Lecture 8 Flashcards
Describe the cytosolic and nuclear phase of signal transduction
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
Signal transduction is a ____ of phosphorylation and dephosphorylation.
cascade
Describe the structure of a signal transduction receptor and the 2 different things their extracellular domains recognize.
Receptors are generally transmembrane proteins present on the PM
The extracellular domains of these receptors recognize “soluble ligands” or “membrane structures of neighboring cells”
compare the processes of “ligand binding” and “signalling” as they pertain to signal transduction (differences and similar actions)
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
Define Nuclear receptors and how they are activated
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.)
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:
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
True or false:
non-receptor tyrosine kinase receptors will synthesize a tyrosine kinase to conduct phosphorylation in order to carry out signal transduction. explain.
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
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:
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”
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?
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
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
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
The acetylation/methylation of the N-terminal tails of histones is meant to modulate which 3 genetic processes in a cell?
Gene expression
DNA replication
DNA recombination
State the 2 components of signalling pathways
Modular signalling proteins
Adaptor proteins
Eukaryotic signal transduction proteins are ____-based architectures. This means that they are built from combinations of what 2 domains?
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)
Define Allosteric Regulation and then describe what form/structure a signal transduction protein must be in, in order to conduct allosteric regulation
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
Compare a protein’s output activity under basal conditions and when a specific set of input effector ligands are present.
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
What group are the following families a part of? what is the main purpose of all of these?
Src family
Syk family
Tec family
Src, Syk, and Tec families are all “tyrosine kinase families”
Tyrosine kinase families are key players in the regulation of immune functions
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)
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)
For the following Domain, state how many AA’s it is comprised of and what molecular structure it is responsible for reacting with.
SH2
SH2 domains are composed of about 100 AA’s
binds to phosphotyrosine-containing peptides
For the following Domain, state how many AA’s it is comprised of and what molecular structure it is responsible for reacting with.
SH3
SH3 domains are composed of about 50 AA’s
binds to proline-rich stretches
For the following Domain, state what molecular structure it is responsible for reacting with.
PH
PH domains recognize specific phospholipids
Which tyrosine kinase family domain is present in the Syk family and binds to phosphotyrosine motifs in the “Ag receptor complex”?
SH2 domains
State and describe the 5 functional domains of the Src
- (N-terminal) SH4 domain: contains a “myristic acid moiety” which is essential for it’s localization to the inner surface of the PM
- U domain (unique domain): provides the functional specificity to each member of the Src family
- SH3 domain: binds to proline-rich sequences to mediate intra- and intermolecular interactions
- SH2 domain: binds to phosphorylated tyrosine residues on Src and other proteins
(a SH2-Kinase linker is between the SH2 and SH1 domains) - SH1 domain: a catalytic domain (also called Y416)
(a small Y527 unit is between SH1 and the C terminus of the Src)
Describe the configuration of Src in it’s inactive and active forms. What causes it to switch between these forms?
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
Between the Tyr527 and the Tyr416 sites on Src, which of these is capable of autophosphorylation? Describe what the significance of the autophosphorylation is.
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
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.
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)
Many of the substrates that an active Src molecule can phosphorylate go on to become part of what process?
the signaling process
which often involve a cascade mechanism of sequential phosphorylation and dephosphorylation of proteins in the cascade
True or False:
Adaptor Proteins play a part in the cascade mechanism of signalling by catalyzing different reactions in the pathway. Explain.
False
Adaptor Proteins DO NOT have any catalytic activity
What type of interaction domains are found on adaptor proteins? Explain how adaptor proteins put these domains to use.
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)
What do LAT and BLNK stand for? where are both LAT and BLNK found?
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
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
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
What process includes an essential role that must be filled by the LAT gene?
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)
The LAT gene has been determined to be indispensable for _-____ ______ via the TCR
T-cell activation
Describe the following Adaptor proteins (all of which are involved in T cell activation)
LAT:
GADS:
SLP-76
VAV
PLCgamma
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
Describe how all of the following adaptor proteins fit into the process of T cell activation.
SLP-76, LAT, GADS, VAV, PLCgamma
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
The TCR complex consists of what subunits? What mediates the association of these proteins with one another?
