RAS/RAF inhibitors Flashcards
What is responsible for the activation of RAS?
GEFs (guanine exchange factor) remove bound GDP from RAS
What is responsible for the inactivation of RAS?
GTPase activating protein initiate reduction of GTP to inactive GDP bound to RAS>
Why do GEFs promote the activation of RAS overall in a cell, given that RAS has the same affinity to bind GTP/GDP
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
What is NF1, how is it implicated in cancer?
It is a GAP, when mutated RAS remains in its GTP bound form.
Which form of RAS is found most commonly mutated in pancreatic and colorectal cancer?
KRAS
Which form of RAS is found most commonly mutated in melanoma?
NRAS
Which form of RAS is found to be mutated most commonly in bladder and head/ neck cancer>
HRAS - at much lower frequencies
In which portion of the protein, are the 3 RAS isoforms identical?
The first half
Where do the 3 RAS isoforms diverge in homology?
The C terminal domain
How do different RAS protein isoforms distinguish the different effector protein that they are going to bind?
They don’t, the effector binding portion of the three RAS isoforms are identical
What does the RAS C-terminal domain determine for the protein.
Directs the proteins membrane localisation and trafficking
What is the CAAX box?
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.
What is the secondary localisation signal?
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.
Is the secondary localisation signal or the CAAX box responsible for deciding where the RAS isoform ends up on the plasma membrane?
The secondary localisation signal is responsible
What is the secondary localisation signal in KRAS4B?
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.
What defines which effector proteins that RAS isoforms interact with?
Depends on where in the cell the RAS is localised due to the palmitoylation/ secondary localisation signal.
What is Sos?
A GEF that associates with GRB2, then activates RAS by causing the dissociation of GDP from RAS.
What kind of kinase is Mek?
Dual specificity, phosphorylates both threonine and tyrosine residues on Erk
How many substrates do Raf and Mek have?
One - very specific kinase’s. Raf = Mek. Mek = Erk
Why, given that Raf to Mek to Erk, would Ras not directly activate Erk to get the same outcome?
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.
What role do phosphatases have in the Ras - Raf pathway
Important regulatory units of the pathway.
How do phosphatases add sophistication to the overall output of Erk?
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
What kind of kinase are RAF proteins?
Serine/ threonine specific protein kinase
Which RAF paralogues is associated with human cancers?
B-RAF
How do C-RAF/B-RAF become dimerised and activated following RAS activation?
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.
What phosphorylation events are required for RAF activation
Phosphorylatuon in the activation segment as well as two sites in the N-region
What difference in B-RAF amino acids makes it more viable to induce cancers
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.
What are the hotspots for B-RAF mutations
The glycine rich loop region in the kinase domain that interacts with ATP. Also the activation segment
What particular site on BRAF is mostly mutated and what is the mutation?
V600E - Negative charged amino acid is phosphomimetic so initiates kinase activity without the normal regulatory steps.
Do the activation segment and the glycine rich loop come into close contact in the kinase domain 3D structure?
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.
Why did melanomas which didn’t have a V600E mutation respond badly to small molecule inhibitors of BRAF?
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.
How do BRAF inhibitors get drug induced acquired tumour resistance.
Multiple mechanisms that activate the Ras-Raf pathway in different ways, unrelated to Raf (upstream or downstream)
What role do scaffold proteins have for cellular localisation of MAPK pathway?
Scaffold proteins spatially regulate the MAPK pathway. Scaffold proteins target MAPK to different parts of the cellular compartments.
What is KSR and how is it regulated?
It is a scaffold protein that regulates MAPK cascade. It is regulated by a number of proteins which add another layer of complexity.
How do scaffold proteins regulate the ERK pathway?
Spatially and temporally
