Lecture 10 and 11 - Signaling in the Immune System

What are the two forms that kinases can have in signal transduction?

Intrinsic receptor tyrosine kinases (ex: FLT3, c-Kit)

Noncovalently associated kinases (ex: TCR, BCR, JAKs)

What ligand class does the protein domain SH2 bind?

phophotyrosine

What ligand class does the protein domain SH3 bind?

proline

What ligand class does the protein domain PH bind?

phosphoinositides

What is the role of adaptor proteins?

they promote the interaction of multiple signaling molecules

ex: LAT in TCR signaling

What is the role of G proteins/GTPases in signaling?

they serve as molecular switches

• Ras family proteins

• GDP bound form is inactive, GTP bound form is active

• GEFs catalyze binding of GTP (ex: Sos)

• GAPs accelerate GTP to GDP hydrolysis

How are signals transduced from outside of the cell to inside the cell?

signaling molecules are recruited to membrane

What allow for cells to be repeatedly responsive?

the signaling must be turned on and off

• phosphatases vs kinases (ex: SHP - phosphatase)

• polyubiquitination → proteasomal degredation (ex: Cbl - ubiquitin ligase) 

• single/di-ubiquitinated proteins are targeted for degredation in lysosome

Amplification cascades

cause large cellular response after signal is initiated

Why does the TCR associate with ITAM containing proteins?

TCR doesn’t have intracellular signaling domains

TCR cannot reach cell surface without CD3 chains

• CD3 chains + ζ chains = 10 ITAM motifs

BCR complex with ITAM proteins

Igα and Igβ each have one Ig like domain and one ITAM domain

• signaling is initiated by Src-family kinase-mediated phosphorylation

What is the role of Lck in TCR signaling?

Lck is a src-family tyrosine kinase that phosphorylates ITAMs on CD3 chains

What happens after CD3 ITAMs are phsophorylated by Lck?

The ITAMs recruit ZAP-70, which is also phosphorylated by Lck

How is Lck activity regulated?

2 regulatory tyrosines: Y505 and Y394

• Y505 is inhibitory when phosphorylated, CD45 phosphatase dephosphorylates it to prime Lck

• Csk kinase can re-phosphorylate Y505

• Y394 is auto-phosphorylated by Lck to become active

What is kinetic proofreading in TCR signaling?

the TCR must be engaged with the MHC for a sufficient “dwell time” in order to initiate signaling

How does Zap70 become activated?

it is initially autoinhibited, but phosphorylated ITAMs recruit it and it uses SH2 domains to interact with the ITAM before being activated by Lck

What types of receptors do ITAM domains activate?

NK cells, macrophages, neutrophils, mast cells, basophils, etc

What does ZAP-70 do after it is activated?

It phosphorylates scaffold proteins LAT and SLP-76

How does ZAP-70 lead to PLC-γ activation?

1. LAT and SLP76 are joined by Gads

2. inactive PLC-γ is recruited to the complex by PIP3 (which comes from PI3K)

3. Itk is recruited by PIP3 and SLP76

4. Itk phosphorylates and activates PLC-γ

How does PLC-γ initiate a large Ca2+ influx into the cell?

• PLC-γ cleaves PIP2 to generate IP3 and DAG

• IP3 binds recptors in the ER membrane that cause the release of calcium

• depletion of ER stores causes aggregation of STIM1

What is the significance of STIM1 in TCR signaling?

it binds to ORAI1 in the plasma membrane to form the CRAC channel, which enables lots of Ca2+ to enter the cell.

What is the significance of IP3?

IP3 binds receptors in the ER membrane to trigger calcium release into the cell

• IP3 comes from PIP2 cleaved by PLC-γ

What is the significance of DAG?

• DAG activates PKCθ and RasGRP (a Ras GEF) which leads to the activation of transcription factors

• DAG activates Ras which initiates a downstream MAPK cascade involving Raf, Mek, and Erk

• Erk then enters the nucleus and activates genes via phosphorylation

• it also causes activation of NFkB

How does transcription factor NFAT become activated?

1. serine-threonine phosphorylation retains NFAT in cytoplasm

2. when Ca2+ binds calmodulin, it then activated calcineurin (serine/threonine phosphatase)

3. calcineurin dephosphorylates NFAT

What does Ras signaling result in?

activation of transcription factor AP-1

• Erk phosphorylates Elk-1, a TF that induces FOS

• JNK is activated by PKC-θ, it then enters the nucleus and phosphorylates c-Jun

• JUN/FOS dimers = AP-1

What 3 transcription factors are critical for turning on genes involved in T cell activation?

