Lecture 7 - Rho GTPases in cell migration

Question 1

Describe the features of small GTPases

Answer 1

• small 21 kDa proteins
• one of the largest groups of signalling proteins: Ras superfamily
• change conformation upon activation
• bind and activate downstream effectors

Question 2

What does GTPase signalling depend on?

Answer 2

depends on the bound nucleotide
- active Arf6 is inhibitory
- “signalling active” and “hydrolysis active” are not the same
-When talking about ‘active GTPase’ we are talking about its signaling status. However, as a GTPase it cleaves the GTP, which makes you think that its hydrolysis that its active, however it isn’t

• Activity state of a small GTPase depends on the bound nucleotide

Question 3

What does the activation of hydrolysis (GTP to GDP) lead to?

Answer 3

Suppression of signalling

Question 4

What is the P-loop?

Answer 4

Coordinate phosphate in bound nucleotide - crucial for controlling activity state by controlling the bonding of the nucleotide

Question 5

Describe features of the 3 phosphates

Answer 5

3 phosphates (very negative charge) - need some positive for nucleotide binding - so have Mg2+ (magnesium ion) for nucleotide binding.

Question 6

Describe features of switch regions

Answer 6

(switch 1 & switch 2) - relaxed loops interact with downstream effectors (switch regions bind effectors).

Question 7

Where do effectors bind?

Answer 7

switch regions

Question 8

Explain the catalysis of GTPase activity

Answer 8

1) Positioning of attacking water - catalytic glutamine 61 stabilizes water
Active mutants:
- Q61L catalytic mutant
- G12V pushes Q61 out of position and disturbs P loop

2) counteracting of negative charge at phosphates - P loop, hydrogen bonds and lysine

Question 9

What do GAPs (GTPase activating proteins) do?

Answer 9

Activate hydrolysis - i.e. turn off signaling

Question 10

What do GEFs (Guanine Nucleotide Exchange factors) do?

Answer 10

Accelerate exchange of GDP for GTP
- stabilize nucleotide-free, Mg2+ -free GTPase
- T17N is a dominant negative
- Dbl-homology domain
- DOCK-family
- Sec7 domain

Question 11

Describe GEF specificity

Answer 11

Rho family:
- Rac
- RhoA
- RhoG
- Cdc42

over 70 Dbl family
- Tiam1:9 residues from body and switch 2 form continguous contact
- Rac W56F is Tiam1 insensitive but ITSN sensitive

Question 12

Describe the downstream effects of GTPases

Answer 12

GTP-RhoA –> Rho Kinase –> Myosin Light Chain –> Actomyosin contraction

Question 13

Describe the antagonistic signals surrounding actin-based motility

Answer 13

• Cdc42/Rac1 are protrusive signals
• Rho A is a contractive signal

These are antagonistic so cooperation between the 2 are key.

Question 14

How does the flexibility of Arg help in the functioning of GAPs?

Answer 14

Arg insets into active site to stabilize transition state and positions Q61 properly.
- mechanism conserved, structure completely different
- numbers vary between GAP.

Question 15

What does an increase in Rac1 activity lead to?

Answer 15

• lack of directionality
• it doesn’t affect migration speed, as migration needs localised signals

Question 16

What is Rac1 important for?

Answer 16

Membrane protrusion