Lecture 8

Question 1

Describe the cytosolic and nuclear phase of signal transduction

Answer 1

Cytosolic phase: occurs after the ligand has bound to the receptor, and a modified transcription factor (which was synthesized at the membrane receptor) travels through the cytoplasm towards the nucleus

Nuclear phase: the modified transcription factor is now in the nucleus and affecting the transcription of the target gene

Question 2

Signal transduction is a ____ of phosphorylation and dephosphorylation.

Answer 2

cascade

Question 3

Describe the structure of a signal transduction receptor and the 2 different things their extracellular domains recognize.

Answer 3

Receptors are generally transmembrane proteins present on the PM

The extracellular domains of these receptors recognize “soluble ligands” or “membrane structures of neighboring cells”

Question 4

compare the processes of “ligand binding” and “signalling” as they pertain to signal transduction (differences and similar actions)

Answer 4

Ligand binding involves a conformational alteration of the receptor

Signaling requires a ligand-induced “clustering of receptors” that is called “cross-linking”

BOTH processes result in changes in the cytosolic portion of the receptor that promotes interactions with other signalling molecules

Question 5

Define Nuclear receptors and how they are activated

Answer 5

Nuclear receptors are intracellular transcription factors that are activated by “lipid-soluble ligands” that can freely cross the PM (ex. estrogen, progesterone, retinoic acid, etc.)

Question 6

Describe the following major categories of receptors that are involved in signal transduction:

Non-receptor tyrosine kinase based receptors:

Tyrosine kinase receptors:

Nuclear receptors:

GPCR receptors:

Delta/Delta Notch receptors:

Answer 6

Non-receptor tyrosine kinase based receptors: Feature a transmembrane non-receptor tyrosine kinase based receptor that will become phosphorylated by a non-receptor tyrosine kinase (which is found in the cytoplasm of the cell) after it binds to a ligand

Tyrosine kinase receptors: features an intracellular tyrosine kinase domain that will conduct phosphorylation to carry out signal transduction. This is one big, connected unit of a receptor and tyrosine kinase (2 in 1 shampoo)

Nuclear receptors: a nuclear hormone freely passes through the PM, binds to a nuclear hormone in the cytoplasm, and then the complex enters the nucleus to affect gene transcription.

GPCR receptors: a GPCR ligand binds to a GPCR (has 7 transmembrane domains) and functions to create cAMP in the cytoplasm from ATP

Delta/Delta Notch receptors: A “notch ligand” on a neighboring cell cleaves the intramembranous “Notch” protein, allowing the newly cleaved IC notch subunit to enter the nucleus and affect gene transcription

Question 7

True or false:
non-receptor tyrosine kinase receptors will synthesize a tyrosine kinase to conduct phosphorylation in order to carry out signal transduction. explain.

Answer 7

False

Non-receptor tyrosine kinase receptors will work together with non-receptor tyrosine kinase molecules that are already present in the cytoplasm of the cell. These 2 are NOT connected, if they were connected, they would be referred to as a tyrosine kinase receptor

Question 8

Of the following protein kinases, state what molecules or subunits they respectively phosphorylate. Why are these phosphorylations important?

Tyrosine kinases:

Serine/threonine kinases:

Lipid kinases:

Answer 8

Tyrosine kinases: phosphorylate specific tyrosine residues

Serine/threonine kinases: phosphorylated Serine or Threonine residues

Lipid kinases: phosphorylate lipid substrates

These phosphorylations are important bc they all phosphorylate the cytosolic portion of the receptor and “initiate signal transduction”

Question 9

There are specific types of phosphatases for all types of protein kinases. what do they do and what type of role do they usually play in signal transduction?

Answer 9

these specific types of phosphatases REMOVE the phosphate residue, effectively modulating the signal transduction

It is no surprise that this usually plays an inhibitory role in signal transduction

Question 10

State 3 protein modifications that facilitate signalling events besides phosphorylation/dephosphorylation. Be sure to include what occurs after these different types of protein modifications are conducted

Answer 10

The covalent addition of ubiquitin molecules either targets the protein for degradation or drives signal transduction.

