24 - The Ras Oncogene

Question 1

What is Ras?

Answer 1

• Ras is the prototypical member of the superfamily of small GTPases
• Ras is a pivotal signalling molecule in RTK-mediated proliferation

Question 2

What does Ras cycle between?

Answer 2

Ras cycles between an inactive, GDP bound state and an activated, GTP bound state

Question 3

What activates Ras?

Answer 3

Guanine nucleotide exchange factors (GEFs) activate Ras by increasing the exchange of GTP for GDP

Question 4

How often do mutations in Ras occur?

Answer 4

Activating mutations of in Ras occur in 30% of human cancers

Question 5

What is mutant Ras insensitive to?

Answer 5

Mutant Ras (most frequently with single amino acid substitutions at Ras codons 12, 13, or 61) is insensitive to down regulation by rasGAP and as a result in constitutively active in the active GTP- bound form

Question 6

What happens when Raf is in the off conformation?

Answer 6

In the off conformation, Ras is in its GDP bound form, when inputs happen this activates a GEF which allows exchange to GDP for GTP, and in the GTP form, the Ras in in the conformation where it can interact with Ras binding domains in effector proteins

Question 7

what is required to switch Ras off?

Answer 7

Catalytic activity of GAP enzymes is required to switch it off, which promotes the hydrolysis of GTP to GDP

Question 8

When is Ras ON and OFF?

Answer 8

GTP bound Ras is ON, and GDP bound Ras is OFF

Question 9

Is Ras on or off in cancer?

Answer 9

In cancer, Ras in always GTP bound so it is always active and stimulating downstream signalling

Question 10

Was does Ras call for (structure)?

Answer 10

Ras calls for a 21 kilodalton protein, it has a lipid modification at its C-terminus wich anchors Ras to its cell membrane, it has two switch regions, switch I binds to effector proteins and switch II is where the GEF binds

Question 11

What does nucleotide biding is Ras regulate?

Answer 11

Nucleotide binding regulates structure of switch 1 and switch 2

Question 12

What does Ras when it is ON have a high affinity for?

Answer 12

GTP bound form has high affinity for downstream targets

Question 13

What happens to the switch domains when GTP is bound?

Answer 13

When GTP is bound there is slight movement of the switch domains, switch 2 more, the pocket is also bigger to allow GTP to be bound

Question 14

Where are switch 1 and 2 in the inactive form?

Answer 14

Switch 1 and switch 2 are relatively far apart in the inactive form
o Thr35 in 1 and Gly60 in 2

Question 15

What is extra in the active form?

Answer 15

In the active form there is an exra phosphate known as the gamma phosphate and gamma phosphate allows for the conformational change which bonds to the Gly60 and Thr35 and forms a bond pulling the switches together

Question 16

What can Ras be activated by?

Answer 16

Ras can be activated by a GEF known as Son-of-sevenless (SOS), first identified in Drosophila

Question 17

What does SOS contain?

Answer 17

SOS contains proline-rich sequences which bind to the SH3 domain the adaptor protein Grb2

Question 18

What does Grb2 associate with?

Answer 18

Grb2, through its SH2 domain, associates with activated RTKs, or with tyrosine phosphorylated Shc

Question 19

What does Shc associate with?

Answer 19

Shc in turn, associates through its SH2 and PTB domains with RTKs or with no receptor tyrosine kinases such a Src

Question 20

What does the Ras activation cascade result in?

Answer 20

This cascade of protein interactions is thought to transmit the signal from auto-phosphorylation receptor to Ras in part by redistributing SOS to the plasma membrane where Ras is located

Question 21

Activation of Ras pathway

Answer 21

1. Ras can be activated by a GEF known as Son-of-sevenless (SOS), first identified in Drosophila
2. SOS contains proline-rich sequences which bind to the SH3 domain the adaptor protein Grb2
3. Grb2, through its SH2 domain, associates with activated RTKs, or with tyrosine phosphorylated Shc
4. Shc in turn, associates through its SH2 and PTB domains with RTKs or with no receptor tyrosine kinases such a Src

Question 22

Where is Ras found?

Answer 22

Ras is at the cell membrane and it attached to a lipid anchor

Question 23

How is Grb2SOS related to RTKs?

