Signal transduction Flashcards
1
Q
Whats a common event in the signal transduction process?
A
Membrane translocation
2
Q
Membrane translocation facts
A
- Volume=4000 um3
- Surface area=1200um2
- Consider that after translocation to the
- Membrane the protein is within the 5nm of the membrane (approx. diameter of a protein) then these proteins experience a volume of
- 1200X0. 005=6um3.that is a 700fold concentration! Binding and enzymatic reactions are dependent on the concentration of components
3
Q
What does FRET show?
A
That proteins that thether to the membrane interact more
4
Q
What does FRET stand for?
A
Fluorescent responance energy transfer
5
Q
What does FRET depend on?
A
The the distance between fluorophores
6
Q
When does FRET only occur?
A
When molecules are in close proximity e.g. 10 nm and they must have the right oritentation as well
7
Q
What drives cell transformation?
A
Forced membrane localisation of PKB drives cell transformation
8
Q
What mechanisms control protein interaction to the membrane?
A
- Phosphobinding motifs: SH2 and PTB
- Uniquitin binding motifs
- AKAP interaction domains (interaction between a cytosolic and a membrane bound protein)
9
Q
What mechanisms control lipid interaction motif to the membrane?
A
- Phosphoinositide interacting motifs: PH, FYVE, PX, PHD and lysine arginine rich patches
- DAG binding motifs: C2 comain
- membrane interacting motifs: C1 domain
10
Q
What mechanisms control lipid tether to the membrane?
A
- Myristoylation
- Prenylation
11
Q
Whats Myristoylation?
A
Myristoylation is a lipidation modification where a myristoyl group, derived from myristic acid, is covalently attached by an amide bond to the alpha-amino group of an N-terminal glycine residue.
12
Q
What does Fatty acylation mainly consist of?
A
The covalent addition of palmitic acid or myristic fatty acids to protiens
13
Q
Tell me about fatty acylation and what happens in this process?
A
- covalent addition of the 14-carbon saturated fatty acid myristate to the N-terminal glycine residue through a stable amide bond.
- Thought to be irreversible.
14
Q
Tell me the steps to co-translational myristoylation
A
- Removal of the N-terminal methionine
- Activation of myristic acid with CoA
- Coupling of the myristic acid to the glycine
- Gly-X3-X4-X5 (Ser/Thr/Cys) 6 where X represents most AA, except for proline, aromatic or charged residues in position X3
- This is co-translational myristoylation
15
Q
Tell me the steps to post-translational myristoylation and its role in apoptosis
A
- Caspase mediated cleavage of Bid exposes a glycine residue
- Bid becomes myristoylated
- Lipid tether induces insertion into mitochondrial membrane
- Recruitment BAK to the mitochondrial membrane
- Cytochome C release
- Downstream apoptosis
16
Q
Where does protein prenylation main occur?
A
Mainly occurs on CAAX proteins (this is a C terminal protein)
17
Q
What do proteins containing a CAAX motif function as?
A
Proteins containing a CAAX motif at their carboxyl termini, in which ‘C’ is the Cys residue that functions as the isoprenoid attachment site, ‘A’ signifies any aliphatic amino acid, and ‘X’ denotes any of several amino acids.
18
Q
How is protein prenylation initiated?
A
prenylation is initiated by the attachment of a 15‑carbon (farnesyl) or a 20‑carbon (geranylgeranyl) isoprenoid lipid to the Cys residue by protein farnesyltransferase (FTase) or protein geranlygeranyltransferase I (GGTase I), respectively.
19
Q
In protein prenylation, how can proteins be further processed?
A
By Ras converting CAAX endopeptidase 1 (RCE1), which removes the -AAX residues
isoprenylcysteine carboxylmethyltransferase (ICMT), ‘caps’ the carboxyl group on the now carboxy‑terminal isoprenoid-modified Cys residue with a methyl group
20
Q
What is the Ras protein important for?
A
Cell proliferation
21
Q
What is
15 carbon farnesyl diphosphate (FPP)
and
20‑carbon geranylgeranyl diphosphate (GGPP)
both built from?
