Lecture 8; Cell signalling 5

Question 1

What is important in cell signalling?

Answer 1

Shutting off pathways, as prolonged activation can be damaging

Question 2

What are some common shutting down signals or methods?

Answer 2

• Receptor down regulation following internalisation i.e GPCR or RTK
• Phosphotases to reverse post translation modifiations
• Phosphodiesterases
• Protein level regulation i.e ubiquination
• Sequesteration and compartmentilisation of proteins

Question 3

Give an example of protein sequestration;

Answer 3

Ser/Thr phosphorylation Can Promote Sequestration by 14-3-3 Protein Which Can Influence Localisation of Protein

i.e Forkhead (FKHR) transcription factors can be phosphorylated by PKB on sites allowing the binding of 14-3-3 which sequesters the FKHR protein in the cytosol.

Question 4

Other Examples Where Sequestration by 14-3-3 Protein Following Ser/Thr Phosphorylation Can Influence Localisation of a Protein?

1/2

Answer 4

BAD - BAD is phosphorylated by Akt(PKB) it interacts with 14-3-3 and its ability to stimulate apoptosis is blocked

Question 5

Another Examples Where Sequestration by 14-3-3 Protein Following Ser/Thr Phosphorylation Can Influence Localisation of a Protein?

2/2

Answer 5

RAF - RAF can be phosphorylated and this stimulates an interaction with 14-3-3 which blocks its ability to activate the Map-Kinase cascade as GTP Loaded Ras cant interact with it.

(sequestering)

Question 6

Following receptor activation what happens?

Answer 6

Signals Are Shutoff By Internalisation of Receptors

Question 7

Why are receptors internalised? example;

Answer 7

EGF receptor and ligand are broken down after stimulation so receptor can only signal once as its important to tightly control growth

Question 8

Describe how receptor internalisation occurs; i.e EGF receptor

Answer 8

1. Receptor dimerizes and signaling initiated
2. Receptor activation also stimulates move to clathrin coated pits
3. Clathrin coated pits internalise, clathrin uncoats and vesicles acidify
4. EGF dissociates from receptor in acid environment
5. Receptor and ligand broken down in lysosomes so signal ended
6. Empty vesicles recycle to membrane

GPCR also do this, but some receptors can recycle to the membrane

Question 9

What is special about kinase signalling pathways?

Answer 9

They can shut themselves down as a part of the signalling

Question 10

Give an example of a kinase signalling pathway that shuts itself down;

Answer 10

Insulin Signaling Leads to Feedback Inhibition of Itself by Ser Phosphorylation of IRS-1

Question 11

Describe how insulin signalling shuts itself down;

Answer 11

p70 S6 Kinase is activated (during the cascade) it continues the cascade and results in protein translation but it also regulates protein translation

by phoshorylating IRS-1 (ser residue) which reduces its ability to be phosphorylated on Tyrosines (by the insulin receptor, imp step in transduction therefore it this feedback stops transduction)

i.e. it is a feedback regulation as the more insulin activates the pathway the more feedback there is

Question 12

How do phosphotases shut down the signal?

Answer 12

By removing phosphates from the signalling molecules

Question 13

How can phosphotases by categorised?

Answer 13

two broad categories, those targeting phospho-tyrosine and those targeting phosphoserine/phosphothreonine

Question 14

What regulates phosphotase activity?

Answer 14

• signalling, i.e those with Cys residues at the catalyitic site are susceptible to ROS (oxidation)
• Location in the cell
• Activated by ions i.e Calcineurin is Ca sensitive

Question 15

What is an important lipid phosphotase?

Answer 15

• those regulating the levels of PIP3 in the cell.

Two major types of PIP3 phosphatase identified

• SHIP’s which are SH2 domain containing phosphatases that are regulated by cytokines and growth factors and remove the 5’ phosphate from the inositol ring
• PTEN which removes 3’ phosphates

Question 16

Describe what happens when PTEN removes the 3’ phosphate from PIP3;

Answer 16

Function of PIP3 changes from Activate and co-localise proteins containing PH domains

to

Localise proteins anchoring cytoskeleton to membrane

Question 17

Whats a shutoff mechanism for cGMP and cAMP?

Answer 17

Phosphodiesterases (PDE)

Question 18

Describe PDE function;

Answer 18

These regulate the breakdown of cAMP and cGMP back to AMP and GMP
This shuts down these signalling pathways e.g PKA and PKG

Question 19

Describe the PDE families in mammals;

Answer 19

• PDE 4, 7 and 8 are specific for cAMP
• PDE 5, 6 and 9 are specific for cGMP
• PDE 1,2,3,10 and 11 can breakdown both cAMP amd cGMP

Question 20

What can regulate PDEs?

Answer 20

PDEs can be regulated by signaling pathways e.g. PDE3 can be phosphorylated by PKB and therefore be regulated by insulin

Question 21

Whats a long term shutoff mechanism?

Answer 21

Longer term effects requiring gene expression

e.g. Cytokines induce expression of the “Supressors of Cytokine Signalling” or SOCS molecules which feed back to inhibit cytokine signalling

Question 22

Describe the insulin signalling pathway at work;

Answer 22

Insulin binds receptor

• Receptor signals IRS to phosphorylate Class 1 PI 3 Kinase (p85 subunit)
• This creates PIP3
• PIP3 activates PDK1
• PDK1 phosphorylates PKB
• PKB phosphorylates AS160 which causes trafficking vesicles to insert GLUT 4 into the membrane

GLUT 4 transports glucose into the cell

Question 23

How does the insulin receptor regulate protein translation?

