MICRB 410 Exam 3

Three phases of cell signaling

1. reception
2. transduction
3. response

Second messengers

small molecules that relay signals from cell-surface receptors to effector proteins in signal transduction
ex: Ca2+

Is recognition of antigen by TCR and BCR enough to transduce a signal?

No, it is insufficient. There are additional signaling molecules that are required to transduce the signal

Do TCR and BCR have ITAMS?

No, other molecules associated with the receptors do

ITAM

immunoreceptor tyrosine-based activation motif

What is the TCR complex made of? Where are the ITAMs located?

TCR
CD3δε--single dimer (2 ITAMs)
CD3γε-single dimer (2 ITAMs)
two ζ chains (3 ITAMS each = 6 ITAMs)

total of 10 ITAMs
TCR + CD3 = TCR complex

What is the BCR complex made of?

BCR associated with CD79a(Igα) and CD79b (Igβ) and they each have one ITAM

How do ITAMs activate signaling pathways?

Each ITAM has two tyrosines (Y) that are phosphorylated by ZAP-70 when the ligand binds

ZAP-70 function and structure

A kinase that phosphorylates ITAMs
Has a kinase domain and two SH2 domains
The SH2 domains bind to the Tyrosine domains on the ITAMs and phosphorylates them

What does signal strength depend on?

1. affinity of receptor for ligand
2. concentration of signaling molecules
3. positive and negative feedback pathways

What are the three amino acids that can be phosphorylated?

tyrosine, serine, and threonine

What are the two ways that receptors are activated by kinases and the signaling pathway is activated?

1. Receptors have kinases as intracellular domain. When the ligand binds, the receptors dimerize and the kinases phosphorylate each other and the activated kinases phosphorylated downstream substrates

2. Kinase is bound noncovalently to intracellular tail of receptor. When the ligand binds, the kinases phosphorylate each other due to close proximity. Activated kinases phosphorylate downstream substrates

Phosphatases

remove phosphate groups, important part of phosphorylation that makes it rapid and reversible

Phosphotyrosines (pY)

binding sites on ITAMs for SH2 domains

Adaptor proteins

Help to assemble signaling complexes
Have many sites of phosphorylation (so they can be activated by kinases) and lack enzymatic activity
Function by recruiting other proteins to signaling complex and those proteins bind to the phosphorylated sites of the adaptor protein

Grb2

adaptor protein with an SH2 domain and 2 SH3 domains
Binds to Sos protein with its SH3 domain and binds to phosphorylated kinase domain on receptor to transduce a signal

Ras

small GTP binding protein (GTPase)
inactive when bound to GDP and active when bound to GTP

GTPase

small GTP binding protein that catalyzes the hydrolysis of GTP to GDP

What activates Ras?

GEF removes GDP so that Ras can bind GTP and become active

What is the GEF for Ras?

Sos

GAPs

GTPase-activating proteins that accelerate the conversion of GTP to GDP

What are the three ways that signaling proteins are recruited to the membrane?

1. Tyrosine phosphorylation of membrane associated adaptor proteins that recruits phosphotyrosine-binding proteins
2. Recognition of activated GTPases
3. PI3K phosphorylates PIP2 to make PIP3 which is recognized by signaling proteins like Akt and Itk

Akt and Itk

signaling proteins that recognize PIP3

PI3K

PI3-Kinase that phosphorylates PIP2 to make PIP3

What are the two parts of signal amplification?

