cell signalling

Question 1

profound consequences that signalling in cells happens on phenotypes

Answer 1

Signaling is the thing that gives the most profound change to cells
- if we can add a ligand and those cells have the receptor.
- So this is an experiment where we’ve got cells that are positive for the pdgf receptor and cells that do not have the pdgf receptor.
- And if we add the ligand PDF, it stimulates migration and growth, the sheet then closes
- if the same cells lack the receptor though, you can see that this closing is no longer able to happen
- this whole phenotypic approach can only be instructed by activation of the receptor by its ligand

Question 2

experiement - profound changes when you add the stimulation of a receptor tyrosine kinase

Answer 2

here are profound changes when you add the stimulation of a receptor tyrosine kinase.
- So this is an experiment looking at all the different anti tyrosine phosphorylations
- these are naive cells that don’t have any signalling going through them
However, if we transform these cells with src you can see that Now there are multiple different proteins at all different sizes and levels and flavors that are now being phosphorylated and altered in their behaviour.

Question 3

RTK signalling

Answer 3

• we have the 2 tyrosine kinase monomers of the receptor. They are sitting at the cell surface.
  ○ They’re moving around, they can dimerize and if they dimerize within the presence of an EDF molecule or any ligand for the receptor tyrosine Kinase, They come together
  ○ this coming together stays permanent.
  ○ This leads to the trans phosphorylation of one another
  so this monomer Here is able to transphosphorylate its partner and vice versa.

Question 4

why is the concentration of the ligand and the receptor important

Answer 4

• the more ligands We have the more chances of this binding occurring
  the more receptors We have the chances of this coming together is increased.

Question 5

RTK trans phosphorylation - phosphorylation of the receptor

Answer 5

after this activation of a receptor tyrosine kinase
-we see the coming together of the catalytic domain of a receptor.
- It has within it this small little catalytic cleft which is going to do all the phosphorylation.
- It’s held in a position where it cannot be accessed by these inhibitory Loops and it’s only when this is phosphorylated that this catalytic cleft becomes fully open
- and once it is open is able to phosphorylate multiple different areas.

Question 6

RTK transphosphorylation - transphosphorylation of its binding Partners.

Answer 6

not only does the receptor cause this transphosphorylation of each other, It also causes the transphosphorylation of its binding Partners.
- So if we have secondary kinase, which is Jak or Tyk2 and they’re bound on to two different receptor arms, they can transphosphorylate each other.
○ So they now brought together into the same location and they can now phosphorylate one another as well as possibly phosphorylating the receptor further
○ and this phosphorylation on the receptor could then cause a change that causes the recruitment of another secondary signaling molecule.

Question 7

how is RTK signalling altered in cancer

Answer 7

• there are several different ways that we can have dysregulation of RTKs
  ○ the most common of which is some form of mutations.
  ○ So a mutation is either within that catalytic cleft - the removal of the inhibitory or extracellular domain which leads to the receptor signaling without any ligand.
  ○ Another option is we end up with over expression or amplification. This just causes so much of the receptor to being on the cell surface, then it can lead to hyperactivation - turning on independently
  ○ If you have a mutation that leads to the activation of the receptor independent of binding or dimerization - This is constitutive Activation - So it is always on

Question 8

what does overexpression of RTK normally lead to

Answer 8

overexpression normally leads to hyper activation which is just means that it’s harder for the cells to have the correct level or it’s hard for it to turn it off.

Question 9

RTK signalling gone wrong common causes

Answer 9

• non small cell lung cancer, mutations in the EGFR approx 10-15%
• glioblastoma, mutations in the RTK gene known as EGFRvlll are found in up to 50% of cases

Question 10

RTK signalling paracrine signalling

Answer 10

paracrine signalling is cell to cell communication acting locally on neghbouring cells
- coordinates cellular responses within tissues and organs by regulating cell proliferation, differentiation, and survival
- examples include neurotransmitters in the nervous system, growth factors in blood vessel growth and repair, and cytokines in immune cell activation

the receptor tyrosine kinase can be activated through ligand signaling and it can have Self creating ligands
- this paracrine signaling is very common within a tumor between both the stroma and the tumor cells but also within different populations of tumor cells themselves.