A
Both built from isopentenyl diphosphate (IPP)
22
Q
During Prenylation and palmitoylation of RAS controlled membrane localisation, what is the prenyl group put on by?
A
farnesyl transferase or geranylgeranyltrasnferase
23
Q
What are Ft-inhibitors developed for?
Tell me about this
A
Ft-inhibitors developed for the clinic
however RAS instead become modified with geranylgeranyl.
Statins used in clinic to control cholesterol levels. Metadata suggest that long-term statin therapy reduces stroke, some chronic inflammatory disorders, osteoporosis and several types of cancer and Alzheimer disease
24
Q
Tell me about signal regulated membrane localisation
A
1. Single Lipid tethers not normally enough to induce stable membrane localisation.
2. Often requires other membrane binding domains.
A. Phosphoinositide interaction domain. Often polybasic regions.
B. Another tether such as a prenylation or palmitoylation
3. As the tether does not induce stable association this enables various control mechanisms to be used.
25
Why is Kras4B targeted to the PM?
By the interaction of its polybasic region with phosphoinositides
26
What are the classes of Ras?
H, N and K
27
What do H, N and K undergo?
H and N undergo Prenylation and palmitoylation to target them stably to the membrane
K only undergoes prenylation. it uses PBR for stable association at the membrane
28
Why is KRas able to attach to the membrane by H and N aren't?
KRz has a polybasic region which means it can adhere to the membrane. H and N don't have this
KRas has a serine residue which can be phosphorylated. this blocks the interaction of the polybasic tail with the membrane causing KRas to fall off the membrane and become inactivated
29
What control KRas localisation at the PM and downstream function?
A phosphor-switch
30
How does KRas lead to apoptosis?
* PKC is activated by bryostatin
* When KRas falls of membrane with group, it binds to the golgi and mitochondria and this leads to apoptosis
31
Activation of the farnesylation switch attenuates tumour growth in vitro and in vivo
32
Give me some background on the disease: Progeria syndrome such as Hutchinson- Gilford (HGPS)
* are caused by abnormal processing of the CAAX protein prelamin A
**Background on disease**
* Characterised by premature accelerated aging beginning between 6-12 months of age
* There are around 100 living patients worldwide at a given time
* Often fatal age of 13 with no known cure
* Mutations in a protein called Lamin. Lamin controls strength of nucleus and hold nucleus together and provide tensile strength. How it controls the strength can affect the downstream processes
33
FTase inhibitors can be used in the clinic to treat what?
HGPS
34
What are the 3 types of Lamins?
A, B and C
35
What do B type lamins maintain?
Isoprenylation
36
How are C type lamins produced?
As mature proteins without CAAX or clevage
37
How are A type Lamins cleaved?
38
What can sequestration be used to inhibit?
downstream signalling
39
Protein phosphorylation is a common event in signal transduction pathways. What are the stages to organelle specific sequestration?
1. ER unfolded protein response: CHOP
2. Lysome: TFEB
3. Plasma membrane: TUBBY proteins
4. Cytoplasm; Steroid hormones; FOXO factors
40
41
Notch in flies control wing shape and embryonic development
42
The notch signalling pathway in mammalian cells
(watch the YT vids close to exam season to understand notch properly)
* 4 different notch receptors in mammals (1,2,3 and 4)
* Have extracellular egf repeats
* Ligands are Dl1,3,4 and Dlk and Jagged 1,2
* Controls structure of organs and often mutated in tumours which causes downstream proliferation, could help with cancers
43
The regulation of protein phosphorylation is a major pathway downstream receptor activation that mediates cell specific output signalling
44
How can phosphorylation change protein conformation?
Phosphorylation can change conformation by either disrupting molecular interactions or by promoting them. Common event in protein kinase activation
45
Phosphorylation creates new binding sites for a phosphor-specific reader molecule that delivers the output
46
What do phosphorylation readers such as PIN1 induce?
Conformational changes
47
Protein phosphorylation induces diverse downstream inputs
48
Writer/ reader modules can be coupled to induce what?
Complex outputs
49
PTMS; what are they good for? its all a matter of states
50
PTM of the p53 tumour suppressor
51
Tell me about general cell signalling cascades?
