Lecture 4 Flashcards
What is Ras?
- Ras is a guanine nucleotide binding protein
- Ras is a GTPase
- GTPase function is compromised in oncogenic forms of ras
Gross et al (1984):
- Expressed and purified recombinant ras in bacteria
- Analysed bound nucleotides by chromatography
- Incubated with radioactive GTP radioactive in the guanine bit not phosphate
- C-Ras (Wt) and Ha-ras (viral mutant)
- Chromatogram
- Expose to film
- In presence of ras GDP is present – telling us its enz that hydrolyses GTP
- But Ha- Ras isn’t a GTPase – viral ras don’t work as enz – capable of binding but not hydrolyzing
- can calculate how many molecules per second it hydrolyses
Erikson
Immunoprecipitated Ras and incubated with radioactive guanine nucleotides
Ed Skolnick
- Radioactive displacement experiment
- Bound to ras
- Incubated with non radioactive nucleotide
- If you’ve got something that binds specifically you could bind a non radioactive substances that displaces the radioactive substance
- Compete away radioactive with different nucleotides to see if competitive with GDP
- Incubate with no radioactive GDP – compete it oit with cold
- CGMP doesn’t compete so ras doesn’t bind cGMP or GMP
Competitive assay
McCormack et al (1988)
Identification of a GAP
- Presence of something theres an increase in activity more GDp when cytosol added
- Used assay as a biochemical purification – protein GAP identified stim GTPAse activity of Ras and other molecules
Macara and wolfson-
- Look to see if you can displace GDP from Ras more quickly
- Can incubate for hours but GDP doesn’t come of Ras easily
- Brain cytosol contains an activity that facilitates dissociation of GDP
- Conc dependent more in more easily GDP off Ras
- First GEF identified was a functional homologue of CDC25
What animal model was used to identify if ras works upstream, downstream or in parallel ?
Drosophila- looking at the ommatidia
- 7cells
- sevenless mutant has 7th cell missing
- looked at son and Bride of sevenless
What strategy
led to the identification of SEM-5
An expression cloning strategy
- Using bacteria to express mammalian genes in each bacterial colony
- Each gene product should be expressed in each
- Individual colonies express 1 polypeptide
- Chemically attach fluorophore to phosphorylated domains
- Transfer to bacterial plot – wash nitrocellulose and wash in solution with fluorophore and see if anything binds it tightly
- Spot where protein expressed tightly should be able to map back to what colony it came from
GRB2 is highly homologous to what ?
To SEM 5
Describe the model for RTL activation of Ras
- see diagram Involves: GRB2 SH2 SOS
GRB2 can only bind after what has occurred to RTK
Phosphorylated
SOS
is a GEF teases out GDP from Ras allowing it to bind GTP
What type of system is RAs?
Two switch system- on and off state
Eventually GTP will hydrolyse and switches itself off which occurs more quickly if GAP present
What type of system is RAs?
Two switch system- on and off state
Eventually GTP will hydrolyse and switches itself off which occurs more quickly if GAP present
- RasGTP adopts an altered conformation
which binds and activates downstream effectors
on one specific surface all protein effectors seem to bind one side only when in GTP bound form