1. signal cascade
2. release of second messenger

Calmodulin

Ca2+ binding protein that binds effector proteins

SHP

a phosphatase that plays a role in shutting down signaling pathways

What can a deficiency in phosphatases cause?

autoimmunity and cancer

Ubiquitination. What does it do in signaling pathways?

covalent attachment of one or more ubiquitin
can be used to activate or inhibit signaling responses

Polyubiquitination

marks protein for degradation in proteasome

E3 ubiquitin ligase

transfers ubiquitin to protein

K48 linkages and K63 linkages for polyubiquitination

K48 = targets proteins for degradation

K63= serves as scaffold for activation

TRAF-6

E3 ubiquitin ligase

Explain the NFkB activating pathway

1. TLRs dimerase and recruit IRAKs which activate E3 ubiquitin ligase TRAF-6
2. TRAF-6 is polyubiquilated and creates scaffold for TAK1 activation
3. TAK1 associates with IKK and phosphorylates IKKβ which phosphorylates Iκβ
4. Iκβ is degraded and NFκB is released into the nuclease to induce expression of cytokine gene

Lck

a kinase that phosphorylates 3 additional sites to fully activate ZAP-70
always associated with CD4 or CD8

What are the two TCR co-receptors?

CD4 and CD8

How do T cell Co-receptors help the phosphorylation of ITAMS in the TCR complex?

1. Lck attached to co-receptor
2. co-receptor associates with MHC and Lck is now close to ITAMs
3. Lck phosphorylates ITAM of TCR complex
4. ZAP-70 binds to phosphorylated ITAMs
5. Lck phosphorylates ZAP-70

How does Lck associate with a co-receptor?

Has a unique amino-terminal motif that has two cysteine residues that bind a Zn ion that is also bound to a similar motif in the cytoplasmic domain of CD4 or CD8

CD45

dephosphorylates the SH2 domain of Lck to make it primed

Csk

phosphorylates SH2 domain of Lck to make it inactive

What are the two functions of activated (phosphorylated) ZAP-70?

1. phosphorylates LAT and SLP-76 (two adaptor proteins)
2. recruits PI3-K to cell membrane

Gads

link LAT and SLP-76 together

Explain the activation pathway of PLC-γ with ZAP-70 and LAT/SLP-76

1. ZAP-70 phosphorylates LAT and SLP-76 which then come together by Gads
2. LAT:Gads:SLP-76 complex and PIP3 accumulate at plasma membrane
3. PIP3 recruits Itk and LAT:Gads:SLP-76 recruits PLC-γ and they bind via phosphorylated sections
4. PLC-γ is activated by phosphorylation by Itk

PLC-γ

adaptor protein that binds PIP3 and LAT:Gads:SLP-76 and is activated by Itk
cleaves PIP2 to make DAG and IP3
activates 3 signaling pathways that activate 3 different transcription factors

What does cleaved PIP2 make and where do these pieces go?

DAG --> remains in membrane and recruits signaling molecules
IP3--> diffuses in cytosol

What are the three signaling pathways that PLC-γ activates?

1. stimulation of Ca2+ entry = NFAT activation
2. Activation of Ras = activates MAPK = induces AP-1 expression
3. Activation of protein kinase C-θ (PKC-θ)= activates CARMA1 and induces NFκB activation

Explain PLC-γ activation of Calcium

1. PLC-γ is activated and cleaves PIP2 to make DAG and IP3
2. IP3 diffuses into cytosol and binds to IP3 receptors on ER
3. These receptors are Ca channels so the binding stimulates the to open and release Ca into the cytosol
4. Low Ca in the ER induces STIM1 which binds to ORAI1 on plasma membrane
5. ORAI1 is Ca channel and opens to let Ca in from extracellular. The influx of Ca activates signaling pathway and replenishes ER ER Ca

Explain activation of NFAT with activated PLC-γ

1. NFAT is phosphorylated and inactive
2. Ca enters cell and binds to calmodulin which binds to NFAT by phosphatase calcineurin. The phosphatase dephosphorylates NFAT
3. NFAT can now enter nucleus and active gene transcription

Explain Ras activation and expression of AP-1 by PLC-γ

1. DAG on membrane binds to RasGRP (GEF) and activates Ras
2. Ras binds to Sos and enhances its activity
3. Ras activates Raf, which phosphorylates Mek, which phosphorylates Erk
4. Phosphorylated Erk enters nucleus and activates transcription factor Erk-1 to produce AP-1

What for activation pathways does ZAP-70 and LAT and SLP-76 initiate?