Answer 23

• PKB phosphorylates RHEB and blocks its GTPase activity so that it remains loaded and active, activating mTOR and thus ensuring protein translation continues

Low PKB = little translation
also RHEB can act as a GTPase = reduced translation

Question 24

How does insulin receptor regulate glucose metabolism?

Answer 24

PKB Phosphorylates GSK3 which inhibits GSK3 activity. This stops GSK from phosphorylating Glycogen Synthase and so allows Glycogen Synthase to become active

Question 25

How does the insulin receptor enhance glucose anabolism (i.e insulin function)?

Answer 25

PKB Phosphorylates members of the FOXO transcription factor family such as FKHR. This allows them to bind to 14-3-3 proteins in the cytosol which retains them in the cytosol. This sequestration in cytosol means that genes normally activated by FOXO’s are switched off. In liver this helps shift from catabolic to anabolic state

Question 26

How does insulin prevent apoptosis?

favouring cell growth?

Answer 26

BCL proteins normally suppress apoptosis by blocking cytochrome C escape from mitochondria. However BCL function is blocked by binding to BAD which allows apoptosis to occur. PKB Phosphorylates BAD which allows it to be sequestered by 14-3-3 proteins. Therefore this blocks BAD binding to BCL and so blocks apoptosis

Question 27

In summery what mediates insulin action?

Answer 27

PKB mediates insulin actions by phosphorylating a range of proteins containing RXRXXS motifs which changes their function by either directly affecting their activity or affecting the proteins they are able to bind to

Question 28

Describe the adenyl cyclase;

Answer 28

G alpha i and G alpha s domains (space between here is the catalytic activity)

These are membrane bound to 12 TM domains

Thus production of cAMP can be localised to the membrane

Question 29

Describe the function of Gαs and Gαi

Answer 29

One of the intracellular domains has Gαs binding site (stimultes AC enzyme activity) while the other intracellular domain has a Gαi binding (inhibit AC activity)

Question 30

Is cAMP simple?

Answer 30

No

1) 9 isoforms of adenyl cyclase
2) 11 different PDEs
3) 3 PKA isoforms

Question 31

Describe how 9 isoforms of adenyl cyclase makes it a complex system;

Answer 31

9 isoforms and each regulated in a different way
i.e different responses to

• the 8 different Gαi isoforms
• to Ca++
• to kinases.

Since different ACs are expressed in different cells this provides different ways to regulate cAMP in different cells

Question 32

Describe how 11 different PDEs make cAMP a complex system

Answer 32

11 PDEs are expressed in different cells.

• Different regulations
• Different Locations

Question 33

Describe how 3 different PKAs make cAMP a complex system

Answer 33

Any cAMP that escapes PDE can activate PKA

3 PKA isoforms

• function differently
• expressed at different levels in different tissues.

Localisation via A-Kinase Anchoring Proteins (AKAPs) is very important as these bring targets together with PKA

Question 34

What does cAMP do?

Answer 34

cAMP Activates a Number of Catabolic enzymes and Processes via PKA

i. e
1. Acetyle CoA carboxylase - blocks lipid synthesis
2. CREB promotes gene expression
3. Hormone sensitive lipase’s (lipolysis)
4. Glycogen synthase (inactive) blocks glycogen synthesis
5. Activates phosphorylase kinase (stimulates glycogen breakdown)

Question 35

When counters the effects of adenyl cyclase?

Answer 35

Insulin Counters Effects of Adenylate Cyclase by Activating a Phosphodiesterase (PDE3b)

PKA not active
in absence of cAMP
so its substrates become dephosphorylated

i.e effects are now all opposite to before

Question 36

What are the general principles of signal transduction;

Answer 36

1. Specific receptor proteins that bind only to one specific hormone span the plasma membrane
2. When a hormone binds to this receptor it “transduces” a signal across the cell membrane and activates a series of signals inside the cell
3. These signals regulate cell processes such as growth, cell division, cell motility and cell metabolism and so allow hormones to appropriately regulate cell function
4. always feedback mechanisms to shut off the signaling pathways

Question 37

What can go wrong in receptor signalling?

Answer 37

Normal signalling ; Action of receptors is properly balanced by feedback pathway

Defects that cause reduced signaling can cause problems e.g. the insulin resistance that leads to diabetes is caused by reduced signaling through insulin receptors

Question 38

Whats a common cause of cancer?

1/4

Answer 38

Defects in the cell signaling pathways
are a very common cause of cancer;
Overxpression of Receptors

e.g. ERB-B positive breast cancer

This results in too much signaling so cells can grow and move inappropriately

Feedback mechanisms become overwhelemed

Question 39

Whats a common cause of cancer?

2/4

Answer 39

2. Receptor gets mutated so it is always super activated (i.e. a classical oncogenic mutation)
   e. g. EGF receptor mutations common in lung cancer

Question 40

Whats a common cause of cancer?

3/4

Answer 40

3. Receptor normal but members of signal pathways mutated
   (i. e. a classical oncogenic mutation)
   e. g. PI 3-kinase mutations discussed in this lecture

Question 41

Whats a common cause of cancer?

4/4

Answer 41

4. Feedback mechanism lost so pathway always active
   e. g. PTEN deletions discussed in this lecture

This results in too much signaling so cells can grow and move inappropriately

If only one copy of the gene is lost then there may or maynot be enough feedback ?

Question 42

What do the top drugs in the world target?

Answer 42

Cell signalling pathways

i.e beta blockers block GPCR / cAMP signalling