The covalent addition of Lipids may promote PM localization of the signalling molecule

The N-terminal tails of histones can undergo Acetylation and Methylation in order to modulate Genetic genetic processes

Question 11

The acetylation/methylation of the N-terminal tails of histones is meant to modulate which 3 genetic processes in a cell?

Answer 11

Gene expression

DNA replication

DNA recombination

Question 12

State the 2 components of signalling pathways

Answer 12

Modular signalling proteins

Adaptor proteins

Question 13

Eukaryotic signal transduction proteins are ____-based architectures. This means that they are built from combinations of what 2 domains?

Answer 13

Component

They are built from combinations of:
“Interaction Domains” and “Catalytic Domains”

(interaction domains put the reactants in the proper alignment while the catalytic domain is what actually pushes the reaction to occur)

Question 14

Define Allosteric Regulation and then describe what form/structure a signal transduction protein must be in, in order to conduct allosteric regulation

Answer 14

Allosteric regulation: alteration of the activity of a protein through the binding of an effector molecule at a specific site

Signal transduction proteins must be “intact”, meaning that they have both their interaction and catalytic domains, in order to conduct allosteric regulation

Question 15

Compare a protein’s output activity under basal conditions and when a specific set of input effector ligands are present.

Answer 15

A protein’s output activity under basal conditions is tightly repressed

A proteins output activity can be robustly activated by specific set of input effector proteins

Question 16

What group are the following families a part of? what is the main purpose of all of these?

Src family
Syk family
Tec family

Answer 16

Src, Syk, and Tec families are all “tyrosine kinase families”

Tyrosine kinase families are key players in the regulation of immune functions

Question 17

State the Domains that compose the molecular structure of the 3 families that are included in the tyrosine kinase families (be sure they are in the proper order)

Answer 17

Src family:
Unique Domain, SH3 domain, SH2 domain, and a Kinase domain
(U, 3, 2, K)

Syk family:
Unique Domain, SH2 domain, SH2 domain, and a Kinase domain
(U, 2, 2, K)

Tec family: Ph domain, Tec homology domain, Proline-rich peptide, SH3 domain, SH2 domain, and a Kinase domain
(PH, T, P, 3, 2, K)

Question 18

For the following Domain, state how many AA’s it is comprised of and what molecular structure it is responsible for reacting with.

SH2

Answer 18

SH2 domains are composed of about 100 AA’s

binds to phosphotyrosine-containing peptides

Question 19

For the following Domain, state how many AA’s it is comprised of and what molecular structure it is responsible for reacting with.

SH3

Answer 19

SH3 domains are composed of about 50 AA’s

binds to proline-rich stretches

Question 20

For the following Domain, state what molecular structure it is responsible for reacting with.

PH

Answer 20

PH domains recognize specific phospholipids

Question 21

Which tyrosine kinase family domain is present in the Syk family and binds to phosphotyrosine motifs in the “Ag receptor complex”?

Answer 21

SH2 domains

Question 22

State and describe the 5 functional domains of the Src

Answer 22

1. (N-terminal) SH4 domain: contains a “myristic acid moiety” which is essential for it’s localization to the inner surface of the PM
2. U domain (unique domain): provides the functional specificity to each member of the Src family
3. SH3 domain: binds to proline-rich sequences to mediate intra- and intermolecular interactions
4. SH2 domain: binds to phosphorylated tyrosine residues on Src and other proteins
   (a SH2-Kinase linker is between the SH2 and SH1 domains)
5. SH1 domain: a catalytic domain (also called Y416)
   (a small Y527 unit is between SH1 and the C terminus of the Src)

Question 23

Describe the configuration of Src in it’s inactive and active forms. What causes it to switch between these forms?

Answer 23

Inactive: balled up near the PM
(beginning at the PM)
The SH1-Kinase linker is bound to the SH3 domain (rolling the Src up)
Then the SH1 (Kinase) domain features an unphosphorylated Tyr(Y)416 site
Then a Tyr(Y)527 linker unit that is linked to the SH2 domain via a phosphate bond

Active: linear
all of the subunits are lined up and not bound to one another
The Tyr416 site is phosphorylated (making room for a functional catalytic pocket)
The Tyr 527 site is NOT phosphorylated (it’s phosphate bond that was linking it to the SH2 domain is now broken)

Phosphorylation is what switches between these 2 states

Question 24

Between the Tyr527 and the Tyr416 sites on Src, which of these is capable of autophosphorylation? Describe what the significance of the autophosphorylation is.