1. Each cell type has a unique repertoire of cell signalling components (signalsome).
2. During differentiation cells express a particular phenotype and a distinctive set of signalling components (a cell-type-specific signalsome) required to control their particular functions.
3. Abnormal remodelling of cellular signalsomes creates signalling defects that have great significance for the onset of many diseases.
52
Reading list and possible exam questions
**_Reading list:_**
* Expansion of signal transduction by G proteins the second 15 years or so: From 3 to 16 α subunits plus βγ dimers
* Cell Signalling by Receptor Tyrosine Kinases
**_Exam questions_**
1. Describe in detail how b adrenergic agonists can induce an increase in myocardial contraction
2. Explain how b adrenergic agonist induce two different responses in different tissues.
3. Compare and contrast alpha- and beta-adrenergic receptor signalling pathways.
4. Illustrate how protein phosphorylation modulates downstream signalling
53
Adrenergic activation induces a variety of responses in different tissues during the flight or right response.
What responses does it produce?
How?
* Increase strength and heart rate
* Increased blood flow to muscle
* Increased systemic and cellular energy supply
* Increased skeletal muscle force
54
Sympathetic nervous system post ganglionic fibres release what at the target organ?
Noradrenaline
55
What does the adrenal medulla (post ganglionic tissue) release into the blood stream?
What are the following of the adrenal medulla...
* Neural crest cells
* Sympathetic supply
* Secretary cells
* Large secretory cells
Releases **adrenaline** into the blood stream (endocrine)
* **Neural crest cells-** secretory cells of the medulla
* **Sympathetic supply-** preganglionic sympathetic neurons
* **Secretary cells-** postganglionic sympathetic neurones: lack axons or dendrites
* **Larger secretory cells-** secrete adrenaline and NA
56
What can adrenegic stimulation be divided into? What are these subtypes then further divided into?
Adrnenergic agonist derived responses can be split according to the activation of at least two different receptor dependent pathways
57
A typical signal transduction pathway...
58
Adrenaline activates the phosphorylase enzyme to induce glycogen breakdown
59
Sometimes you just have to get your hand dirty...
60
Whats is cAMP what does it lead to?
cAMP is the critical adrenaline induced second messenger that leads to the activation of phosphorylase
61
When is adenylate cyclase activated?
in response to adrenaline stimulation to generate cAMP
62
What are the classes of adenylate cyclase?
There are 6 classes
Class III is the major one in mammalian cells
63
What are the subunits in adenylate cyclase?
64
Are the enzymes in adenylate cyclase membrane bound?
yes
65
In adenylate cyclase, the N terminus C1 and C2 regions, appear to be important for regulation by what?
Galpha-GTP complex signalling in mammalian cells
66
How many isoforms of adenylate cyclase are there in mammalian cells?
10 isoforms
67
Adenylate cyclase isoforms III, V and VIII are stimulated by what?
What are the isoforms I and VI inhibited by?
What does this suggest?
**Isoforms III, V and VIII** are **stimulated** by **Galpha subunits** and can be stimulated by the **Ca2+/calmodulin**
While **Isoforms I and VI** are **inhibited** by **Ca2+**
Suggests cross talk between different signalling pathways
68
Medicine, 1994. Alfred Gilman and martine Rodbell: “for their discovery of G-proteins and their role in signal transduction in cells”
69
Tell me the stages to the heterotrimeric G-protein activation cycle
1. GPCR interact with their cognate ligand which induces a conformational change in the receptor
2. GPCR-ligand stimulates the exchange of GDP to GTP on the Ga subunit
3. GTP exchanges stimulates the dissociation of Galpha from the betagamma subunits (note they all remain at the membrane through lipid modification that inserts into the membrane).
4. Ga subunit stimulates an effector (adenylate cyclase)
5. Intrinsic or stimulated GTPase activity of Ga returns the subunit to resting state and association with betagamma
70
What is the inactive form of protein Kinase A and tell me about its structure
Protein kinase A occurs in an inactive form **(R2C2)**
consisting of **two regulatory (inhibitory) subunits** and **two catalytic subunit**s, and in an **active form (2C)**
71
How does cAMP activate protein kinase A?
