MCDB 436 (12): Intracellular Signaling (II)

transcription factors

proteins that regulate the transcription of genes
- turn on/off specific genes by binding to the DNA
- activator+enhancer and repressor+silencer

activators

bind to enhancer; activate the downstream gene expression. binds with specific DNA sequences and recruits RNA polymerase or other activators/protein complexes to make RNA

repressors

bind to silencers; TF acts as a repressor to prevent downstream transcription

combinatorial regulation

TFs need to bind in a specific combination to turn on/off gene expression
- use of multiple TFs to regulate a gene means that different sources of info can be integrated into a single outcome

NFkB TFs

central regulators of innate and adaptive immune functions; consist of NFkB1, NFkB2, ReIA, ReIB, and c-Rel

NFkB1 and NFkB2

p50/p52; lack transcriptional activation domains; their homodimers act as repressors

Rel-A, Rel-B, c-Rel

carry transcriptional activation domains
- can form homo- and heterodimers with other members of Rel (except Rel-B)

activation of NFkB signaling pathway

most common inducible NFkB binding activity: p50/p65 homodimers (NFkB1 and RelA)

- unstimulated cell: NfkB dimers inactive in cytoplasm because of IkB (kB inhibitors)

- ligand binding (TNFa, IL-1, CD40L, LPS) -> begin recruitment and activation of IKKs -> phosphorylate IkB -> degradation of IkB by ubiquitin -> NFkB activation

IKKs

IkB kinases; phosphorylate IkB to signal for ubiquitin degradation, allowing for NFkB activation

IkB

kB inhibitors; present in unstimulated cells on NFkB dimers

NFkB - epithelial cells

IL-8, adhesion molecules, IL1, IL2, TNFa, IL-12
- important components of innate immune response to invading microorganisms; required for ability of inflammatory to migrate where NFkB is activated

T-bet

immune cell-specific member TF
- induced in T cells (with many methods)
- induced in B cells through STAT1 after BcR and/or IFNyR ligation

regulates TH1 differentiation; balances terminal differentiation and memory cell potential in CD4 and CD8 T cells; in CD8, prevents cell exhaustion

immune receptor types

- Ig: ligand binding
- transmembrane signaling protein: with ITAM/ITIMs, proximity to Src family kinase

ITAMs

activating tyrosine containing motif attached to cytoplasmic tails of transmembrane signaling proteins

contains TWO tyrosine residues

ITIMs

inhibiting tyrosine containing motif attached to cytoplasmic tails of transmembrane signaling proteins

TcR complex

- signaling: initiated by ITAMs in CD3 epsilon, gamma, delta, and zeta (3) chains - 10 total
- motif also on BcR, NKcR

CD4 and CD8

- CD8: alpha-beta heterodimer (or alpha-alpha), each contains one Ig domain
- CD4: monomeric, contains 4 Ig domains
- cytplasmic tail binds to Lck

Lck

mediates ITAM phosphorylation and downstream transcription regulation
- Src family kinase
- attached to CD4 and CD8 cytoplasmic tails

ITAM phosphorylation pathway

TcR and co-receptor brought together by binding to peptide:MHC complexes ->
recruit Lck (on CD4/8 tail) ->
phosphorylation of ITAMs in CD3 chains (y, d, e, z) ->
recruit PTKs + ZAP-70 ->
ZAP-70 binds to phosphorylated ITAMs through SH2 domains ->
enables ZAP-70 phosphorylation and Lck activation ->
phosphorylates SLP-76 and Lat ->
joined by Gads to create LAT:Gads:SLP-76 complex ->
downstream effects

ZAP-70

PTK Syk family protein associated with the zeta chain of CD3

phosphorylates LAT and SLP-76

ITAM:SH2 domains

ITAM has 2 tyrosine residues, providing sites for recruitment of SH2 domains - such as ZAP-70

ZAP-70 phosphorylation + LAT:Gads:SLP-76 complex

phosphorylates LAT and SLP-76; linked by Gads
LAT:Gads:SLP-76 complex

1) Akt activation -> increased cellular metabolic activity
2) PLC-gamma activation -> TF activation
3) Vav activation -> actin polymerization and cytoskeletal reorganization
4) ADAP recruitment -> enhanced integrin adhesiveness and clustering

LAT

linker for activated T cells; transmembrane protein with large cytoplasmic domain (scaffold proteins)
- multiple docking sites; can serve as a docking site for other proteins

PLC-gamma signaling

splits into
1) stimulation of Ca2+ entry
2) activation of Ras
3) activation of protein kinase C (PKC)

PLCgamma1 - Ca2+ and PKC signaling pathway

Lck -> ZAP-70 -> LAT -> PLCgamma1 -> PIP2 breakdown

1) IP3 -> Ca2+ -> stimulates STIM1, CRAC/ORAI1, calmodulin (CaM), calcineurin (CaM-dependent protein phosphatase), NFAT (nuclear factor of activated T cells)

