Structure of Class I Receptors

Cytokine Growth Factor Rev 2001 12:19

One chain for Ligand Binding

One chain for Signal transducing

Nat. Rev. Immunol.2001 1:200

Critical Roles of C Family in Lymphocyte Development and Function

N. Eng. J. Med. 2000 343:1313

SCID Resulting from Defects in IL-7R, JAK3 or c Chain

Nat. Rev. Mol. Cell Biol. 2002 3:651

Phenotypes of JAK-Knockout Mice

Prevention of Organ Allograft Rejection by a Specific Janus Kinase 3 Inhibitor

Science 2003 302:875

NHP: non-human primates

Low dose

high dose

Nat. Rev. Mol. Cell Biol. 2002 3:651

Phenotypes of STAT Knockout Mice

Nature Genet. 2003 33:388 (U. Paris)

Impaired Response to IFN/ and Lethal Viral Disease in Human STAT1 Deficiency

Negative Regulation of the JAK-STAT pathway

Receptor-mediated endocytosis and degradation

Dephosphorylation by tyrosine phosphatases

Naturally occurring dominant negative STATs such as STAT1 and STAT3 that don’t have transactivating domain

Suppressor of cytokine signaling (SOCS) family

Protein inhibitor of activated Stats (PIAS)

Cell 2002 109:S121-S131

Negative Regulations of Cytokine Signaling

Nat. Rev. Immunity 2003 3:900

Structural and Functional Domains of SOCS Family

Nat. Rev. Immunol. 2002 2:410K: Kinase inhibitory region

Inhibitory Mechanisms of SOCS Molecules

Trend. Immunol. 2003 24:659

SOCS Family Members Target Signaling Proteins for Degradation by Proteasome

Trends Biochem. Sci. 2002 27:235

SOCS Targeting Key Signaling Proteins for Degradation by the Proteasome

Sci. STKE, Vol. 2003 169:pe6

Liver Degeneration and Lymphoid Deficiencies in Mice Lacking SOCS1

Proc. Natl. Acad. Sci. 1998 95: 14395

SOCS1-/-

SOCS1+/+

SOCS1 Is a Critical Inhibitor of IFNSignaling and Prevents Fatal Neonatal Actions of this Cytokine

Cell 1999 98:597

JBC 2006 281:11135

Nat. Rev. Mol. Cell Biol. 2002 3:651

Phenotypes of SOCS Knockout Mice

Hypersensitivity of SOCS-1 KO Mice to LPS In Vivo

Immunity 2002 Vol. 17:583

SOCS1 Negatively Regulates TLR Signaling by Mal/TIRAP Degradation

Nat. Immunol. 2006 7:148 Forward

TLR Signaling Pathways

Cell Death Diff. 2006 13:816

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SOCS1 Is a Suppressor of Liver Fibrosis andHepatitis-induced Carcinogenesis

JEM 2004 199:1701

Nat. Rev. Immunol. 2005 5:593

SAP: Scaffold Attachment factor A and B, Acinus, and PIASRLD: RING finger-like Zinc binding domainAD: Acidic domain SIM: SUMO- interacting motifS/T: Serine and Threonine-rich

Structural and Functional Domains of PIAS FamilySIM

Nat. Rev. Immunol. 2005 5:593

PIAS-protein Regions Involved in Protein–protein Interactions

Nat. Rev. Immunity 2003 3:900

Proposed Mechanisms for Inhibiting the JAK–STAT Pathway by PIAS Proteins

Negative Regulation of PIAS on Gene Transactivation

Nat. Rev. Immunol. 2005 5:593

Positive Regulation of PIAS on Gene Transactivation

Nat. Rev. Immunol. 2005 5:593

Some but not All IFN-inducible Genes Are Enhanced in Pias1-/- MEF

Nat. Immunol. 2004 5:891

Enhanced Antiviral Response and Sensitivity to LPS-induced Toxic Shock in Pias1-/- Mice

Nat. Immunol. 2004 5:891

Enhanced Expression of NF- B-Regulated Genes in Response to TNF- and LPS in Pias1 null Cells

Mol. Cell. Biol. 2005, 25:1113

PIAS1 Affects the Binding of p65 to the Endogenous Promoters Revealed by ChIP Assays

Mol. Cell. Biol. 2005, 25:1113

Nat. Rev. Immunol. 2005 5:593

Specificity of PIAS1-mediated Transcriptional Repression in STAT1 and NF-B Signaling

Proc. Natl. Acad. Sci. USA 2004 101:99

PIAS1/PIAS3 Activates Smad Transcriptional Activity

Suggested Readings

1. JAK-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins Science 1994 264:1415

2. STAT: transcriptional control and biologic impact. Nat. Rev. Mol. Cell Biol. 2002 3:651

3. Regulation of gene-activation pathways by PIAS proteins in the immune system

Nat. Rev. Immunol. 2005 5:593

Textbook:

Immunobiology Janeway 6th ed. 2005, Chapter 6

Cellular and Molecular Immunology 5th Ed. 2003, by Abbas and Lichtman, Chapter 11

R-SMAD: Receptor-regulated SMAD ex. SAMD2 and SMAD3

Co-SMAD: Common-mediated SMAD ex. SMAD4

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Cross-link protein to DNA

Break cells and sonicate chromatin

Add pre-blocked protein A/G beads

Add primary Ab

Immunoprecipitate and enrich chromatin

Degrade protein and reverse crosslinks

Detect specific DNA with PCR

Chromatin Immunoprecipitation (ChIP) for Detecting Protein-DNA Interactions in Vivo

http://www.activemotif.com/catalog/cell_biology/chipit

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