By dissociation of the R-subunits from the R2C2-complex (allosteric activation).
72
What do the R subunits in protien Kinase A contain?
What does this bind to?
The R-subunits contain a **pseudosubstrate sequence**: Arg - Arg - Gly - Ala - Ile, Which binds to the catalytic site of the C-subunits.
Binding of cAMP allosterically moves the pseudosubstrate sequence out of the catalytic sites.
73
What does protein kinase A modulate the activity of?
Protein kinase A modulates the activity of many proteins by phosphorylating them.
74
What is phosphorylase kinase a direct target for?
Phosphorylase kinase is a direct target for cAMP dependent PKA and couples receptor activation with increased glycogenolysis
75
Phosphorylation of phosphorylase induces what?
**A conformational change in the active site**
Shown is the phosphorylase dimer. Phosphorylase is activated by a change of shape. The shift between the two shapes is controlled by phosphorylation of serine 14 (coloured pink). Note the change in shape around the active site (left-hand side of the upper subunit).
76
Structure of phosphorylase
* Yellow: AMP allosteric site
* Orange: ser14 phosphorylation site
* Blue: glycogen binding site
* Red: catalytic site
77
What is the structural change of phosphorylase associated with?
What does this change increase?
* Ser14 is close to the subunit interface.
* The structural change associated with phosphorylation, and with the conversion of phosphorylase b to phosphorylase a, is the rearrangement of the originally disordered residues 10 to 22 into α helices (tower helices).
* This change increases phosphorylase activity.
78
In muscle, AMP is a potent activator of what?
* In muscle AMP is a potent activator of **phosphorylase** in the absence of phosphorylation
* In liver however enzyme completely inactive unless phosphorylated and cannot be activated by AMP
79
cAMP activation of PKA and phosphorylation of different target coordinates a cellular response to increase glucose synthesis and release
80
When switching off GPCR and cAMP signalling, tell me what happens with receptor desensitisation
* GPCR C-terminal tail phosphorylation
* Beta-adrenergic receptor kinase
* Phosphorylated tails recruit beta-arrestin which...
1. Attenuates heterotrimeric G protein activation
2. induces receptor internalisation
81
When switching off GPCR and cAMP signalling, tell me what happens with Galpha inactivation?
* Intrinsic or GAP stimulates GTPase activity (RGS proteins)
* GTP hydrolysis which...
1. Stops activation of downstream targets
2. Causes re-association of the Galpha subunit with the beta gamma subunits
82
When switching off GPCR and cAMP signalling, tell me what happens with cAMP inactivation?
* Activation of phosphodiesterase activity (PDE)
* hydrolysis of cAMP to generate AMP
83
How does beta-adrenergic signalling stimulate the release of the free fatty acids into the blood stream?
What function does phosphorylation inhibit?
PKA phosphorylate
1. HSL, leads to translocation to the lipid droplet and its activation
2. Perilipin acts as a barrier to lipid hydrolysis
3. Phosphorylation inhibits this function
84
How does beta-adrenergic signalling increase heart rate and force?
**Localised activated PKA phosphorylates**
1. The Cav1.2 ion channel which increases Ca2+ influx in response to depolarisation
2. The ryanodine receptor (RYR) stimulates increased calcium induced calcium release from SR
3. Phospholambam and prevents it from inhibiting SERCA mediated Ca2+ uptake. Enable faster relaxation for next contraction
* AKAPs (a-kinase anchoring proteins) contribute to spatial and selective restriction of PKA signalling
85
Cell specific targets of PKA define specific outputs in response to beta adrenergic receptor stimulation
86
Adrenergic agonist derived responses can be split according to the activation of at least two receptor dependent pathways
87
What can GPCR, including the alpha 2-adrenoreceptor, also couple?
G proteins that inhibit cAMP synthesis
88
What can bacterial toxins differentiate between?
Types of G protein signalling
89
What do Alpha2 receptors couple to?