Answer 24

Tyr416 can be autophosphorylated,

This autophosphorylation activates Src by displacing the Tyr416 out of the binding pocket so that the substrate can access the binding site

Question 25

Between the Tyr527 and the Tyr416 sites on Src, which of these is the “Critical Site”? describe what the significance of the critical site is.

Answer 25

Tyr527 is the more critical site

Phosphorylation of the Tyr527 site, inactivates Src bc it adds a phosphate bond that links Tyr527 and the SH2 domain of Src. (this folds the Src into an inaccessible bundle)

Question 26

Many of the substrates that an active Src molecule can phosphorylate go on to become part of what process?

Answer 26

the signaling process

which often involve a cascade mechanism of sequential phosphorylation and dephosphorylation of proteins in the cascade

Question 27

True or False:

Adaptor Proteins play a part in the cascade mechanism of signalling by catalyzing different reactions in the pathway. Explain.

Answer 27

False

Adaptor Proteins DO NOT have any catalytic activity

Question 28

What type of interaction domains are found on adaptor proteins? Explain how adaptor proteins put these domains to use.

Answer 28

adaptor proteins have protein-proteins interaction domains

Adaptor proteins function as molecular hubs that physically link different enzymes and promote the assembly of complexes of signaling molecules(LAT and BLNK)

Question 29

What do LAT and BLNK stand for? where are both LAT and BLNK found?

Answer 29

LAT: Linker for the Activation of T cells
it is an integral membrane protein so it is found in the PM

BLNK: B cell LiNKer
it is a cytosolic protein so it floats around the cytoplasm

Question 30

Which of the following are found on adaptor proteins? explain why these are/are not found on adaptor proteins.

SH2

SH3

Tyrosine residues

Proline-rich stretches

Answer 30

All of these (SH2, SH3, Tyrosine residues, and Proline-rich stretches) are found on adaptor proteins

Tyrosine residues serve as docking sites for other signalling proteins with SH2 domains

Proline-rich stretches can bind to other proteins that contain SH3 domains

SH2 and SH3 on an adaptor protein can obviously bind to their respective sites

this ability to bind to/be bound to that is possessed by adaptor proteins is what allows them to participate in a complex, ever-adjusting system of signal transduction

Question 31

What process includes an essential role that must be filled by the LAT gene?

Answer 31

T cell signal transduction

(Jurkat cells with a knockout of the LAT gene showed no TCR-mediated signalling, but the TCR signalling was restored when these cells were transfected with the LAT gene)

Question 32

The LAT gene has been determined to be indispensable for _-____ ______ via the TCR

Answer 32

T-cell activation

Question 33

Describe the following Adaptor proteins (all of which are involved in T cell activation)

LAT:

GADS:

SLP-76

VAV

PLCgamma

Answer 33

LAT is an integral membrane adaptor protein

GADS is a cytosolic adaptor protein that belongs to the Grb2 family

SLP-76 has a proline-rich domains and SH2 binding domains

VAV proteins are GEFs (guanine nucleotide exchange factors)

PLCgamma (phospholipase C) leads to Ca2+ signalling, when it is recruited

Question 34

Describe how all of the following adaptor proteins fit into the process of T cell activation.

SLP-76, LAT, GADS, VAV, PLCgamma

Answer 34

When LAT is phosphorylated, it recruits PLCgamma and the GADS adaptor protein (an adaptor protein, recruiting other adaptor proteins)

PLCgamma goes off to trigger Ca2+ signalling after phosphorylated LAT recruits it

The proline-rich stretch on SLP-76 then associates with the SH3 domain on GADS

SLP-76 then recruits VAV, after being tyrosine-phosphorylated by GADS

VAV is a GEF that activates actin cytoskeletal rearrangements and transcriptional alterations

Question 35

The TCR complex consists of what subunits? What mediates the association of these proteins with one another?