2) DAG -> PKC, NFkB, Ras

ORAI1

plasma membrane calcium channel (aka Ca release-activated Ca channel)

PLC-gamma -> catalyze PIP2 breakdown -> DAG + IP3 -> less ER Ca2+ -> STIM1 binds to ORAI1 -> Ca2+ enters cytosol from ECF

DAG

confined to the membrane; product of PIP2 by PLC-gamma catalyzation
- diffuses in the plane of the membrane and serves as a molecular target that recruits other signaling molecules to the membrane

IP3

diffuses into the cytosol; product of PIP2 by PLC-gamma catalyzation
- binds to IP3 receptors on ER

STIM1

transmembrane protein that clusters in the ER membrane as a result of low Ca2+ in the ER
- binds to ORAI1 -> Ca2+ channel opening -> Ca2+ enters cytosol from ECF

NFAT

nuclear factor of activated T cells; regulated by Ca2+ signaling
1) phosphorylation on Ser and Thr residues keep NFAT in cytoplasm of unstimulated cells

2) Ca2+ entry activates Ser/Thr phosphatase calcineurin -> dephos's NFAT

3) dephos'd NFAT enters nucleus -> activate gene transcription

inhibited by cyclosporin A and FK506

Ras activation and MAPK pathway

Lck -> mediate ITAM phos -> ZAP-70 + LAT -> Grb2 + SOS (adaptor protein) -> Ras activation -> MAPK cascade -> AP-1

AP-1

transcription heterodimer composed of c-Fos and c-Jun
- participates in turning on transcription of many genes important for T-cell activation (ex: IL-2)

T cell signaling process

- receptor-ligand binding
- dimerize (cross-link or conformational change)
- signaling pathways
- activate transcription factors
- translocation to nucleus
- affect gene transcription

T cell transcription factors

NFkB, NFAT, AP-1

B cell signaling

BCR complex with co-receptor

- cross-linking of BCR; co-receptor of BCR; phosphorylation of ITAMs tyrosines by Src-family kinases (Lyn, Fyn, Blk)
- include adaptors and scaffold proteins

- PLC-gamma activation -> IP3 -> Ca2+ dependent enzymes
+ DAG -> PKC

- Ras pathway -> ERK, JNK

BCR complex

CR2-CD19-CD81 (C3d fragment attached to Ag)
cytoplasmic tail recruits Src family kinase; NOT Lck

B cell transcription factors

Myc, NFAT, NFkB, AP-1

co-stimulatory receptors

modulate Ag receptor signaling
- naive T: CD28
- naive T: TNFr CD40
- goal: enhance Ag receptor signals -> PI3K activation -> PIP3 -> T&B cell activation

CD28 signaling

CD28 with CD80/86 -> PI3-kinase -> PIP3 ->

(1) recruit PDK1 to phosphorylate and activate Akt
(2) recruit Itk to phosphorylate PLC-gamma
(3) recruit Vav -> Cdc24 activation

CTLA-4

inhibitory receptor signal competing for CD80/86 over CD28
- induced on activated T cells
- function controlled largely by regulation of its surface expression

PD-1

programmed death-1; inhibitory T cell receptor

BTLA

B and T lymphocyte attenuator; inhibitory receptor on T and B cells

Ag-receptor signaling pathways - summary

tyrosine kinases -> adaptors and scaffold proteins -> phospholipases and lipid kinases -> GTPases, ser/thr kinases, phosphatases -> TFs, cytoskeletal changes, adhesion, metabolism

cytokine receptors

- type I: hematopoietin family
- type II: IFN family
- TNFr family
- IL-1R family: Ig superfamily
- chemokine receptors: GPCR

JAK-STAT signaling

JAK = signal transducer
STAT = transcription activator

1) cytokine-mediated receptor dimerization
2) JAK phosphorylates tyrosine residues of cytokine receptor
3) STATs recruitment and JAK-mediated phosphorylation
4) dimerization of STATs
5) translocation into nucleus

JAK-STAT signaling in the immune system

- abnormal signaling = immune disorders

- regulated at different steps, such as suppressor of cytokine signaling (SOCS) proteins, protein inhibitor of activated STAT (PIAS) proteins, and protein tyrosine phosphatases (PTP)

- also regulated by post-translational modifications (phosphorylation, ubiquitylation)

STAT1 and STAT4

TH1 cell differentiation

IFN-gamma -> STAT1 -> T-bet -> TH1
IL-12 -> STAT4 -> T-bet -> TH1

STAT6

TH2 cell differentiation

IL-4 -> STAT6 -> GATA-3 -> TH2