Question 11

what is one of the most common dysfunctional phenotypes of cancer cells

Answer 11

expressing receptors not associated to their tissue of origin

Question 12

autocrine signalling in cancer cell

Answer 12

the cancer cells themselves producing the ligand for the receptor they are already expressing.
- And this is known as autocrine signaling.

Question 13

how does RTK signalling drive autocrine signalling

Answer 13

you have a feed-forward mechanism of ligand activating the same receptor causing the production of a different ligand activating the same or a different receptor and then the cycle begins again.

Question 14

RTK domain homology

Answer 14

tyrosine kinase domains are highly conserved even between non receptor kinases

Question 15

RTKs that require co-factors to activate them

Answer 15

some receptor tyrosine kinases require not just their ligand, butalso other cofactors to activate them.
-e.g the fgf receptor 1 and one of its ligands fgf receptor2 but for it to complete its binding it also requires Heparin. - Heparin is a molecule that’s found in The extracellular Matrix and without fgf receptor 2 cannot fully stimulate the receptor.
- this cofactor activation can help tune cells to respond to the ligands in the right place.

Question 16

how does requirement of cofactors make the signal more specialised

Answer 16

○ So it could be that fgf receptor is found within different areas of the body, but by requiring Heparin, it means that the cell may need to be in a particular orientation whether it’s polarized within an epithelial sheet, to actually be able to be able to access Heparin as well as the ligan
○ so it’s a way of making Making the signal more specialized than just having the ligand there itself.

Question 17

RTK signalling gone wrong - gene fusion

Answer 17

So one of the other ways the receptor tyrosine kinase signaling can go wrong is by Gene Fusion.
- Gene Fusion is when we have two genes, they’re translocated, So that one part of the gene is now fused to the other part of another Gene.
- This happens fairly regularly - is not as common as mutations or amplifications, but it’s a very common process.

Question 18

example of gene fusion

Answer 18

in this example is the protein fig and a receptor tyrosine kinase ros
- They get fused. So now on the extracellular side There are two halves of fig
- These two halves wants to naturally come together because they have a natural partnership
- when they then form this dimerization independent of the EGF receptor, because the ligands are now close enough together They will then be able to transphosphorylate each other Other and then lead to the activation of the downstream signaling.

Question 19

RTK signalling - So what happens on the receptor that enables the secondary molecules to come and bind to it

Answer 19

one of the main secondary adapter molecules is sos, son of sevenless.
- we have a tightly controlled docking mechanism, So they all have domains within them - sh2 and sh3 domain.
-sh2 is a particularly folded protein structure.
○ So it’s made up of these beta sheets And these Alpha helices making two different groups
○ these two negatively charged spaces enables The Binding of phospho tyrosines, but also Proline Rich sh3 domain.
○ so there is the ability to recognize a phosphotyrosine but also amino acids that are flanking it.

Question 20

RTK signalling - docking

Answer 20

a receptor gets activated and it’s not just the canonical Pathways that can be activated, There’s also the ability to dock with multiple different internal molecules.
- These are different signaling partners that will lead to the activation of different pathways.

Question 21

what is found on all secondary adapter proteins

Answer 21

this common structure- SH2 domains and sh3 domains are found on all these secondary adapter proteins.

Question 22

how do we get activation once these these secondary molecules are binding - self inhibited step

Answer 22

this is a Well defined protein structure interactions and how something can be controlled within itself and through signaling.
- we have src held within It’s normally Inactive structure.
- we’ve got the sh3 domain binding to its own Linker segment
- the sh2 domain which binds to phosphotyrosine is held by a tyrosine within its own structure.
- this tyrosine s27 is phosphorylated normally So it’s in an inactive State, the sh2 domain is binding less the sh3 domain is then binding its own Linker region and this produces a closed structure.
- Means the kinase domain is not able to be activated.
- And the activation Loop is in the wrong position.