Answer 35

the AlphaBeta TCR non-covalently linked to the CD3 and Zeta proteins (2 CD3’s and 2 Zeta proteins)

The association of these proteins with one another is mediated by “charged residues in their transmembrane regions”

Question 36

Describe why the AlphaBeta TCR must work together with the CD3 and Zeta proteins in order to achieve a signal transduction?

Answer 36

The cytoplasmic tails of the AlphaBeta TCR are too short to transduce signals, so both CD3 proteins and both Zeta proteins must serve as the “signal transducing subunits” of the TCR complex

Question 37

Compare the structures of the CD4 and CD8 coreceptors.

Answer 37

CD4 has:
4 extracellular Ig-like domains
A hydrophobic transmembrane region
A highly basic cytoplasmic tail that is 38 AA’s long

CD8 has: (it is composed of 2 related chains called CD8alpha and CD8beta)
1 extracellular Ig domain
A hydrophobic transmembrane region
A highly basic cytoplasmic tail that is 25 AA’s long

(both CD4 and CD8 have a hydrophobic transmembrane region and a highly basic cytoplasmic tail, but they differ in length)

Question 38

Which type of MHC molecules do CD4 and CD8 interact with?

Answer 38

CD4 interacts with MHC class II molecules

CD8 interacts with MHC class I molecules
(the Beta2 microglobulin in MHC class I molecules is also included in this reaction)

Question 39

Compare the structure of activating and inhibitory immune receptors

Answer 39

activating receptors have separate polypeptide chains for recognition and associated signalling polypeptide chains that contain cytosolic ITAMS (Immunoreceptor tyrosine-based activating motifs)

inhibitory receptors in the immune system typically have ITIMS (Immunoreceptor tyrosine-based Inhibitory motifs) on the cytosolic portion of the SAME CHAIN that uses it’s extracellular domain for ligand recognition.

Question 40

What is FCgammaRIIB and on what 2 cell types is it found?

Answer 40

FCgammaRIIB is a inhibitory receptor that is found on B cells and myeloid cells

Question 41

State how many types of signaling chains and how many ITAMs are found to compose the TCR complex (name them)

Answer 41

The TCR complex has 6 types of signaling chains (Gamma, Epsilon, Alpha, Beta, Delta, and Zeta)

The TCR complex has 10 ITAMs (3 on each Zeta signaling chain and 1 on each Gamma, Epsilon, and Delta signaling chain)

Question 42

What is considered to be “a cytosolic interpretation of the Ag affinity to the TCR”? explain why this is the case.

Answer 42

the number of ITAMs that are phosphorylated is a cytosolic interpretation of the Ag affinity to the TCR

The stronger or prolonged binding of Ag to the TCR results in “increasing numbers of phosphorylated ITAMs”

Question 43

What can influence the nature of the cellular response at different stages of differentiation and activation?

Answer 43

The Ag affinity of a TCR can influence the nature of the cellular response at different stages of differentiation and activation

Question 44

What causes positive and negative selection of T cells in the thymus?

Answer 44

Weak TCR signals are required for the positive selection of T cells in the thymus

Strong TCR signals in the thymus result in the negative selection of T cells and their death via apoptosis
(these need to be destroyed bc they are reacting to self antigens)

Question 45

What is the function of an immunologic synapse?

Answer 45

it stabilizes T cell-APC interaction a promotes the migration of adhesion molecules within the T cell membrane

Question 46

True or False:

The cytoplasmic tails of the TCR can sometimes have ITAMs. explain.

Answer 46

False

The cytoplasmic tails of the TCR lack ITAMs. This is why they must cooperate with the CD3 and Zeta intracellular signalling structures in order to initiate a signaling cascade.

Question 47

How many AA’s do ITAMs feature? be sure to include which 2 AA’s always flank the AA sequence of ITAMs.

Answer 47

ITAMs feature 4 AA’s and are flanked by Tyrosine and Lysine (Tyr-X-X-Lys)

Question 48

True or False:

Multiple ITAMs are located at 1 0- to 1 2-AA intervals along the cytoplasmic tail. explain.

