RAS/RAF inhibitors

Question 1

What is responsible for the activation of RAS?

Answer 1

GEFs (guanine exchange factor) remove bound GDP from RAS

Question 2

What is responsible for the inactivation of RAS?

Answer 2

GTPase activating protein initiate reduction of GTP to inactive GDP bound to RAS>

Question 3

Why do GEFs promote the activation of RAS overall in a cell, given that RAS has the same affinity to bind GTP/GDP

Answer 3

GTP is at a ten fold higher concentration inside the cell compared to GDP. Therefore it is more likely to bind to GTP following the removal of GDP by a GEF

Question 4

What is NF1, how is it implicated in cancer?

Answer 4

It is a GAP, when mutated RAS remains in its GTP bound form.

Question 5

Which form of RAS is found most commonly mutated in pancreatic and colorectal cancer?

Answer 5

KRAS

Question 6

Which form of RAS is found most commonly mutated in melanoma?

Answer 6

NRAS

Question 7

Which form of RAS is found to be mutated most commonly in bladder and head/ neck cancer>

Answer 7

HRAS - at much lower frequencies

Question 8

In which portion of the protein, are the 3 RAS isoforms identical?

Answer 8

The first half

Question 9

Where do the 3 RAS isoforms diverge in homology?

Answer 9

The C terminal domain

Question 10

How do different RAS protein isoforms distinguish the different effector protein that they are going to bind?

Answer 10

They don’t, the effector binding portion of the three RAS isoforms are identical

Question 11

What does the RAS C-terminal domain determine for the protein.

Answer 11

Directs the proteins membrane localisation and trafficking

Question 12

What is the CAAX box?

Answer 12

A motif found in the C-terminus of RAS, recognised by farnasyl transferase which covalently attaches a lipid group to the cysteine which will act to anchor RAS into the lipid bilayer of the plasma membrane.

Question 13

What is the secondary localisation signal?

Answer 13

Cysteine residues that lie upstream of the CAAX motif that are recognised by palmitoyltransferase. This adds a palmitoyl groups which define the way it is transported (what post-translational modifications it accumulates) and where in the plasma membrane it localises.

Question 14

Is the secondary localisation signal or the CAAX box responsible for deciding where the RAS isoform ends up on the plasma membrane?

Answer 14

The secondary localisation signal is responsible

Question 15

What is the secondary localisation signal in KRAS4B?

Answer 15

No cysteine residue present. Instead, it has a stretch of positively charged lysine residues which have an electrostatic interaction with the negative phospholipds of the lipid membrane.

Question 16

What defines which effector proteins that RAS isoforms interact with?

Answer 16

Depends on where in the cell the RAS is localised due to the palmitoylation/ secondary localisation signal.

Question 17

What is Sos?

Answer 17

A GEF that associates with GRB2, then activates RAS by causing the dissociation of GDP from RAS.

Question 18

What kind of kinase is Mek?

Answer 18

Dual specificity, phosphorylates both threonine and tyrosine residues on Erk

Question 19

How many substrates do Raf and Mek have?

Answer 19

One - very specific kinase’s. Raf = Mek. Mek = Erk

Question 20

Why, given that Raf to Mek to Erk, would Ras not directly activate Erk to get the same outcome?

Answer 20

Ras - Raf - Mek - Erk allows for the amplification of a single activated Ras - Raf which can then activate multiple Mek - Multiple Erk.
Also for more points of regulatory points of control which gives more complexity and sophistication the signal has.

Question 21

What role do phosphatases have in the Ras - Raf pathway

Answer 21

Important regulatory units of the pathway.

Question 22

How do phosphatases add sophistication to the overall output of Erk?

Answer 22

S/T phosphatases and tyrosine kinases can act on the threonine and tyrosine residues that become phosphorylated on Erk by Mek. Both are required for full activation of the enzyme. Therefore, if one is removed by a phosphatase then the signal is turned down, modulated. There are also dual specificity phosphatases which could switch off the signal entirely

Question 23

What kind of kinase are RAF proteins?

Answer 23

Serine/ threonine specific protein kinase

Question 24

Which RAF paralogues is associated with human cancers?

Answer 24

B-RAF

Question 25

How do C-RAF/B-RAF become dimerised and activated following RAS activation?

Answer 25

In their inactive state, B/C-RAF are associated with 14-3-3 dimers that occlude the catalytic domain of the RAF proteins via an intramolecular interaction. When Ras becomes activated RAF is recruited to the membrane. At the membrane a phosphatase removes a specific phosphorylatuon site. 14-3-3 can no longer bind a single RAF so forms an intermolecular interaction between B and C-RAF.

Question 26

What phosphorylation events are required for RAF activation

Answer 26

Phosphorylatuon in the activation segment as well as two sites in the N-region

Question 27

What difference in B-RAF amino acids makes it more viable to induce cancers

Answer 27

In the N-region; two residues are required to be phosphorylated for the RAF protein to be activated. In the N-region of B-RAF has an aspartic acid residue rather than tyrosine seen on A/C RAF. This residue can’t be phosphorylated but is negatively charged so is a phosphomimetic. As a result B-RAF is primed for activation (only one phosphorylation even required) and has a high basal activity.

Question 28

What are the hotspots for B-RAF mutations

Answer 28

The glycine rich loop region in the kinase domain that interacts with ATP. Also the activation segment

Question 29

What particular site on BRAF is mostly mutated and what is the mutation?

Answer 29

V600E - Negative charged amino acid is phosphomimetic so initiates kinase activity without the normal regulatory steps.

Question 30

Do the activation segment and the glycine rich loop come into close contact in the kinase domain 3D structure?

Answer 30

Yes, normally they interact to keep the protein in an inactive state. When mutated there is a disrupted interaction between the two loops which lets the protein adopt its active conformation.

Question 31

Why did melanomas which didn’t have a V600E mutation respond badly to small molecule inhibitors of BRAF?

Answer 31

Related to the ability of RAF to dimerize. BRAF inhibition induced RAF dimerisation. The small molecule only inhibits one RAF in the dimer. The inhibitor then leads to pathway activation.

Question 32

How do BRAF inhibitors get drug induced acquired tumour resistance.

Answer 32

Multiple mechanisms that activate the Ras-Raf pathway in different ways, unrelated to Raf (upstream or downstream)

Question 33

What role do scaffold proteins have for cellular localisation of MAPK pathway?

Answer 33

Scaffold proteins spatially regulate the MAPK pathway. Scaffold proteins target MAPK to different parts of the cellular compartments.

Question 34

What is KSR and how is it regulated?

Answer 34

It is a scaffold protein that regulates MAPK cascade. It is regulated by a number of proteins which add another layer of complexity.

Question 35

How do scaffold proteins regulate the ERK pathway?

Answer 35

Spatially and temporally