MICRB 410 Lecture 22-23 ch 7 exam 3

Three phases of signaling

reception
transduction
response

What is an important part of transduction?

amplification of the signal

Signal Transduction
fn/purpose
in general, how it works
how is the effect induced?
exs. in eukaryotic cells

Signal transduction pathways relay information from the cell surface to the nucleus
There is a cascade of events that provide a pathway from the original event to activation of a gene (see figure)
To induce the effect, the signal is amplified during signa

3 different ways in which a signal can be induced inwards

1)You can receptor binding and a single receptor activating one or more pathways,
2)you can two receptors that must function together to activate a single pathway,
3)You can have two signal pathways that are activated by different receptors and that cross

what is required for inward signaling?

amplification of the signal

General principles of signal transduction and propagation: RECEPTION
what happens

Transmembrane receptors convert extracellular signals into intracellular biochemical events

General principles of signal transduction and propagation: SIGNAL TRANSDUCTION
how is it mediated?
how does this occur?

Intracellular signal propagation is mediated by large multi-protein signaling complexes.
- This occurs through a variety of mechanisms (outlined on slide 16)

General principles of signal transduction and propagation: CELLULAR RESPONSES
3 possible responses

- altered gene expression and/or metabolism
- Functional activation
- Cell differentiation (results from altered gene expression) (from immature B or T cell to mature B or T cell)

General Scheme of T and B cell signal transduction
5 steps

Is recognition of an antigen by TCR or BCR enough to transduce a signal?
how is this fixed?

No, so TCR and BCR exist in complex with other signaling molecules

where do TCRs bind antigens?
BCRs?

T cell receptor binds peptide-antigens presented on MHC molecules
B cell receptor binds antigen in solution

Antigen-binding is translated into an effect via....
what is necessary for expression?
what is required to transduce a signal?

signaling molecules associated with TCR and BCR
assembly of the entire complex is necessary for efficient expression a t the cell surface
entire complex is required to transduce a signal inwards

T cell receptor complex
what does it consist of? (3 + 1)
what is it associated with? how?
what do these signaling modules have?
what allows for it to stay in the membrane?

T-cell receptor complex:
- TCR-? and TCR-? chains,
- CD3
- ?-chain (zeta) accessory molecules
�Short cytoplasmic tail
�Specifically ?? TCR is noncovalently associated with signaling modules: i) CD3?? - single dimer
ii) CD3?? - single dimer
iii) ?? - two c

what is in the transmembrane regions of TCRs that allow them to stay in the membrane?

Transmembrane regions have + or - changes allowing for interactions

B cell receptor complex
what does it associate with?
what do they each have?
what does a BCR recognize?
what does activation lead to?

� B cell receptor associates with invariant chains, CD79a and CD79b
�CD79a (Ig alpha) and CD79b (Ig beta) each have one ITAM
�BCR recognize and bind Ag
�Induces signaling cascade
�B Cell activation leads to clonal expansion and antibody production

does the cytoplasmic tail of BCR have signaling activity?

no

B cell receptor complex
what are BCRs associated with?
what causes changes in them?
what does this allow?

�BCRs are associated with two invariant chains, CD79a and CD79b
�Aggregation or conformational changes in the Ig molecule causes changes in the CD79a and CD79b heterodimer
�This allows antigen binding signal at surface to be transduced into the cell

7 signaling strategies used to transduce the signal

1)Dimerization leading to phosphorylation via kinases
2)Signaling molecules contain different binding domains that allow them to form large multi-protein complexes
3)Use of scaffolds and adaptor molecules to generate large signaling complexes
4)Small G-pr

_____ leads to phosphorylation via _____

dimerization leads to phosphorylation via kinases

3 steps of receptors with intrinsic kinase domains

1. in one class of recepors the kinase domain is an intrinsic part of the receptor
2. ligand binding dimerizes the receptor, activating the kinases which phosphorylate each other
3. the activated kinases phosphorylate downstream substrates

3 steps of noncovalently associated kinases

1. in another class of receptors, a kinase is noncovalently associated with the receptor
2. ligand binding dimerizes the receptor, activating the associatd kinases, which phosphorylate each other
3. the activated kinases phosphorylate downstream substrate

addition of phosphate group to an amino acid by proteins = ______

kinases

3 amino acids that can be phosphorylated

tyrosine
serine
threonine

2 ways kinases can interact with a receptor

kinase protein can be part of receptor or can be associated with receptor

what type of activity to kinases have?

kinases have intrinsic activity

what does receptor binding do in terms of kinases?

receptor binding results in close association of kinases and cross phosphorylation

what does phosphorylation by kinases produce?

