Ras oncogene lecture

Question 1

what is RAS?

Answer 1

a GTPase

- guanine nucleotide binding protein

Question 2

Is ras a kinase? how was this shown?

Answer 2

• ras is not a kinase
• when incubated with radioactive ATP no band formed which was the size of RAS
• Ras does not bind/hydrolyse ATP

Question 3

what happened when Ras was precipitated with radioactive GTP?

Answer 3

On the autoradiogram, a protein the size of ras is labelled. (takes P from GTP)
- but this form of RAS is the ONCOGENIC mutant form
(p binds to ras and gets stuck)

Question 4

what is the oncogenic mutant form of RAS?

Answer 4

Glycine 12 - valine

Question 5

what can RAS bind and how was this shown?

Answer 5

GTP , GDP

• doesn’t bind GMP, ATP or cGMP
• shown by a competition experiment (add excess of non-labelled nucleotide to see if it outcompetes labelled nucleotide (watch RA nucleotide go down))

Question 6

what did gross et al show abount c-ras and ha-ras in 1984?

Answer 6

• showed that c-ras has GTPase activiry

- showed that harvey (oncogenic/viral) rasdoes not have GTPase activity

Question 7

what was the experiment which showed that c-ras is a GTPase but oncogenic RAS is not

Answer 7

• express and purify recombinant c-ras and h-ras in bacteria
• incubate H-ras and c-ras with radioactive nucleotides - isolate guanine nucleotides and run 2 ways on a gel
• start bound to GTP and watch if GDP appears
• with c-ras GDP increases over time - shows c-ras is converting GTP into GDP (GTPase)
• with h-ras GDP does not appear - not hydrolysing GTP

Question 8

what was the control is the gross et al experiment 1984?

Answer 8

• have a GTP only lane

- ensure that GDP is not forming due to degradation/time

Question 9

what was identified in the expreiment by mccormack et al in 1988?

Answer 9

A ras-GAP

Question 10

what was the experiment that found ras-GAP by mccormack in 1988?

Answer 10

• took mcf7 cell line extract in assay
• when cell line was added more GDP forms
• shows there is something in this tumour cell line which increases the GTPase activity of RAS

Question 11

what did macara and wilson find out about cdc25 in 1990?

Answer 11

• cdc25 is a GEF

Question 12

what experiment did wilson and macara do in 1990 which showed that cdc25 is a GEF?

Answer 12

• did the opposite experiment to maccormack
• looked at GDP release - when brain cytosol was added there is something in the brain cytosol that causes GDP dissociation from RAS - GEF
• GEF homologous to cdc25

Question 13

where abouts in the ras cycle is blocked by an oncogenic form of RAS?

Answer 13

RasGTP cannot be inactivated into RASGDP

• Ras remains active
• issues in GAP activity

Question 14

what did genetic enhancer and supressor screens and epistatic analysis show about RTKs and RAS?

Answer 14

they showed that there is homology between drosophila and c.elegans - RTK and RAS homology

Question 15

what is the pathway in c.elegans in vulval induction which shows hierachy of RTK and RAS

Answer 15

• lin3 (GF)
• let23 (EGF-R - RTK)
• sem5 ??? (later found to be grb2)
• let60 (RAS)

Question 16

what is the pathway in drosophila in eye development which shows hierachy of RTK and RAS

Answer 16

• bride of sevenless (GF)
• sevenless (EGF-R- RTK)
• son of sevenless (cdc25, ras-GEF)

Question 17

what did the FRET experiment by Matsuda et al show?

Answer 17

• showed that only ras-GTP can bind downstream proteins which have a Ras-BD
• RAS-GDP does not bind Ras-BD proteins

Question 18

what does FRET stand for?

Answer 18

fluorescence resonance energy transfer

2 molecules align and donor molecule transfer to acceptor molecule

Question 19

what is sem 5 homologous to?

Answer 19

homologous to GRB2

binds phosphorylated domain on RTK

Question 20

what downstream signalling does active RAS turn on?

Answer 20

• PI3K
• Raf
  -Ral GEF
  ( enhance cell growth, cell morphology and movement )