Rho GTPases in cell migration

L7

Why is the Ras family termed a superfamily?

one of the largest groups of GTPase signalling proteins, with over 100 members of Ras superfamily in humans, and is further divided into 5 main families 

How do GTPases function and what is their role?

change conformation upon activation and hydrolyse GTP (guanosine triphosphate) into GDP + bind and activate downstream effectors

What other molecular structure do small GTPases act like? How do they differ?

like the alpha subunit of heterotrimeric G proteins - hydrolase enzyme

differ bc can act independently

What is the size of small GTPases?

21 kDa

Are small GTPases kinases? 

no! don’t add phosphate groups to other proteins but cycle between GTP and GDP states, interacting with effector proteins, some of which can be kinases 

How does the shape of the GTPase impact its function and activity?

dictates it - the extra phosphate group bound to the guanosine + diphosphate group will activate the GTPase ie GTP = on, GDP = off

What bound molecule does GTPase signalling depend on?

bound nucleotide - GDP = off, GTP = on

What distinguishes signalling active from hydrolysis active?

signalling active means GTPase is in on form, ie bound to GTP // hydrolysis active means hydrolysis is occurring, separating the extra phosphate group from the 2 others, bringing the bound nucleotide from a GTP back to a GDP ie the off form of the GTPase

How does the GTPase Arf6 activity effect downstream signalling?

Arf6 when active is an inhibitory GTPase

Do different GTPases have different hydrolysis speeds? Which GTPase is fastest?

yes! rac is fast

How does cycling regulation of GTPases impact speed of these molecular switches going from on to off forms?

very fast! just depends on hydrolysis of GTP

What is the role of GEF?

guanine nucleotide exchange factor with accelerate GDP for GTP exchange by 10×10^7 fold, allows rapid bindign of GTP to GTPases = active form of the latter

Why is the regulation of GTPases termed “cyclic“? 

cycle between GTP and GDP bound states under influence of GEF and GAP

What is the role of GAP?

GTPase Activating Proteins stimulate GTPase activity, accelerate phosphate group removal by 2000×10^5 fold, turning the protein’s activity off

Does GEF or GAP lead to the active/on form of a small GTPase?

GEF

What is the role of GDI?

Guanine Nucleotide Dissociation Inhibitor maintain small GTPases in their inactive state ie maintain the small GTPase bound to GDP

What are the 4 main structures of a GTPase driving their activity?

• switch regions 1 and 2 - bind effectors through subtle shape changes

• p loop for phosphate coordination (GTP/GDP)

• magnesium 2+ for nucleotide binding

Which side of the GTPase has active structures essential to the enzyme’s role?

the right side - containing nucleotide and effector-binding sites (switch regions, p loop and Mg2+)

What is the most accurate way of measuring GTPase activity?

measure effector binding (measuring phosphorylation or antibody binding not as informative/reliable)

Define hydrolysis and list molecules involved

positioning of attacking water

requires water molecule and catalytic glutamine that brings in water and holds it in the correct place

How can hydrolysis of GTP be catalysed ?

• positioning of attacking water thanks to the anchoring by the catalytic glutamine - brings in the water and maintains it in the correct position = hydrolysis

• Counteracting of negative charge at phosphates- P-loop (¹²GxxGKT¹⁷), hydrogen bonds and lysine (both positive)

• arginine residue on p50RhoGAP will also accelerate hydrolysis

How can the hydrolysis of GTP be affected and what does this lead to?

mutations affecting the glutamine residue of the GTPase slowing hydrolysis leading to constitutive signalling (Q61L catalytic mutant -turns glutamine into lysine = water not brought in OR G12V pushes Q61 out of position and disturbs glutamine, pushing it out and inhibiting water molecule from being in the right place)

How do active mutants like Q61L affect GTP hydrolysis?

glutamine becomes lysine - impairs GTP hydrolysis by blocking access to a catalytic water molecule = no hydrolysis occurs = GTPase stays in active/on form

Name an example of a GAP involved in activating hydrolysis ?

p50RhoGAP (p50)

What do GAPs activate?

