Rho GTPase in cell migration

Small GTPases of the Ras superfamily typically have what molecular size?

They are typically 21kDa in size.

Small GTPases function as molecular switches by oscillating between an active, GTP-bound state and an inactive, _ state.

GDP-bound

What are the five primary families of the Ras superfamily?

Ras, Rho, Rab, Arf, and Ran.

During the 'active signaling' phase of a GTPase, what is its primary action?

It binds to effector proteins to trigger cellular changes like growth or movement.

The 'active hydrolysis' phase of a GTPase involves breaking the chemical bond of which part of the GTP molecule?

The third phosphate group.

Which type of regulatory protein promotes the 'active signaling' state of a GTPase?

Guanine Nucleotide Exchange Factors (GEFs).

Which type of regulatory protein promotes the 'active hydrolysis' state of a GTPase?

GTPase Activating Proteins (GAPs).

The 'active signaling' phase of a GTPase is considered the _ phase, whereas active hydrolysis is the 'timer' phase.

work

What two components coordinate the binding of GTP in a small GTPase?

The P-loop and a magnesium ion (Mg2+).

GTP binding forces which two regions of the GTPase into a rigid, 'loaded' conformation?

The switch regions, specifically Thr17 and Th35.

What is the functional consequence of the switch regions adopting a rigid, 'loaded' conformation?

They are able to bind to effector proteins, triggering downstream cellular responses.

During active hydrolysis, what is the role of the catalytic glutamine-61 (Q61)?

It helps to position a water molecule to attack the GTP phosphate bond.

How does a GAP (GTPase Activating Protein) facilitate hydrolysis?

It inserts an arginine residue into the active site to neutralize the negative charge buildup during the transition state.

What happens to the switch loops of a GTPase after hydrolysis is complete?

They relax and change shape, causing them to no longer be able to bind to effectors.

Function: Ras family of GTPases

Primarily involved in cell proliferation, differentiation, and survival signaling.

Function: Rho family of GTPases

Primarily involved in cytoskeletal organization, cell motility, and cell shape.

Function: Rab family of GTPases

Primarily involved in vesicle trafficking, docking, and fusion.

Function: Arf family of GTPases

Primarily involved in vesicle budding and the formation of transport coats.

Function: Ran family of GTPases

Primarily involved in nucleocytoplasmic transport, moving RNA and proteins in or out of the nucleus.

A strictly conserved lysine residue (e.g., Lys16 in Ras) provides the electrostatic glue that holds the GTP molecule in place by interacting with which parts of the nucleotide?

The beta and gamma phosphates.

What is the primary function of Guanine Nucleotide Exchange Factors (GEFs)?

They accelerate the exchange of GDP for GTP by facilitating the release of the tightly bound GDP.

What is the molecular mechanism of the G12V mutation in GTPases?

It creates steric hindrance by pushing Q61 out of position, preventing the hydrolytic machinery from working.

Why is the Q61L mutation highly oncogenic in GTPases?

It completely disables the protein's ability to hydrolyze GTP, thus turning itself off.

To facilitate GDP release, a GEF physically distorts the GTPase and displaces the _, which normally anchors the phosphate groups.

Mg2+ ion

What is the molecular effect of the T17N mutation in a GTPase?

The mutant GTPase has a much higher affinity for GEFs but cannot complete the exchange process.

How does the T17N mutant GTPase act as a dominant-negative inhibitor?

It soaks up all the available GEFs in the cell, preventing them from activating healthy wild-type GTPases.

The Dbl-homology (DH) domain is the most common catalytic domain for GEFs that activate which GTPase family?

The Rho family.

The DOCK-family of GEFs uses the _ domain to activate Rho GTPases.

DHR2

The Sec7 domain is characteristically found in GEFs that activate which family of GTPases?

ARF GTPases.

Tiam1 is a GEF that specifically activates which GTPase?

Rac.

What is the effect of the Rac W56F mutation on its interaction with GEFs?

It makes Rac insensitive to the GEF Tiam1 but leaves it sensitive to the GEF ITSN.

Which of the three primary Rho GTPases acts as the 'navigator' responsible for cell polarity and forming filopodia?

CDC42.

Which of the three primary Rho GTPases acts as the 'driver' that induces lamellipodia and drives forward protrusion?

RAC1.

Which of the three primary Rho GTPases acts as the 'engine/brake' regulating stress fibers and pulling the rear of the cell forward?

RhoA.

Activated RhoA directly binds to and activates which downstream effector kinase?

Rho kinase (ROCK).

What is the immediate molecular target of active Rho kinase (ROCK)?

Myosin light chain phosphatase (MLCP).

How does ROCK regulate the activity of MLCP?

ROCK phosphorylates MLCP, which inhibits it.

What is the normal function of Myosin Light Chain Phosphatase (MLCP)?

It acts as a brake by removing phosphate groups from myosin to prevent contraction.

By inhibiting MLCP, the RhoA-ROCK pathway leads to an increase in the level of phosphorylated _, triggering contraction.

Myosin Light Chain (MLC)

What is the cellular consequence of increased phosphorylated MLC due to the RhoA pathway?

It triggers actomyosin filament assembly, leading to cell contraction and retraction of the cell's rear.

What happens to a cell's migration speed if RAC1 activity becomes excessive or imbalanced?

The cell can slow down or lose its direction entirely.

According to the provided diagram, by how much does a Guanine Nucleotide Exchange Factor (GEF) accelerate GDP/GTP exchange?

It accelerates the reaction 10-10^7 fold.

According to the provided diagram, by how much does a GTPase Activating Protein (GAP) accelerate GTP hydrolysis?

It accelerates the reaction 2000-10^5 fold.

What protein is shown in the diagram to prevent the dissociation of GDP from Rac1?

Guanine Nucleotide Dissociation Inhibitor (GDI).

According to the diagram, Rac1 bound to GTP activates which downstream effectors?

PAK / wave2.