produces binding sites for other proteins (adaptor molecules)

proteins that phosphorylate other proteins = ___

kinases

important feature of phosphorylation

it is rapid and can be reversed by phosphatases

what is phosphorylation?
(def)

addition of a phosphate

what kind of activity do kinases have?

intrinsic activity

what is intracellular signal propagation mediated by?
how do these complexes form?
ex of one of these domains?

intracellular signaling is mediated by large multiprotein complexes
These complexes form via the interaction of different proteins via domains within each protein. ?
ex. SH2 domain (a phosphtyrosine domain)

Signaling proteins contain ________ that allow them to interact with signaling molecules at different stages of the signal cascade

domains

Assembly of signaling complexes is mediated by ______

scaffold and adaptor proteins

scaffold fn
adaptor prot fn

Scaffolds function to bring many different signaling proteins together
Adaptor proteins function to bring two different proteins together

goal of scaffolds and adaptor prots

build large protein subunits (complexes) that transduce signal

scaffold protein steps to activation
3

unphosphorylated scaffold
activation of protein kinase results in phosphorylation of a scaffold
the phosphorylated scaffold recruits signaling proteins that bind to it

adaptor protein ex: Grb2
activation steps (3)

the adaptor Grb2 binds to the signaling protein Sos via its SH3 domain
an activated receptor becomes tyrosine phosphorylated
Grb2 binds to the phosphotyrosine via is SH2 domain, bringing Sos to the receptor

G Proteins
1. fn
2. inactive state
3. what activates them
4. other activity they have and what they do with this

1. Small G proteins also play a prominent role in signaling and often act as molecular swtiches
2. These proteins are bound to GDP in their inactivate state
3. When GEF proteins are activated, they act on the small G proteins and catalyze the exchange of

activation and inactivation of G proteins
4 steps

Ras
1. what type of protein is it
2. where does it play a role/what it does
3. related genes where?
4. mutant forms are often found where?

1. small GTP binding protein
2. oncogene in tumor inducing viruses
3. related genes in all euk cells
4. mutant forms found in many tumors

what are GTP binding proteins activated by?

by GEF (Guanine-nucleotide Exchange Factor)

what are GTP binding prots key components of?
ex?

Key components of several signaling pathways
ex. MAP kinase pathway (Mitogen Activated Protein kinases)

3 mechanisms by which signaling proteins are recruited to the membrane

1) Binding to P sites on membrane associated proteins (this could also be done by an adapter or scaffold protein)
2) Recognition of activated small G proteins embedded in the membrane
3) binding of membrane lipids that are produced during signaling.

Ras activation stimulates the ______ and induces expression of _____

Ras activation stimulates the mitogen-activated protein kinase (MAPK) relay and induces expression of the transcription factor AP-1.

ubiquitination of proteins
in what ways can this be used?

activating or inhibitory--can be used to activate or inhibit different signaling cascades

Ubiquitin conjugation of proteins be used to both ____ and ____ responses

inhibit and activate signaling

3 types of ubiquitination

mono, di, multi

effect of polyubiquitination
effect of mono or di ubiquitination

Polyubiquitination-->Degradation in proteasome
Modification of membrane receptor receptors with mono or di-ubiquitination targets them for lysosomal degradation

Ubiquitination
what does it occur on?
where can it occur?

occurs on Lysine (K)residues
can occur at K48 or K63

K48 vs K63 fn

K48 targets proteins for degradation
K63 serves as scaffold for activation

effect of dephosphorylation

dephosphorylation inhibits signals

Recall: ubiquitin scaffolds play a role in ________
how?

activating NFkB
scaffold associates with proteins, bringing them in close proximity and allowing for phosphorylation

2 mechanisms of signal amplification

kinase cascades
generation of secondary messengers

Signal Amplification
2 ways and expl of each

1)Kinase cascades - one kinase phosphorylating and activating several others
2)Release of secondary messengers (i.e. Ca2+) = activate many other proteins

what is missing from this picture?

CD4 or CD8

antigen binding leads to ______ which leads to _____
What do ITAMS contain that allow them to function, and what is this function?
What do ITAMs serve as?
what part of the signaling cascade is this? what does it lead to?

Antigen-binding leads to phosphorylation of ITAMs which leads to activation of several signaling pathways
ITAMS contain tyrosines that can be phosphorylated and bind to other proteins that can be phosphorylated in turn
ITAMS serve as docking sites for oth

Engagement of ____ leads to phosphorylation of ITAMs in TCR complex
Recall: TCR cannot..

co receptors
recall: TCR can not signal alone

CD4 and CD8 are always associated with ___
what is this?