Alpha2 receptors couple to a pertussis toxin sensitive G1/G0 to inhibit cAMP synthesis
90
Gas and Gai activation can coordinate downstream responses though modulating cAMP synthesis and other pathways
Tell me about beta3/2 and alpha 2
And how the sympathetic and parasympathetic systems work?
* beta2-adrenergic stimulation induces glycogenolysis in the liver and alpha2 in the beta-cells of the pancreus attenuate glucose induced insulin secretion.
* Adrenline acts on both the b3 and a2 receptor in adipose to to control adenylate cyclase and cAMP synthesis and lipolysis.
* Sympathetic and parasympathetic systems work antagonistically.
91
Pertussis toxin sensitive signalling through betagamma subunits
GIRK signalling:
What are the physiological affects?
* Autaptic (release of neurotransmitter at a synapse autoregulates its own release). Dopamine and epinephrine release
* Neuron to neuron inhibition
* Pain perception
92
Analgesia. Many pharmacological agents that are used in pain relieve target the activation of Gi/Go GPCR. E.g. opioid, serotonin, dopamine.
93
Alpha1 adrenoreceptor couples to Gaq and the activation of phospholipase C to regulate downstream smooth muscle cell contraction.
94
Tell me the structure of: **phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2)**
Whats the parent lipid?
* Favoured chair structure has 5 equatorial OH and 1 axial oh.
* Consider a turtle with its head as the axial OH then the glycerol is connected to the right flipper which is position 1.
* Phosphatidylinositol is the parent lipid
95
With Phosphoinositides ...
1. ... where can phosphorylation occur and what does this generate
2. What is phosphorylation controlled by?
1. Phosphorylation can occur on the 3, 4, and the 5 position to generate seven different phosphoinositides.
2. The phosphorylation is controlled by an array of lipid kinases and phosphatases (about 80 in the human genome)
96
Can Phosphoinositide’s be used as signal transducing messengers?
yes
97
What does GPCR or a1-adrenergic receptors act as and what does this lead to?
GPCR (acetylcholine (muscarinic) or a1-adrenergic) act as a **GEF for Gaq** which leads the activation of a **phospholipase C and PtdIns(4,5)P2 hydrol**ysis
98
For the phospholipase C (PLC) family...
* What is the core enzyme composed of?
* Where is the active site?
* What is the active site interrupted by?
* What can the PH domain interact with?
* What can the C2 domain interact with?
* Core enzyme composed of the **PH domain, four tandem EF domains, a split TIM barrel (X Y) and a C2 domain.**
* The active site is in the **TIM**, which is interupted by an auto-inhibitory loop insert.
* The PH domain can interact with **phosphoinositides, rac, bg**. Brings the enzyme to the membrane where it is active
* The C2 domain interacts with **calcium and with the membrane surface** and contributes to Gaq binding (b –isoforms) .
99
Tell me about PLC enzymes?
* PLC are very active enzymes that are autoinibited and activation mechanisms essentially move the loop out of the cleft.
* In PLC-z the loop is activatory and its removal inhibits the enzyme
100
Tell me the specific PLC isoforms couple receptors and stimuli to PtdIns(4,5)P2 hydrolysis?
* PLC is activated in response to the activation of many different stimuli
1. **Tyrosine kinase receptors (PDGF)**
2. **G Protein** coupled receptors (Histamine)
3. Specificity of signalling is driven by specific interaction domains :for example
a. PLCb1 activated by **Gaq**, PLCb isoforms also activated by bg signalling (Gi/o)
b. Tyrosine phosphorylated receptor interacts with SH2 domain of PLCg1. PLCg1 is then tyrosine phosphorylated and activated
c. During fertilisaiton sperm specific PLCz injected into the egg. Drives Calcium oscilations
101
GPCR activate PLC mediated hydrolysis of PtdIns(4,5)P2 to generate two new second messengers Diacylglycerol (DAG) and Ins(1,4,5)P3
102
What is Ins(1,4,5)P3 ?
Ins(1,4,5)P3 is a water-soluble molecule and diffuses across the cytoplasm to the IP3 receptor which is found on the membrane of the endoplasmic reticulum
103
What is the Ins(1,4,5)P3-receptor ?