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”
Describe why the AlphaBeta TCR must work together with the CD3 and Zeta proteins in order to achieve a signal transduction?
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
Compare the structures of the CD4 and CD8 coreceptors.
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)
Which type of MHC molecules do CD4 and CD8 interact with?
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)
Compare the structure of activating and inhibitory immune receptors
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.
What is FCgammaRIIB and on what 2 cell types is it found?
FCgammaRIIB is a inhibitory receptor that is found on B cells and myeloid cells
State how many types of signaling chains and how many ITAMs are found to compose the TCR complex (name them)
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)
What is considered to be “a cytosolic interpretation of the Ag affinity to the TCR”? explain why this is the case.
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”
What can influence the nature of the cellular response at different stages of differentiation and activation?
The Ag affinity of a TCR can influence the nature of the cellular response at different stages of differentiation and activation
What causes positive and negative selection of T cells in the thymus?
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)
What is the function of an immunologic synapse?
it stabilizes T cell-APC interaction a promotes the migration of adhesion molecules within the T cell membrane
True or False:
The cytoplasmic tails of the TCR can sometimes have ITAMs. explain.
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.
How many AA’s do ITAMs feature? be sure to include which 2 AA’s always flank the AA sequence of ITAMs.
ITAMs feature 4 AA’s and are flanked by Tyrosine and Lysine (Tyr-X-X-Lys)
True or False:
Multiple ITAMs are located at 1 0- to 1 2-AA intervals along the cytoplasmic tail. explain.
True
Verbally describe the events of the TCR-dependent cascade (please note that there are 3 outcomes from 1 stimulus)
(Draw this to learn it)
- TCR engages pMHC class II, and that complex goes on to engage with CD4
- The TCR, pMHC class II, and CD4 complex work to activate LCK
- 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
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)
- PKC phosphorylates IKK, which activates it
- Activated IKK phosphorylates IKB proteins on 2 conserved Serine residues and induces IKB “polyubiquitination”
- Once IKB is polyubiquitinated (UUUUU) the proteasome recognizes it and degrades it
- Degrading the IKB then allows the NF-KB dimers to be released and they enter the nucleus to activate gene transcription
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
- LCK phosphorylates the ITAMs of Zeta chains
- ZAP-70 then binds to these phosphorylated Zeta chains and becomes activated by becoming “self-phosphorylated”
- Activated ZAP-70 then phosphorylates the membrane-associated LAT adaptor protein
- Activated LAT then binds to GADS, which goes on to “dock” SOS (Son of sevenless, which is a GTP/GDP exchange factor)
- SOS converts Ras-GDP to Ras-GTP, which consequently activates MAP kinase AND ERK (early response kinases)
- The ensuing MAP kinase cascade then activates AP-1
Define Clonal Anergy. What is this condition characterized by?
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.
Describe what Anergic T cells do in response to an appropriate Ag stimulation
They DO NOT proliferate
True or False:
Anergy can be broken by exogenous IL-2. explain.
True
Explain how Anergy can be induced. (Hint: 2 methods)
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
Compare what occurs when TCR engagement occurs with and without CD80/CD28 costimulation
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.
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
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
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)
- 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 - Calcineurin dephosphorylates cytoplasmic NFAT, which then enters the nucleus
- 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
What initiates the immunologic synapse? (include which cells these are found on)
TCR (on a CD4 or CD8 T cell) recognition of pMHC (on an APC)
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.
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)
Describe the bond that occurs between the CD4/CD8 molecule and the corresponding class of MHC molecule.
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
Describe the 2 respective molecules that are involved in the adhesion molecule interaction between T cells and APC’s at an immunologic synapse.
LFA-1 expressed by the T cell interacts with ICAM-1 on the APC cell
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
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
Describe not only what provides the “2 signals” that occur at an immunologic synapse, but also what effect each of the signals cause.
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)
Describe what ITAMs are and what process they help conduct between T cells and APC’s.
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
Describe the structure of CD3 and the process that it plays a part in.
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
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.
The TCR complex would be in the middle and the LFA-1 proteins would be surrounding it