Lck
Lck = Src-family kinase

Phosphorylation of ITAMs in TCR complex
1. CD4 and CD8 are always associated with ___
2. what is this?
4 steps

1. CD4 and CD8 are always associated with Lck
2. Lck = Src-family kinase
Steps:
1. Binding of CD4 or CD8 to MHC brings Lck into close proximity with ITAMs
2. Lck phosphorylates the ITAMS
3. Co-receptor binding also stabilizes the interactions between TCR

Lck activity is itself regulated by ______

phosphorylation

where is Lck in inactive form?

Normally bound to CD4 and CD8 in inactive form

Lck
1. when/how is it in its inactive form?
2. what is responsible for this?
3. when is it primed?
4. what does full activation require?

1. When SH2 domain is phosphorylated, Lck remains in a folded inactive conformation
2. C-terminal Src kinase (Csk) is responsible for this phosphorylation (not depicted)
3. Lck is primed when CD45 dephosphorylates the SH2 domain (unclear when this occurs)

Phosphorylated ITAMs recruit and activate ______
4 steps

the tyrosine kinase ZAP-70
1. ZAP-70 has two tandem SH2 domains that bind phosphorylated ITAMs
2. After binging ITAM, ZAP-70 is phosphorylated by Lck at three sites
3. ZAP-70 is now fully active
4. ZAP-70 is now in close proximity to the cell membrane

TCR signalin initial steps video slide 32

...

2 essential functions of activated ZAP-70

1)ZAP-70 phosphorylates two scaffold proteins:
2) PI-3 Kinase is recruited to cell membrane and activated

Activated ZAP-70 fn: ZAP-70 phosphorylates two scaffold proteins
what are the two proteins?
how are these proteins linked?
what does this form and what is its function?

1. transmembrane LAT (linker of activated T cells)
2. adaptor protein SLP-76
LAT and SLP-76 are linked by adaptor proteins Gads
- form LAT:Gads:SLP-76 complex�this will form a scaffold that will bind other prots

Activated ZAP-70 fn: PI-3 Kinase is recruited to cell membrane and activated
potential mechanism
what does PI-3 do?

unknown mechanism, but potentially via recruitment of Ras to LAT
PI-3 phosphorylates PIP2 to PIP3 (these lipids are bound in the cell membrane)

where are PIP2 and PIP3 located?

bound in the cell membrane

4 different signal cascades that get activated in a T cell

AS A RESULT OF ZAP-70 acting on LAT and SLP-76 to form the LAT-GADS-SLP-76 complex, and the activation of PI-3 Kinase several down stream signaling pathways are activated!!!! These include...
1) Activation of PLC-?? = altered transcription via NFAT, NFkB,

T Cell Signal Cascade 1: PLC-??
3 things it leads to

production of secondary messengers:
induces calcium influx into cell and leads to NFAT activation
leads to Ras activation and the expression of AP-1 transcription factor
results in activation of protein kinase C-?? (PKC-??) which induces NFkB activation

T Cell Signal Cascade 1: PLC-??
Activation of PLC-?? leads to the production of _____
steps 1-4 (of 6)

secondary messengers
1.Phospholipase C-?? (PLC- ??) binds PIP3 on membrane via PH domain, SH2 and SH3 domains interact with SLP-76
2.Itk also binds PIP3 and binds SLP-76,
3.Ikt phosphorylates PLC- ??
4.Activated PLC- ?? cleaves PIP2 into diacylglycerol (D

T Cell Signal Cascade 1: PLC-??
Activation of PLC-?? leads to the production of _____
steps 5 and 6

5.DAG remains in membrane and recruits signaling molecules
6.IP3 diffuses into cytosol

T Cell Signal Cascade 1: PLC-??
At this stage, activated PLC-y activates ____ which each activate ____
and what are they?

At this stage, activated PLC-?? activates 3 signaling pathways which each activate 3 different transcription factors
i) Stimulation of Calcium entry = leads to NFAT activation
ii) Activation of Ras = activates MAPK = induces AP-1 expression
iii) Activatio

T Cell Signal Cascade 1: PLC-??--pathway 1: stimuation of calcium entry
6 steps

1.IP3 diffuses into cytosol and binds IP3 receptors on ER membrane
2.IP3 receptors = stimulates opening of calcium channels, that open to release Ca into cytosol
3.Low Ca in ER, induces STIM1 conformation change and clustering (shown in red)
4.STIM1 oligo

T Cell Signal Cascade 1: PLC-??--pathway 1: stimuation of calcium entry
1. PLC-?? induced Calcium influx into cell leads to activation of _____
2. phosphorylation of NFAT = ?
3. what does influx of calcium induce?
4. what does this molecule do in turn?
5.