1. activation of Akt
2. Activation of PLC-γ
3. Vav activation
4. ADAP activation

Explain how PLC-γ activation results in activation of PKC-θ (C-θ) and induces NFkB production

1. PKC-θ binds to PH domain of DAG which is in membrane
2. PKC-θ phosphorylates CARMA1 which creates a scaffold for TRAF-6 which is polyubiquitinated
3. TAK1 phosphorylates IKKβ, then IκB which induces IκB ubiquitination and degradation which releases NFκB which activates transcription in the nucleus.

What does the activation of these 4 proteins do?

PLC-γ
Akt
Vav
ADAP

PLC-γ = transcription factor activation
Akt = increased cellular metabolic activity and promotes cell survival
Vav = actin polymerization and cytoskeletal reorganization
ADAP = enhanced integrin adhesiveness and clustering

Explain Akt activation from ZAP-70 and LAT/SLP-76

1. Akt and PDK1 bind to PIP3 in the cell membrane
2.PDK1 phosphorylates Akt
3. Phosphorylated Akt phosphorylates Bad which releases Bad from Bcl-2.
4. Bad binds 14-3-3, which promotes cell survival
5. Akt phosphorylates a GAP for Rheb which allows Rheb to bind to mTOR for increased cellular metabolic activity

Explain ADAP activation from ZAP-70,SLP-76 and LAT

1. ADAP recruited to LAT:Gads:SLP-76 complex and binds to phosphorylated parts
2. ADAP recruits other proteins that activate Rap1 (GTPase)
3. RAP1 induces LFA-1 aggregation and conversion to the high-affinity binding state which increases adhesiveness and promotes stability of immune synapse

Explain Vav activation from ZAP-70, SLP-76 and LAT

1. Vav recruited to cell membrane and binds to PIP3 and the LAT:Gads:SLP-76 complex
2. Vav activates Cdc42 (small GTPase) which binds to and activates WASp
3. Activated WASp recruits others proteins that lead to actin polymerization

What does defects in WASp cause?

Wiscott-Aldrich syndrome - defect in the formation of immune synapse and defects in antibody response

What happens to ADAP deficient cells?

impaired proliferation and cytokine production upon TCR stimulation

B7.1

Co-stimulatory ligand that binds to CD28 on T cells that help with activation

B7.2

Co-stimulatory ligand that binds to CD28 on DCs

What does B7 binding with CD28 cause?

PI3-kinase activation and recruitment of Lck

IL-2

-essential for T cell activation
-TCR activation leads to low IL-2 and CD28 activation leads to increased IL-2
-NFAT, AP-1, and NFkB all bind to promoter of IL-2 to induce expression

What proteins phosphorylate ITAMs in BCR activation?

Blk, Fyn and Lyn

What protein transduces the signal in BCR activation?

Syk

What forms the B-cell co-receptor complex?

CD19, CD21 and CD81

Bkt

Binds to PIP3 on membrane and activates PLC-γ

What does a deficiency in Bkt cause?

X-linked agammaglobulinemia, characterized by lack of antibodies

Explain BCR activation by CD40 and CD40L

CD40 L on T cell and CD40 on B cell bind which ultimately activated NFkB

CTLA-4

inhibitory receptor on T cells for B7
has slightly higher affinity for B7 than CD28, so it reduces CD28 co-stimulation

PD-1

-inhibitory receptor on T and B cells
-has ITIM on tail (inhibitory motif)

What happens when ITIM is phosphorylated

recruits SHP and SHIP, two phosphatases

SHP

tyrosine phosphatase that turns off signaling

SHIP

inositol phosphatase that removes phosphate from PIP3 to produce PIP2 to reduce Akt recruitment and activation

Checkpoint Blockade

inhibiting inhibitory receptors to enhance T cell responses

Where are B and T lymphocytes made?