Answer 48

True

Question 49

Verbally describe the events of the TCR-dependent cascade (please note that there are 3 outcomes from 1 stimulus)

(Draw this to learn it)

Answer 49

1. TCR engages pMHC class II, and that complex goes on to engage with CD4
2. The TCR, pMHC class II, and CD4 complex work to activate LCK
3. LCK phosphorylates ITAMs to activate ZAP-70
   4a. ZAP-70 phosphorylation LAT, which goes on to activate ras and rac
   5a. ras and rac initiate MAP phosphorylation cascades that activate AP-1 family transcription factors (in nucleus)
   4b. ZAP-70 phosphorylates LAT, which goes on to activate PLCgamma
   5b. PLCgamma cleaves PIP2, in order to form DAG and IP3
   6a. IP3 first moves to the ER to promote an increase in Cytoplasmic Ca2+, THEN it works with Ca2+ to activate Calcineurin
   7a. Calcineurin then activates NFAT, which goes to the nucleus to activate genes
   6b. DAG and Ca2+ activates PKC, which goes on to phosphorylate IKB
   7b. IKB then causes NFKB to migrate to the nucleus and activate genes

Question 50

Beginning with PKC, explain the steps that occur in order to activate NF-KB dimers to enter the nucleus and activate gene transcription
(This is just for the TCR-dependent cascade. There are many other stimuli besides the PKC portion of the TCR-dependent cascade that can initiate the NF-KB pathway by phosphorylating IKK)

Answer 50

1. PKC phosphorylates IKK, which activates it
2. Activated IKK phosphorylates IKB proteins on 2 conserved Serine residues and induces IKB “polyubiquitination”
3. Once IKB is polyubiquitinated (UUUUU) the proteasome recognizes it and degrades it
4. Degrading the IKB then allows the NF-KB dimers to be released and they enter the nucleus to activate gene transcription

Question 51

Beginning with CD4/CD8 recognizing an antigen and activating LCK, explain the steps that occurs in order to activate AP-1 via the Ras-MAP kinase pathway

Answer 51

1. LCK phosphorylates the ITAMs of Zeta chains
2. ZAP-70 then binds to these phosphorylated Zeta chains and becomes activated by becoming “self-phosphorylated”
3. Activated ZAP-70 then phosphorylates the membrane-associated LAT adaptor protein
4. Activated LAT then binds to GADS, which goes on to “dock” SOS (Son of sevenless, which is a GTP/GDP exchange factor)
5. SOS converts Ras-GDP to Ras-GTP, which consequently activates MAP kinase AND ERK (early response kinases)
6. The ensuing MAP kinase cascade then activates AP-1

Question 52

Define Clonal Anergy. What is this condition characterized by?

Answer 52

Clonal Anergy: describes T cell unresponsiveness at the cellular level

Clonal Anergy is a hyporesponsive state that is characterized by a reduced capacity to synthesize IL-2.

Question 53

Describe what Anergic T cells do in response to an appropriate Ag stimulation

Answer 53

They DO NOT proliferate

Question 54

True or False:

Anergy can be broken by exogenous IL-2. explain.

Answer 54

True

Question 55

Explain how Anergy can be induced. (Hint: 2 methods)

Answer 55

Anergy can be induced by substimulatory levels of Ags in the ABSENCE of:

CD28:CD80/86 interactions between costimulatory receptors on T cell and counter receptors on APCS

A costimulatory signal provided by soluble cytokines

Question 56

Compare what occurs when TCR engagement occurs with and without CD80/CD28 costimulation

Answer 56

TCR engagement by itself causes calcium-mediated signals to induce the activation of NFAT only.
NFAT alone elicits the expression of a distinct set of “anergy-inducing genes”.
This inhibits T cell function at different levels and causes a status of T-cell unresponsiveness

TCR engagement alongside CD80/CD28 costimulatory molecules creates a “productive immune response” by activating the NF-KB and AP-1 transcription factors in the nucleus.

Question 57

What do the following processes all have in common?