1. NFAT
2. �Phosphorylation of NFAT = inactivate state
3. influx of Ca induces conformation change in calmodulin
4. Calmodulin activates calcineurin which dephosphorylates NFAT
5. NFAT is activated and moves to the nucleus to induce gene expression

T Cell Signal Cascade 1: PLC-??--pathway 2: Ras activation and expression of AP-1 transcription factor
5 steps

PLC-?? activation leads to Ras activation and the expression of AP-1 transcription factor
1. DAG in membrane recruits RasGRP which is a GEF
2. RasGRP binds and activates Ras in the cell membrane
3. Ras activates first protein in mitogen-activated protein

T Cell Signal Cascade 1: PLC-??--pathway 3: activation of protein kinase C-?? (PKC-??) which induces NFkB activation
6 steps

PLC-?? signaling pathway results in activation of protein kinase C-?? (PKC-??) which induces NFkB activation
1. PLC-?? leads to activation of PKC-?? (isoform of protein kinase C)
2 PKC-?? is only found in T cells and muscle cells
3. PKC-?? via PH domain b

what does activation of PLC- lead to?

0

what does Akt activation lead to?

alters metabolism

what does recruitment of ADAP do?

upregulates adhesion

what does activation of Vav do?

initiates actin polymerization

T Cell Signal Cascade 2: Akt activation
what does it do?
2 steps to Akt activation
2 things activated akt does

Akt activation upregulates cellular metabolic pathways
1. Akt and PDK1 bind PIP3 in membrane (PIP3 is produced by PI-3 kinase)
2. PDK1 phosphorylates Akt, activated Akt:
1)Inhibits cell death via phosphorylation of Bad, this frees Bcl-2 allowing it to pro

T Cell Signal Cascade 3: recruitment of ADAP
what does this lead to?
4 steps

Recruitment of ADAP leads to enhanced integrin-mediated cell adhesion
1. ADAP is recruited to LAT:Gads:SLP-76 complex
2. ADAP recruits RIAM and SKAP55,
3. ADAP:RIAM:SKAP complex binds small GTPase, Rap1 and activates it
4. Activate Rap1 promotes LFA-1 agg

T Cell Signal Cascade 3: recruitment of ADAP
why is this impt?

Enhanced binding is required to promote signal transduction and activation of the T cell

T Cell Signal Cascade 4: activation of Vav
what does this do?
7 steps

1. Formation of immune synapse (clustering of activated receptors and co-receptors on both T cell and APC) is required for activation
2. Upon TCR activation, Vav is recruited to cell membrane via PIP3 binding
3. WASp is also recruited via Nck which binds

Summary of TCR activation downstream effects

what else is required for full activation of T cells? (in general and specifically)

co stimulatory signals
specifically, CD28 co stim receptor

What does CD8 bind on dendritic cells?

CD28 on T cells binds B7.1 (CD80) and B7.2 (CD96) on dendritic cells

steps of T cell activation after CD28 binds B7
3 steps

1. After binding B7, Lck phosphorylates the cytoplasmic tail of CD28
2. Phosphorylated CD28 recruits and activates PI-3 kinase (unknown mechanism) to generate PIP3
3. This activates 3 of 4 signaling pathways in TCR activation (in pic)

which 3 pathways does CD28 co stimulation activated?

CD28 Co-Stimulatory Receptor is Required for full activation of T cells video slide 50

...

what, together, induce IL-2 gene expression?

NFAT, AP-1, and NFkB act together to induce IL-2 gene expression

what is IL-2?

a cytokine

what is fundamental to T cell activation?

IL-2 expression

IL-2
1. contains several...
2. what binds the promoter?
3. what else induces expression?
4. what else leads to increased IL-2?

1. IL-2 promoter contains several regulatory elements
2. Oct1 produced constitutively in lymphocytes and binds IL-2 promoter
3. Other sites bound by NFAT, AP-1 and NFkB to induce IL-2 expression
4. TCR activation leads to low level IL-2, CD28 activation l

By further promoting ___, ___, and ____, ____increases IL-2

NFAT, AP-1 and NFkB, CD28

BCR
what else is attached to it?
what is necessary for expression?
what is required to transduce signal inwards?
where are ITAMs?

Surface Ig
with CD79a and CD79b
Assembly of the entire complex is necessary for efficient expression at the cell surface
Entire complex is required to transduce a signal inwards
ITAMs in CD79a/b

B cell Receptor Complex
1. what it associates with
2. where ITAMs are/how many
3. what does BCR do/recognize?
4. what leads to activation?
5. what does activation lead to?