Question 23

how do we get activation once these these secondary molecules are binding - structurally rearranged step

Answer 23

receptor tyrosine kinase has now been activated.
○ There is now a rich region for the sh3 domain to bind to as well as a new phosphp tyrosine group
○ this conformation enables the unfolding and the structural rearrangement of Src so now the kinase domain and the activation Loop is now accessible.
- This then leads to the receptor tyrosine kinase being able to phosphorylate tyrosine.
- This time there is y416 within the activation Loop this enables the activation Loop to change its position, leaving this catalytic cleft open which then leads to src being able to signal to its Downstream molecules.
○ So this is a very nice system by which the spatial and structural Arrangements of two domains as well as having an inhibitory phosphorylation could hold a protein within a closed structure

Question 24

how do we get activation once these these secondary molecules are binding - activation

Answer 24

if however there is a structure that has spatially similar Dynamics, so there’s a phosphotyrosine just the right distance away from a Linker region, these two domains can unfold, be open, enabling the access for a different kinase to then come along and activate an activation Loop.
○ So this phosphorylation, once the src is unfolded on an active receptor tyrosine Kinase can be activated by the receptor tyrosine kinase itself, but could also be activated by other adaptor kinases

Question 25

RAS

Answer 25

one of the most common things that is activated Downstream of RTK is ras
- ras can get to a receptor in a more direct way or an indirect way.
-for example, Grb2 contains within it an sh2 domain and 2 sh three domains which can then bind sos (which is a GEF itself)
○ So this is what’s going to exchange GDP for GTP to activate RAS
○ this could bind directly to receptor tyrosine kinase or it could bind indirectly through for example shk, which is another sh2 domain, which would then be phosphorylated on one of its tyrosine residues, which would then go up to sos And then Ras

Question 26

RAS activation downstream signalling

Answer 26

Ras activation Downstream signaling acts as a slightly different node.
- So it acts in a cycle or between an active and an inactive State depending on its GDP or GDP status.
- it is therefore regulated by gaps - things that want to remove GDP, hydrolyze it and produce GDP within its domain or GEFs which want to exchange GDP for GTP leaving it in an active state.

Question 27

why does RAS activation act as a node

Answer 27

Ras activation acts as a node because it can signal through the major different
Pathways.
- So once RAS is activated it can lead to the activation of the map kinase pathway, raf, MEK and erk
- Erk can then phosphorylate multiple different transcription factors
- or it can signal through pi3 kinase which could lead to activation of AKT
- and then it’s control of several different survival Pathways such as apoptosis

Question 28

RTK signalling and RAS - activation of PI3 kinases

Answer 28

pi3 Kinases are slightly different to kinases that phosphorylate proteins
- instead of proteins They are phosphorylating lipids
○ within the bilayer we have this phosphatidyl inositol lipid. It has within it a six carbon ring.
○ This is where phosphorylation will happen.
- So in the cytoplasm this normally exists in a PIP2 State on the intracellular surface
- pi3. Kinase can phosphorylate one of these secondary structures Once it gets cleaved to make PIP3
- PIP3 has phosphorylations on 3 4 and 5 and this structure here can directly interact with Pro proteins to make them activated

Question 29

PI3 kinase converting PIP2 to PIP3

Answer 29

pi3 kinase taking PIP2 to PIP 3 is a reversible process.
- So P10 can dephosphorylate the third carbon producing pip2 again
- this negative regulating or P10 means that it’s a tumor suppressor.
○ So if you lose P10 there is an accumulation of pip3 -
§ The more PIP3 there is, the more activation of its main Downstream kinase.
-this Pip 3 can be bound within a pH domain of the kinase akt, once it’s activated. AKT signals directly onto key Regulators of Pathways associated with the Hallmark of cancer

Question 30

when PI3 kinase is activated by a receptor tyrosine kinase what does it lead to

Answer 30

leads to the activation of AKT through the production of PIP3
- and this directly inhibits the cyclin-dependent kinase Inhibitors p21 and p27.
- these would normally inhibit cdk2 complexes as well as CDC2 complexes.
- so these are normally naturally inhibited but in the presence of AKT These Inhibitors get turned off and cell cycle is able to progress freely.

Question 31

what is happening on PI3 kinase to make it mutagenic and in cancer

Answer 31

• pi3 kinase can be mutated in multiple different parts of its domain structure.
• there are two major areas of the pi3 kinase that is phosphorylated
  ○ one is within this helical domain and the other part here is within the kinase domain.