Ins(1,4,5)P3-receptor is a ligand-gated ion channel which allows the influx of calcium cations upon activation
104
How many forms of the Ins(1,4,5)P3-receptor exist?
What is the receptor made of/ its domains?
Three isoforms of the receptor exist
The receptor is a tetramer made of the following domains:
* N-terminal Ins(1,4,5)P3binding domain
* Coupling domain
* Transmembrane domain
* Gatekeeper domain
105
How is the Ins(1,4,5)P3-Receptor activated?
* IP3 binds to binding domain (cytosolic face)
* Conformational changes in other domains
* Channel is opened
* Influx of calcium into the cell from internal stores (in the ER or SR)
106
The Ins(1,4,5)P3-Receptor can be activated by IP3 binding, what is the signals for IP3 binding transferred through?
The signal for IP3 binding is transferred through both the N-terminal and internal coupling domains to the gatekeeper domain, which triggers a conformational change in the activation gate formed within the transmembrane
107
Phospholipase C (PLC) mediated hydrolysis of PtdIns(4,5)P2 generates two second messengers: Diacylglycerol (DAG) and inositol(1,4,5)P3 (ins(1,4,5)P3
108
Protein kinase C is a major downstream target activated in response to what?
increases in Diacylglycerol (DAG)
109
What are the PKC family?
Serine threonine kinases
DAG and Ca2+ dependent (cPKC) or DAG dependent (nPKC)
110
What do Pseudosubstrate (like PKA) inhibit?
enzyme activity
111
When Pseudosubstrate interact with DAG at the membrane, what does it cause?
Interaction with DAG at the membrane and Ca2+ induces conformational change and relieves pseudosubstrate inhibition.
112
PKC are also regulated by what?
PKC are also regulated by a family of binding proteins called RACK (receptor for activated C-kinase) which akin to AKAP regulate subcellular distribution and downstream phosphorylation
113
Activation of Gaq and PLC underlie smooth muscle cell contraction in response to adrenergic stimulation through the a1-receptor
114
Adrenergic stimulation through the a1-receptor also (remember b-receptor) regulates glycogen breakdown through Gaq and PLC activation
115
Give 9 reasons why GPCR can respond to a large range of diverse molecules with very diverse sizes
1. 7 transmembrane
2. Largest family in the genome (approx 1000)
3. Fast (some involved in vision, smell, taste)
4. Ligand interaction induces a conformational change in receptor
5. GEF activity of the receptor induces a switch in GTP binding of the alpha subunit
6. Both alpha and the beta/gamma subunits can signal
7. Alpha subunits can be stimulatory and inhibitory
8. Switched off by intrinsic GTPase
9. Receptor phosphorylation switches receptor off
116
Multiple diverse inputs are transduced by a limited number of transduction cascades but can lead to a diverse array of output responses.
Give 2 possible responses
* Different GPCR activation in the same cell that increase cAMP can elicit the same downstream output
* The same GPCR in different cell types can elicit cell type specific outputs. This might be the consequence of tissue specific expression of downstream targets (phosphorylase kinase (liver) versus HSL (adipose))
117
Other effectors of cAMP signalling: more than just PKA
118
What does EPAC couple?
cAMP signalling to regulation of cell adhesion and secretion
119
What is EPAC?
What does it induce?
EPAC is a **guanine nucleotide exchange factor (GEF)** that binds cAMP
which induces a conformational change to activate its GEF activity
120
Tell me about EPAC affinity for cAMP?
EPAC directly EPAC has a lower affinity for cAMP than PKA: might only be activated at high concentration
121
What does EPAC activate?
It activates the small molecular weight g protein **RAP** (not the same as a heterotrimeric g protein. It is like Ras)
122
What does RAP do?
RAP acts as a molecular switch to regulate cytoskeletal dynamics, cell adhesion and secretion
123
GPCR signalling leads to the activation of diverse reversible protein phosphorylation cascades
124
GPCR that regulate cAMP synthesis, some targets phosphorylated by PKA and specific tissue outputs
125
Examples of GPCR ligands that activate Gaq to regulate PLC signalling
126
Differential signalling outputs can be attained by tissue selective receptor subtype expression
