What was discovered when the immunoprecipitated Ras was incubated with 32P-ATP
No phophorylation occured - When they used GTP instead there was incorporation into Ras - however - this was because the Ras they were using was mutated and had a threonine at position 59. This threonine was being phosphorylated and didn't represent Ras' normal action.
What experiment was done in E.coli to determine Ras' mechanism of action?
The Ras gene was expressed and purified in E.coli. The purified Ras was then incubated with radioactive guanine nucleotides and produced a time course chromatography. This showed that over time that Ras makes GDP by hydrolising GTP.
What effect does the oncogenic Ras (H-Ras) have on its function>
There is no GTPase activity - therefore Ras is constituitively active and signaling to downstream effector molecules
What is the cycle of Ras activity?
What does the crystal structure of Ras tell us about its function?
1. Its not a kinase
2. Ras binds to the terminal phosphate of GTP via two amino acids - Gly12 and Gln61
3. These two residues are often mutated in oncogenic Ras and hydrolysis of GTP cannot occur
4. The hydrolysis of GTP is needed to turn off Ras into the non-proliferating form
What model system first showed the link of Ras to cancer? How?
Drosophila - High resolution SEM was used to look at the 7 rhabdomeres of the ommatidia. Mutants were generated, one of these was sevenless (missing seventh cell). Genetic enhancer/ supressor screens and epistatic analysis revealed that there was something epistatic (Upstream) to sevenless - bride of sevenless
Also found something downstream of sevenless (Son of sevenless)
How was C.elegans investigated to discover the Ras pathway like drosophila
Looked at genes involved in vulval induction. There was a mutant called Let-23. Through similar genetic analysis he discovered Lin-3 was upstream, SEM5 was downstream and Let-60 was further downstream. This had clear similiarites to drosophila when compared and led to the following conclusion
Growth factors = BOS and Lin 3
EGFR = Sevenless and Let-23
This proved that Ras lied downstream of RTK signalling
How was GRB-2 and its function discovered
An expression cloning strategy led to the identification of SEM-5. Then bacteria expressing a library of mammalian genes, with individual colonies expressing a single polypeptide. The bacteria were lysed and probed to see if any of the proteins expressed would bind the EGF receptor. (only the tyrosine kinase domain of the EGFR was used and it had a fluorophore atached. GRB2 bound to the EGFR selectively, the plasmid was extracted from the bacteria colony, GRB2 is highly homologous to SEM-5 which had been shown to act upstream of Let-23 in C.elegans. Suggested GRB2 to bind RTKs and be upstream of Sos.
What is the full mechanism of Ras signalling including RTKs
The SH2 domain of Grb2 binds the phosphotyrosine on active RTKs. Sos interacts with the two SH3 domains on Grb2. Sos is a GEF so turn Ras on. Ative Ras initiates cell proliferation.
Any of these components can become mutated to cause cancers.
Which downstream Ras targets are responsible for increasing cell growth, gene expression and cell morphology/movement?
Cell growth = PI3K
Gene expression = Raf (MAPKKK)
cell morphology/ movement = Ral-GEF