Answer 23

Grb2SOS is inactive in the cytoplasm and when we get RTK activation we get autophosphorylation of the receptor and we get recruitment of Grb by its SH2 domain, its interacting with SOS by its SH3 domain and SOS then allows Ras to exchange GDP for GTP
* GTP now renders Ras in its active conformation

Question 24

What does GEF do to Ras?

Answer 24

The GEF lowers the activation energy of Ras
* Without a GEF you need to put in a lot of energy in order to exchange GDP for GTP

Question 25

Describe the path of activation energy of Ras with GEF

Answer 25

The GEF breaks down the activation energy into two areas, the first being GDP, then there is a drop where there is a combined complex and then a small activation hump where we lose GDP

Question 26

Describe a model of SOS and Ras

Answer 26

We have this model where SOS enzyme interacts with switch 2, we think of the two alpha helices of SOS as two fingers of a hand, and the two helices project into switch 1 effectively pushing the switch 1 open and widening the binding site so that GTP can enter
* The concentration of GTP in the cell is much higher than GDP so by mass action, GTP entering due to its high concentration

Question 27

What does genetic and biochemical evidence support?

Answer 27

Genetic and biochemical evidence supports a role for GTPase activating protein (GAP) in the negative regulation of Ras activity (off-switch)

Question 28

The Ras GAP is associated with what?

Answer 28

Ras GAP is associated with a number of RTKs through its SH2 domains

Question 29

How does Ras GAP regulate Ras activity?

Answer 29

GAPs down regulate Ras activity by promoting the conversion of bound GTP to GDP
o The GAP interacts with Ras activating the Ras catalytic activity which hydrolyses the gamma phosphate returning the GTP back to GDP

Question 30

Why is inactivation of Ras important?

Answer 30

Inactivation of Ras is extremely important because cancer is associated with mutations that render Ras permanently active
* Such mutations are found in 30% of all metastatic tumours

Question 31

Where do Ras-associated mutations occur?

Answer 31

These mutations occur at amino acid positions 12, 13 and 61 of the protein, and prevent the GAP for Ras (RasGAP) from doing its job

Question 32

How does RasGAP work?

Answer 32

RasGAP works by inserting an arginine amino acid into the nucleotide-binding project of Ras, completing the catalytic machinery

Question 33

What is the function of the arginine amino acid?

Answer 33

This amino acid has been dubbed the ‘arginine finger’ and it has two functions
1) It neutralised the negative charge that develops at the g-phosphate the phosphate group in GTP that will be removed during hydrolysis into GDP
2) The arginine finger fixes the conformation of the glutamine at position 61 is Ras. Glutamine 61, in turn, positions a water molecule ready to attack the g-phosphate

Question 34

What does the conversion of active Ras back to inactive Ras involve?

Answer 34

The conversion of active Ras back to inactive Ras involves the loss of a phosphate, so this is done by the GAP interacting with Ras completing the catalytic machinery allowing the hydrolysis of GTP back to GDP
* The GAP can then dissociate

Question 35

What does the CAP contribute in small G proteins like Ras?

Answer 35

In small G proteins like Ras, the GAP contributes an arginine which fits into the nucleotide binding pocket completing the nucleotide machinery, this is different to large proteins as the large G proteins already have the arginine present in the active pocket meaning they are not so reliant on GAP activity in the cell

Question 36

What happens in the arginine-finger hypothesis?

Answer 36

• ‘arginine finger’ neutralised the negative charge that develops at the b-phosphate, by contacting this and the gamma-phosphate
• ‘arginine finger’ in the GAP helps to stabilise the glutamine in Ras, Fixing H2O
• water molecule attacks the gamma-phosphate of GTP

Question 37

What does the arginine finger hypothesis explain?

Answer 37

The arginine finger hypothesis explains the cancer promoting nature of mutations at position 61:
- The arginine finger doesn’t have the same effect on amino acids other than glutamine, and those other amino acids can’t position the water molecule correctly

In addition, any amino acid other than glycine at positions 12 and 13 in Ras interferes with the hydrolysis reaction by displacing the arginine finger and thereby glutamine 61