L8: RAS pathways Flashcards
(10 cards)
extracellular signal sources
xtracellular signals come from a variety
of sources
- cell:cell contact (ICAMs, cadherins) - cell:matrix contact (integrins) - Hormones - growth factors (PDGF, EGF, IGF) - cytokines (IL’s, TNF, SDF) - osmotic shock, heat etc.
Src
Proteins which contain a Src homology 2 (SH2) domain bind to tyrosine phosphorylated residues.
Proteins which contain a Src homology 3 (SH3) domain bind to PXXP residues.
EGFR Ras pathway
EGFR → RAS Pathway Breakdown
Growth factor (like EGF) binds to its receptor (EGFR, a receptor tyrosine kinase).
This causes receptor dimerisation and autophosphorylation of tyrosine residues on the intracellular tail of EGFR.
🔗 Signal Transmission via SH2 & SH3 Domain Proteins
SHC (an adaptor protein) is recruited first:
SHC has an SH2 domain, which binds specific phosphorylated tyrosines on EGFR.
This specificity is due not only to the phosphorylated tyrosine, but also to the surrounding amino acid context — SH2 domains recognize particular motifs.
Once SHC is bound, it is phosphorylated by EGFR’s kinase activity.
Phosphorylated SHC then acts as a docking platform for GRB2.
GRB2 binds to phosphorylated SHC via its SH2 domain.
GRB2 has two SH3 domains on either side of its SH2 domain.
SOS (a guanine nucleotide exchange factor) binds to GRB2 via its polyproline motifs, which are recognized by GRB2’s SH3 domains.
⚙️ Downstream Activation
SOS is now brought to the membrane, where it activates RAS by promoting the exchange of GDP → GTP.
Active RAS-GTP initiates the MAPK signalling cascade → RAF → MEK → ERK → transcriptional changes promoting cell proliferation, survival, and differentiation.
ras family
RAS FAMILY;
3 closely related proto-oncogenes:
H-ras, K-ras (4a/4b), N-ras
Bound to inner surface of the plasma membrane by a c-terminal lipid tail
Bind GTP/GDP
Have intrinsic GTPase activity
Play a central role in signal transduction
events at pm after egf
Events at the plasma membrane after EGF:
SOS CAN TURN RAS gdp to ras gtp turn it on,
Raf is a key effector (binding protein) of RAS/
Raf:
Ras binding domain
serine/threonine kinase domain
This protein is also a kinase.
Now moved from tyrosine phos events to serine/threonine phos events
Binds to the active ras gtp. Not in plasma membrane in cytoplasm in closed conformation. Gets attracted up to plasma membrane to bind toa active ver of ras here via ras binding domain.
For ras to be fully actived: raf dimers.
Heteroologous dimers are preferred. Often bc raf
Three isoforms A-RAF, B-RAF and C-RAF
B-raf/C-raf most frequently isolated
Next: raf activates a kinase called MEK BY PHosphorylating a serine in mek. Mek ican phosphorylate both serines, threonine? and tyrosines- dual kinase. Delivers 2 phosphorylations here on Erk (serine and threonine kinase). causes erk to transocate into the nucleus. It actovates trasncriptioj factors and drives trasncriptin of cyclinD1 gene (reg cell cycle).
MAP kinases
What happens when signalling goes wrong?
Map kinases:invonvelemt in cancer development
MAP kinase family members originally identified from studies of mutated
Study of many different cancer cell lines in culture indicates ERK activity is often higher in cancer cells
proteins in aggressive cancers
Most erk phosphorylated in cancer cells and not normal cells? Proportion of erk in cells thats phorphorylated has changed. Changed in amount of phsohprulated erk not a change in the amount of total protein int he cells.
She showed images of western blots and analysed them and i think this is important to know how to do for the exam
Ras is mutated in 30% of all human cancers
Raf is mutated in 60% of malignant melanomas
Mutations in ras:
Amplification of ras proto-oncogenes and mutations leading to constitutively active Ras present in 30% of all human cancers (12, 13 and 61 mutants most common)
Reason those image is that anything that mutates has to confer advantage to the cells. Mutations means ras doesnt get turned off. Mutations int hat region reg gtp binding and unbinding, delivers ras that is always on.
Frequency of significant kras mutations: alot of mutations on codon 12 and a little bit in codon 13.