Central (primary) lymphoid tissues (PLO)
B cells: bone marrow
T cells: thymus

Where do mature B and T cells go after PLO?

Secondary/Peripheral Lymphoid Organs (SLO) such as the lymph nodes, spleen, etc.

Where are antigens? SLO or PLO?

SLO

What is the ancestor cell of B and T cells?

Multipotent hematopoietic stem cell

Negative v. Positive selection of lymphocytes

Negative selection = BCRs and TCRs that interact strongly with self-antigens are eliminated to prevent autoimmune reactions

Positive selection = BCRs and TCRs that interact weakly with self-antigens survive

Where does BCR and TCR selection occur?

selection for BCR in bone marrow and selection for TCR in thymus

What cells do activated B cells give rise to?

plasma cells or memory B cells

Explain the B Cell Life Cycle

1. B cell precursor (bone marrow), Ig genes rearrange to make BCR
2. negative selection and immature B cell formation
3. migration to SLO and mature B cell forms by binding to antigen
4. activated B cell differentiates into plasma cell or memory cell

Stromal Cells

specialized network of non-lymphoid connective tissue that are essential in lymphocyte development

Steps of B-Cell Development

1. Lymphoid-Myeloid primed multipotent progenitor binds to FLT3 to ligand on bone marrow stromal cell to make CXCL12 and Il-7 receptor
2. CLP (on stromal cell) binds to VCAM-1 and CAM
3. Kit on B cell binds to SCF on stromal cell which activates kinase and proliferation of B cell progenitors
4. Late pro-B cell
5. Pre-B cell
6. Immature B cell release from stromal cell with IgM as receptor

What does a deficiency in IL-7 and the receptor cause?

block in B cell development and less T cells

During B cell development, which chains of the receptor rearrange first and second?

Heavy chain first and then light chain

Heavy chain VDJ rearrangement

D-J
V-DJ
VDJ

Light chain VJ rearrangement

V-J
VJ

What does the B cell receptor do to test for correct rearrangement?

Makes two 'surrogate' proteins that have a structural resemblance to the light chain and can pair with μ chain

What happened to the child that inherited defective alleles of the λ5 gene?

-no pre-B-cell receptor development
-B-cell immunodeficiency so many infections

What do BLNK and Btk do in B-cell signaling?

suppress RAG-1 and RAG-2 expression

Deficiency of BLNK

block of B-cell development at pro-B cell stage

Mutation in Btk

Bruton's X-linked agammaglobulinemia (XLA)- no mature B cells

Steps of making sure Ig genes were rearranged correctly in B cells

1. pre-B cell tests heavy chain gene
2. VpreB and λ5 made and bind to heavy chain to make pre-B cell receptor
3. complex associates with other receptors and dimerizes by surrogate invariant chains
4. dimerization phosphorylates ITAMs
5. BTK and BLNK suppress RAG 1 and 2

Allelic exclusion

only one allele of a gene is expressed while the other allele is silenced

Results of Heavy chain and light chain rearrangement. What can be done if non-productive rearrangement occurs in either?

Heavy chain produces both productive and non-productive rearrangements (50% of pre-B cells survive)

Light chain produces non-productive and can rescue it by further rearrangement (multiple attempts)

Isotypic exclusion

only one type of light chain is used

What are the two checkpoints to determine fate of B cell

1. capacity of heavy chain to bind the surrogate light chain and make pre-B cell receptor
2. capacity of binding the light chain to pre-B cell heavy chain and then asses B-cell receptor

What are glycoproteins, proteoglycans, and glycolipids

self-antigens

What is tolerance

reactivity to self-antigens

What does the fate of the immature B cell depend on when it comes to tolerance?

signal delivered from sIgM
-no strong reactivity to self-antigen --> mature
-strong reactivity to self-antigen --> clonal deletion or receptor editing