Lecture 16 - Signalling Pathways Flashcards
Mitogenic Signalling pathways
How does growth factor signalling occur?
EGFR dimerisation and phosphorylation leads to the recruitment of signalling molecules such as Grb2 and SOS that bind to phosphorylated residues on the EGFR.
SOS recruits an important molecule, Ras.
Ras activates a downstream kinase Raf.
Raf phosphorylates and activates another downstream kinase such as MEK.
The signalling cascade continues till a target molecule such as a transcription factor is phosphorylated and activated to transcribe genes.
e.g. cell cycle genes such as c-myc
Growth factor signalling (MAPK pathway)
What is the role of Ras, GEF and GAP?
Ras is a small GTPase
Ras is farnesylated at the C-terminus - this allows it to anchor at the plasma membrane where it is in close proximity to Sos.
GEF - GTP exchange factor that promotes the exchange of GDP to GTP in Ras.
This changes Ras-GDP (inactive) to Ras-GTP(active).
Sos that binds to Grb is a GEF.
GAP - GTP activating protein that activates the GTPase activity of Ras.
GTP is hydrolysed and Ras-GDP results from this.
This essentially “switches off” the effects from the ligand.
The Ras-GAP is known as p21 protein activator 1
Why does Ras have an interesting transport path?
Ras is in fact a soluble protein but needs to be at the plasma membrane for its activation to occur.
We learnt in protein trafficking that plasma membrane proteins are inserted at the RER during co-translational translocation.
BUT for Ras, post-translational modification is needed to enable Ras to associate with the plasma membrane.
How does Ras get targeted?
The C-terminal hypervariable domain (amino acids 165—188/189) specifies membrane localization through post-translational modifications that include the farnesylation of each isoform on the C-terminal CAAX motif.
If the amino acid in the X position of CAAX is not Leu as is the case for all RAS proteins, then farnesyltransferase (FTase) modifies the CAAX Cys with a 1 5-carbonfarnesyl lipid
SECOND SIGNALS - PALMITOYLATION I POLYBASIC LYS RESIDUES
Although CAAX processing is necessary for the delivery of RAS proteins to, and their stable association with, the plasma membrane, it is not sufficient for these events.
Elements within the HVR that are upstream of the CAAX sequence are also required. What are they?
These elements have been called ‘second signals’ for plasma membrane targeting.
There are two types of second signal: One consists of Cys residues that serve as acceptor sites for palmitoylation (as found in HRAS, NRAS and KRAS4A) and the other consists of polybasic regions rich in Lys residues (as found in KRAS4B)
How can Ras ‘not be switched off’?
When Ras is mutated such that it is constitutively bound to GTP because its GAP cannot bind, GTP is NOT hydrolysed.
Without hydrolysis of GTP to GDP, Ras-GTP is in the “ON” state.
How can colony formation assay be used as a technique for studying transformation?
Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis.
The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells.
This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions.
What is the Wnt signalling pathway for?
Wnt is required for maintenance of intestinal epithelium
Inhibition of Wnt signaling leads to progressive degradation of intestinal epithelial structure
Signalling pathway in the colon - adult stem cells
What happens when Wnt is absent?
Cell division is suppressed in the absence of growth factor Wnt.
- transcription factor ß-catenin is phosphorylated by kinase GSK3-ß.
This triggers the ubiquitination of ß-catenin by a SCF-type of ubiquitin ligase.
Ubiquitinated ß-catenin is degraded by the proteasome.
APC = adenomatous polyposis coli (scaffolding protein)
- large protein that binds to a number of other proteins
Signalling pathway in the colon - adult stem cells
What happens when Wnt is present?
When Wnt is present, the effector molecule Dishevelled (Dsh) inhibits the kinase GSK3.
This prevents the ubiquitination of ß-catenin.
ß-catenin is stabilised and enters the nucleus to drive the transcription of cell division genes, eg c-myc.
What is Familial Adenomatous Polyposis Coli (FAP)?
This is a cancer syndrome that predisposes afflicted individuals to colorectal cancers.
The critical tumour suppressor gene APC was discovered through the study of a familial
colorectal cancer known as FAP.
The distinctive feature of this syndrome is the appearance of large number of tumours known as polyps in colon of individuals that inherited defective tumour suppressor genes APC or Axin
Individuals inheriting deletions or mutation in the APC gene develop numerous benign growth called polyps in the colon.
The polyps are not present at birth, but by late adolescence there maybe more than 1000 polyps. Some of these polyps develop into malignant colon tumours by the 40s.
Without intervention all affected persons develop colon cancers.