CD28:CD80/CD86 costimulation via phosphorylation, release, and degradation of IKB

CD28:CD80/CD86 costimulation via MAP kinase, SAP kinase pathways

TCR signal via dephosphorylation of cytoplasmic NFAT

Answer 57

All 3 of these will induce gene expression of IL-2 mRNA (which will go on to produce IL-2 proteins, duh)

IL-2 goes on to promote the development of T cells

Question 58

Describe the 3 different processes by which CD28: CD80/CD86 costimulation and TCR signaling stimulates the production of IL-2 (which causes the development of T cells)
(each process accounts for a part of the whole that is needed)

Answer 58

1. phosphorylates the complex of Inactive NF-KB and IKB, which causes IKB to be degraded.
   This then frees up NF-KB to go enter the nucleus
2. Calcineurin dephosphorylates cytoplasmic NFAT, which then enters the nucleus
3. MAP and SAP kinase pathways each initiate their own pathways the produce the components that compose Active AP-1, which is formed in the nucleus

The final product that is formed to induce gene expression of IL-2 mRNA = Active NF-KB, Active NFAT, and Active AP-1

Question 59

What initiates the immunologic synapse? (include which cells these are found on)

Answer 59

TCR (on a CD4 or CD8 T cell) recognition of pMHC (on an APC)

Question 60

True or False:

CD8 and CD4 immunologic synapses are identical besides the fact that one uses a CD4 coreceptor to recognize Class I MHC’s and the other uses CD8 coreceptors to recognize Class II MHC’s. explain.

Answer 60

False

CD8 and CD4 immunologic synapses are identical besides the fact that one uses a CD4 coreceptor to recognize Class II MHC’s and the other uses CD8 coreceptors to recognize Class I MHC’s.

(the previous statement had CD8 and CD4 mispaired to the appropriate class of MHC molecule)

Question 61

Describe the bond that occurs between the CD4/CD8 molecule and the corresponding class of MHC molecule.

Answer 61

it is a “weak bond” that is stabilized by the noncovalent interaction between a CD4/CD8 and the proper non-polymorphic region of the MHC molecule

Question 62

Describe the 2 respective molecules that are involved in the adhesion molecule interaction between T cells and APC’s at an immunologic synapse.

Answer 62

LFA-1 expressed by the T cell interacts with ICAM-1 on the APC cell

Question 63

For the following complexes that all occur at an immunologic synapse, describe the ways they “move toward or away from one another”.

LFA-1 : ICAM-1 complexes

pMHC : TCR : CD4/CD8 complexes

CD2 : LFA-3 complexes and costimulatory CD28 : CD80/CD86

Answer 63

LFA-1 : ICAM-1 complexes move away from the pMHC : TCR : CD4/CD8 complex at the immunologic synapse.

At the same time, CD2 : LFA-3 complexes and costimulatory CD28 : CD80/CD86 move toward the pMHC : TCR : CD4/CD8 complex at the immunologic synapse

Question 64

Describe not only what provides the “2 signals” that occur at an immunologic synapse, but also what effect each of the signals cause.

Answer 64

The “1st signal” is triggered by the formation of the pMHC : TCR : CD4/CD8 complex and is necessary but not sufficient to stimulate a naive T cell to proliferate/differentiate

The “2nd signal” (or group of signals) is provided by one or more costimulatory molecules and is also required for T cell activation

(T cell activation will only occur if both signals occur)

Question 65

Describe what ITAMs are and what process they help conduct between T cells and APC’s.

Answer 65

ITAMs are the regions of signaling proteins that are phosphorylated and dock other signaling molecules

The play a major role in the conduction of signal transduction between T cells and APC’s

Question 66

Describe the structure of CD3 and the process that it plays a part in.

Answer 66

CD3 is composed of 3 polypeptide chains (gamma, delta, and epsilon) and CD3’s are always arranged in pairs of gamma-epsilon and delta-epsilon

it plays a part in the conduction of signal transduction between T cells and APC’s

Question 67

Describe the arrangement of LFA-1 and the TCR complex in an immunologic synapse if you were to take a cross-sectional slice of the cylindrically shaped synapse between the T cell and the APC.

Answer 67

The TCR complex would be in the middle and the LFA-1 proteins would be surrounding it