1. B cell immunoglobulin associates with invariant chains, CD79a and CD79b
2. CD79a (Iga) and CD79b (Igb) each have one ITAM
3. BCR recognize and bind Ag
4. signaling cascade induces B cell activation
5. B Cell activation leads to clonal expansion and Ab

what does BCR use to phosphorylate ITAMs?
to transduce signal?

BCR activation utilizes Blk, Fyn, and Lyn to phosphorylate ITAMS and Syk to transduce the signal

BCR activation
1. Blk, Fyn, Lyn = ?
2. normally associate with what? when do they become activated?
3. what antigen binding induces?
4. syk = ?
5. what syk does
6. and 7. last 2 steps

1. Src family kinase
2. Normally associated w ITAMs and become activated upon antigen binding by BCR
3. Associated with CD79a/b, antigen binding induces clustering and cross phosphorylation of ITAMs
4. Syk - tyrosine kinase binds ITAMs in CD79 and becomes

what forms the B cell co receptor?

CD19, CD21, and CD81

B cell co-receptor
1. what is the complex? what does it bind?
2. results in what?
3-5 next steps

1. Co-receptor complex of CD19, CD21, and CD81 bind to complement C3 cleavage product C3dg on pathogen
2. Results in clustering of co-receptor with BCR, Blk can then phosphorylate CD19 cytoplasmic tail
3. Syk binds ITAMs in CD79
4. Syk phosphorylates SLP-

BCR and B cell co receptor complex activation
1. activates similar pathways as in what?
2 -5. . steps of activation
6. in T cells vs B cells

1. BCR and B-cell co-receptor complex activation activates similar pathways as in T cells
2. PIP3 is bound by PDK1 and Akt. PDK1 activates Akt = ? cell death via Bcl-2 and? biosynthesis via mTOR
3. Bkt recruited to membrane via PIP3 and SLP-65. Bkt phosph

components of T cell vs B cell signaling pathway
1. what phosphorylates ITAMS
2. what are the co receptor?
3 others

B cell activation is facilitated by _____ cells
1. what cells receive these signals?
2. what is one signal?
3. what is another signal?
4. CD40 function

CD4+ T cells
1. Na�ve B cells receive additional activation signals CD4+ T cells:
2. One signal= TCR-MHC engagement = cytokines
3. Another signal = stimulation of CD40 on B cells by CD40 ligand on T cells
4. CD40 increases NFkB expression and PI 3-kinase

B cell activation facilitated by CD4+ T cells
1. CD40 analogous to what on T cells?
2. central role of CD40
3. the role of what CD40 activates
4. Akt role/fn
5. as shown previously, CD40 also does what?

1. CD40 on B cells is analogous to CD28 on T cells
2. Activating PI 3-kinase is the central role of CD40
3. Major effects of PI 3-kinase are exerted via activation of Akt through PIP3 production that recruits PDK1 and Akt
4. Akt enhances survival via Bcl-

2 mechanisms by which T an B cell activation is regulated

1) Inhibitory Receptors
2) Immunoreceptor Tyrosine-based Inhibitory motifs (ITIM)

Regulation of immune response via inhibitory receptors
1. how do they do this?
2. two receptors that can regulate signaling
3. which is most well characterized?

1. Inhibitory receptors on lymphocytes downregulate immune response by interfering with co-stimulatory signaling pathways
2. Two receptors can regulate signaling:
-CTLA-4 on T cells
-PD-1 on T and B cells
3. CTLA-4 is most well characterized

CTLA-4
1. location
2. where does it move and why?
3. what does it have high affinity for? therefore what is its function?
4. overall function

1. CTLA-4 located in intracellular vesicles
2. CTLA-4 moves to cell surface after appropriate signals
3. CTLA-4 has higher affinity for B7.1 than CD28, causing it to cluster and preventing interaction with CD28
4. Reduces CD28 co-stimulation

______is expressed transiently on T and B cells and contains an _____

PD-1
ITIM motif

PD-1 and ITIM Motif
1. what is PD-1?
2. what does they cytoplasmic tail contain?
3. what happens when ITIM is phosphorylated?
4. SHP =?
5. SHIP = ?
6. what PD-1 binds and what the result of this?

1. PD-1 is an inhibitory receptor expressed transiently on T and B cells
2. Cytoplasmic tail contains immunoreceptor tyrosine-based inhibitory motif (ITIM)
3. When ITIM is phosphorylated recruits SHP & SHIP
4. SHP = tyrosine phosphatase, removes phosphate

summary slides 67 and 68

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study questions slide 69

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