Single amino acid subsitituon that is having a huge impact on the functionality of this protein.
specific mutations
Ras isoform codon mutation bias
Hras- codon 12 and 61
Kras- most at codon 12
Nras- codon 61
Pancreatic cancer: almost exclusively (90%): kras mutation
Mutations in raf:
More recently discovered to be important in cancer progression - Ras always considered to be reason for tumour development in MAPK signals
B-Raf frequently mutated in certain cancers (60% in melanoma) and this is independent of Ras mutations
Suggested that B-Raf mutations develop throughout tumour progression as opposed to Ras which is present throughout
What we see in raf: mutations alot are in kinase or regulatory domain. As mutations are delivering raf that cant be turned off so constantly phsophorylating mek. See raf mutations develop throughout tumour progression while ras is present throughout? Dont see both in same tumour or same cell often.
V600 mutation
V600E is constitutively active
(generally monomer)
leads to increased
Turns raf contitulley active. Doesnt need to dimerise. Prevalent in melanoma.
Colony forming growth in soft agar assay:
Cancer cells good at growing in suspension dont need as much adhesion
development of metastasis
Lots of cytoplasmic substrates are proteins involved in cell adhesion, cytoskeleton, reg mysoin activity? So dyereg pathway that causes high levels of erk is not only drive proliferation but prime those cells to e better at migration.
We know the Ras > Raf >MEK > Erk pathway leads to proliferation
BUT
Increased Raf and MEK phosphorylation might also lead to migration through Erk activity in the cytoplasm
therapeutics
kinases are relatively easy to inhibit with small molecule inhibitors
Kinases sit in families. Sometimes cant get specificity to a single kinase.
Can think of targeting atp binding site, preventing kinase from picking up atp, needs atp to get phosphate to put on protein.
Can compete with the substrate, design compound to sit where substrate sits so substrate cant dock on kinase and get phosphorylated
Or compound can bind outside or inside kinase domain and causes some sort of conformational change that prevents kinase activity. More specific than the others. Harder to do. Allosteric inhibitors. In regulatory region or kinase region>
Oncogene addiction as a basis for targeting
Oncogene addiction refers to the experimental observation that a tumour cell, despite its plethora of genetic alterations, can seemingly exhibit dependence on a single oncogenic pathway or protein for its sustained proliferation and/or survival.
Evidenced by the profound response
in vitro or in vivo
when treated with inhibitors
Ras oncogene in mapk pathway and cells can become addicted to that pathway.
Melanoma- high prevalence of raf mutation and we know it is addicted to that raf pathway so driving tumourgenesis.
Drug in image- recognises mutated ver of raf protein the v6100e mutation. Recognises raf with mutated residue. Mutant protein doesnt need to be dimerised, acts as monomer, circumventing requriement for ras activity? Inhibitor blocks that activity. Blocks the mutant raf.
But people can acquire resistance.
resistance mechanisms
Resistance Mechanisms to Vemurafenib (a BRAF V600E inhibitor)
Vemurafenib is designed to inhibit mutant BRAF V600E, but cancers can develop resistance through various mechanisms that reactivate the MAPK pathway via wild-type (WT) RAF or upstream components:
PDGFRB Overexpression
Cancer cells overexpress PDGFRB (Platelet-Derived Growth Factor Receptor Beta).
This activates an alternative survival pathway that bypasses mutant BRAF and reactivates WT RAF, keeping the cancer cells alive.
Vemurafenib-Induced WT RAF Dimerization
In the presence of wild-type RAF, vemurafenib can promote dimer formation (e.g., BRAF–CRAF dimers), which paradoxically activates the MAPK pathway.
This is an unexpected effect of the drug—it blocks mutant BRAF but may activate WT RAF in some cells.
NRAS Mutation
Mutations in NRAS (e.g., Q61K) can occur during treatment.
Mutant NRAS continuously signals to RAF → activates WT RAF dimers, bypassing BRAF inhibition.
This is commonly observed in patients who relapse.
Hepatocyte Growth Factor (HGF) from the Tumor Microenvironment
Stromal cells in the tumor environment secrete HGF, which activates the MET receptor.
This triggers survival pathways (e.g., PI3K/AKT) and can help bypass BRAF inhibition.
🔷 Targeting KRAS Directly: Sotorasib (AMG 510)
KRAS mutations have long been considered “undruggable,” but new drugs like sotorasib target specific mutations.
Sotorasib is approved for non-small cell lung cancer (NSCLC) with KRAS G12C mutation.
It works by binding irreversibly to the mutant cysteine residue and locking KRAS in its inactive GDP-bound state, preventing downstream signaling.