Question 32

how do we know that PI3 kinase is oncogenic

Answer 32

• there have been several different experiments that have been done
• one here where they’ve taken P10 or a P10 null. So this is the thing that’s deregulating pip3 production
• when we remove P10 you can see we end up with hyperplasia - multiple cells growing and no longer one single layer

Question 33

what happens when we have got excess pip3 and excess akt or pi3 kinase activity

Answer 33

We have an oncogenic event that drives both cell survival and cell cycle.

Question 34

in what cancer is this excess PIP3/ akt found

Answer 34

So this is found in lots of different cancers.
- So melanoma has got over an 80% hyperactivation of AKT found within it.
- P10 is quite regularly lost. So that’s either mutantly inactivated or it’s repressed - This Is 40 to 50% in melanoma.

Question 35

what is another way we can end up with hyper activation of PI3 kinase or MAP kinase

Answer 35

is to lose the ability to regulate Ras activity.
- NF1 is normally part of The feedback on Ras that removes GTP hydrolyses it to GDP, causing it to be inactive
- if NF1 is not there we end up with ras activation with a very slow turnover back to the inactive form.
- The more active Ras there is the more signaling we get through pi3 kinase and the map kinase pathway.

Question 36

how can NF1 be lost

Answer 36

can quite often be lost, its naturally turned over by pkc activity.
○ So if you get pkc mutations, you can end up with a loss of NF1
- but it’s also found in other genetic disorders. So if NF1 is mutated or lost you can end up with a genetic disorder called neurofibromatosis type 1.
○ It’s characterized by benign tumors in the nervous system.
○ It’s a familial genes so it can lead to familial cancers, but it is associated with gliomas and different types of nerve sheath tumors.

Question 37

how should RTK signalling be working normally

Answer 37

• we’ve got receptor tyrosine kinase secondary structures binding through their tightly-controlled domains
• this leads to Sos
• The GEF activity of sos is changing GDP to GTP
• this activation of Ras should then signal through different Pathways including the map Kinase Pathways and then eventually this is turned off.
• So this system is normally turned off by sprouty.

Question 38

sprouty

Answer 38

○ sprouty is a Protein dephosphorylate
§ the ability to deactivate RAF kinases and sh2 by removal of the phosphorylation sites.
§ Once they are removed they are then able to no longer bind because this phosphorylation site does not exist, SH2 can’t be engaged, therefore SOS isnt there, when sos isnt being bound RAS is deactivated

Question 39

how does RAS axtivate RAF

Answer 39

ras gets activated,
- when ras is activated, the rafs which come in three different flavors (ARAF, BRAF, CRAF) They’re able to bind to ras.
- Once they are bound, They can form either homo or hetero dimers.
- They then Auto phosphorylate each other, This allows the phosphorylation of other sites and BRAF only needs one in this n-terminal region

Question 40

structure of a typical RAF protein

Answer 40

• We’ve got the kinase domain
• We’ve got the P Loop - This is the activation area.
• We’ve got different binding domains
• We’ve got inhibitory proteins in this n-terminal region

Question 41

difference between N terminal region between BRAF, CRAF and ARAF

Answer 41

There is a distinct difference between b-raf and c-raf and a RAF and that is that it lacks these tyrosine residues here.
- This means that BRAF only requires one phosphorylation event within this region
- the lead to its ability to become active and this is important because b-raf is the RAF kinase that’s normally found to be mutated the most
○ and it’s thought to be because if you end up with just one mutational change, this inhibitory domain only requires one further signal to lead to activation rather than 2 so it is able to become mutated and activated easier than the other two forms because of this n region change.

Question 42

how many cancers is MRAF mutated in

Answer 42

50-70% of malignant melanomas, 40% of thyroid carcinomas, 30% of ovarian tumours, and nearly 100% of hairy cell leukaemias

• mutations affecting the valine residue at amino acid position 600 are most prevalent, with V600-BRAF accounting for 80-90% of mutations in melanoma

Question 43

what do v600 mutations in BRAF allow

Answer 43

allow BRAF to adopt an active kinase conformation in the absence of dimerisation and, as a result, these mutants can signal as RAS independent monomers