NFAT, AP-1, and NFκB

What are the 4 things that TCR activation induces?

1. Transcription factor activation

2. metabolic changes and increased T cell survival

3. tight integrin adhesion

4. cytoskeletal reorganization

What is the significance of AKT?

AKT leads to enhanced T cell survival

• it phosphorylates Bad, which releases pro survival Bcl2

AKT alters cellular metabolism

• cause release of GAP from Rheb, active Rheb drives mTOR activation

• induces expression of nutrient transporters and activity of glycolytic enzymes supporting cell growth

• metabolic change allows lymphocytes to proliferate once every 4-8 hrs when active

How is AKT activated?

1. activation of PI3K generates, PIP3

2. PIP3 recruits PDK1 which activates AKT

What 4 things result from mTOR activation?

• increased lipid production

• increased ribosome biosynthesis

• increased mRNA synthesis

• increased protein translation

How does TCR signaling promote tight integrin adhesion?

Rap1 activation

1. LAT:Gads:SLP-76 recruits ADAP

2. ADAP activates Rap1

3. Rap1 causes accumulation of the integrin LFA1 and conversion to a high affinity conformation (allows tight adhesion of T cell to APC)

What is the significance of Vav in TCR activation?

Vav causes changed in actin via Cdc42

1. Vav (a GEF) is recruited to PIP3 and LAT:Gads:SLP-76 complex

2. WASp  is recruited to the complex

3. Vav activates g protein Cdc42, which activates WASp

4. WASp recruits proteins that induce actin polymerization

what does a WASp deficiency cause?

• Wiskott-Adich syndrome

• absence of WASp causes low platelet counts, Ig deficiency, snd viral susceptibility

What is the cause and effect of the immunological synapse?

Cause = TCR stimulation

Effect = directional secretion

• both actin cytoskeleton and microtubules must reorganize in order to point at target cells

What is the cSMAC and pSMAC

cSMAC = central supermolecular activation complex

pSMAC = peripheral supermolecular activation complex

How is the BCR complex similar to TCR?

• the signaling process is homologous

• BCR associates with ITAM containing Igs (alpha and beta)

• syk = ZAP-70 homolog

• ITAMs phosphorylated by Blk, Fyn, or Lyn

Summary of Antigen receptor signaling

TCR/BCR → Tyrosine kinases → adaptors and scaffold proteins → phospholipases and lipid kinases → GTPases, serine/threonine kinases, phosphatases → transcription factors, cytoskeletal changes, adhesion, metabolism

What is required for PI3K activation?

T cell co-stimulation through CD28 is required for PI3K activation

What is the significance of CD28?

• CD28 binds B7.1 and B7.2 on APC and is phosphorylated by Ick

• CD28 recruits and activates PI3K, which phosphorylates PIP2 to generate PIP3

What are the roles of PIP3?

1. recruits AKT and PDK1 leading to AKT activation → survival and metabolic changes

2. binds Itk, which activates PLC-γ → TF activation

3. binds Vav, enabling Cdc42 activation and Wasp activation → cytoskeletal changes

What happens when TCR and CD28 work together?

They transduce all signals needed for IL-2 transcription

IL-2 is critical for T cell proliferation

How does the TNFR superfamily augment B cell activation?

CD40L on T cells activates CD40 on B cells

• in absence of CD40, NIK is K48 ubiquitinated and degraded (CD40 ligation causes NIK stabilization)

• NIK activates IKKα, which phosphorylates p100 (NFkb precursor)

• P100 is cleaved to form p52, which makes a dimer with relB

• CD40 activation is essential for activation of naive B cells

What is the significance of CTLA-4?

CTLA-4 is an inhibitory receptor that inhibits T cell activation

• it isn’t recruited to the plasma membrane until TCR is activated

• has a higher affinity for B7.1 and B7.2 than CD28

• outcompetes CD28 binding for B7 to inhibit T cells

• critical to prevent uncontrolled T cell proliferation

What receptors are related to CD28, except they are inhibitory?

CTLA-4, PD-1, and BTLA

What does it mean when a receptor has ITIM or ITSM motifs?

ITIM containing receptors inhibit activation of multiple immune cells

• recruit SHP and SHIP (phosphatases)

• long isoforms of KIR inhibit NK cells by binding self MHC-I molecules

• PD-1 recruits SHP2, which dephosphorylates CD28

What molecule is inhibitory for BCR signaling?

FcγRIIB - keeps antigens present as immune complexes from activating naive B cells

What are CAR T cells?

Chimeric antigen receptor T cells

• understanding lymphocyte signaling has led to their design

• they are used for treatment of immune system diseases