GTPase hydrolysis activity (GTP to GDP)

What do GAPs inactivate?

GTPase signalling activity

What is the role of GAPs?

to stabilise GTPases and maintain glutamine in the correct position + reduce freedom (reduced entropy barrier ie reduced barrier towards breaking down the molecule) → leads to catalysed hydrolysis

What is the role of Arg 85 in p50 activity?

arginine finger turns off GTPase by stabilising the transition state of the protein and positioning Q61 and gamma phosphate correctly on the GTPase = correct position of water = hydrolysis

Describe the role of p50 on the small GTPase Rac1 and how it specifically catalyses hydrolysis?

p50, specifically its Arg 85 residue will:

• stabilise the GTPase, including the glutamine 61 which allows the correct positioning of water and the gamma phosphate at the p loop

• restricts freedom of the molecule = reduced entropy barrier = easier to break GTP to GDP

• counteract negative charge at p - loop = easier to break down molecule 

What do GEFs accelerate ?

Guanine Nucleotide Exchange factors accelerate exchange of GDP for GTP 

What is the impact of a T17N mutation in a GTPase such as Rac1 ? On GEFs?

dominant negative mutation in a GTPase = GTPase can’t bind and hydrolyse GTP but has a free magnesium so stays tightly bound to GDP but still attracts GEFs that would usually activate other GTPases = pathways blocked

How is the T17N mutation used in research?

blocks specific pathways bc dominant negative mutation affecting GTPases’ and GEFs’ function = study role of Wild Type GTPase 

Name GEFs responsible for activating Rho, Rac, ARF, Cdc42 GTPase families and how they do so

• Dbl-homology domain (diffuse b-cell lymphoma): GEF that binds to Rho GTPases activating them

• Sec7 domain: GEF that catalyses GDP to GTP in Ras and ARF GTPases, critical for vesicle trafficking and Golgi function

Role of Dbl-homology domain

catalyses GDP release form RhoGTPases

Name a family of GEFs that activate Rac/Cdc42 + how they usually activate these small GTPases

DOCK family proteins activating Rho GTPases like Rac1 and Cdc42 by catalysing GDP for GTP

What types of GTPases are stabilised by GEFs?

nucleotide-free, Mg2+ - free ones

What distinguishes dock family GEFs from dbl homology GEFs? 

both activate Rho GTPases but in different ways, Dock being an atypical GEF use the DHR-2 catalytic domain // dbl homology GEFs use the DH domain making them typical GEFs 

How many Dbl family GEFs exist? Describe their specificity and what this is due to? Give an example of gef sensitivity changing

over 70 family GEFs

very specific molecules, one single mutation can make a Rac into Cdc42 like the Rac W56F which is Tiam1 insensitive but ITSN sensitive

What 5 main residues define the specificity of GEFs?

• tiam1

• itsn1

• vav 1/2/3 - very promiscuous

• dbl

• Ect2

What is the consequence of the W56F mutation in a Rac GTPase?

becomes insensitive to the usual GEF, Tiam1 but sensitive to the ITSN GEF

What is the Tiam1 GEF specific to? How do they interact?

Rac1 GTPase

9 residues from tiam1 body interact with Rac1’s switch 2 site - when bound they form a contiguous contact

Which GEFs have a diffuse b-cell lymphoma domain? What do these Guanine Exchange Factors catalyse?

Dbl (RhoA, Cdc42, Rac1), Vav (Rac1, Cdc42), Tiam1 (Rac)

Which GTPases drive actin-based cell motility? Which GEF?

Cdc42, Rac1 and RhoA

Dbl (diffuse b-cell lymphoma)

What is the role of Cdc42 in actin-based motility? Which GEF is specific to this GTPase?

drives filopodia through protrusion

Rho GTPase Cdc42 activated by DOCK family and Dbl, Asef, intersectin 2 and other GEFs 

What is the role of RhoA in actin-based motility? Which GEF is specific to this GTPase? 

body contracted through stress fibre and cytoskeletal movements = last step, moves body forward 

GEF H1

What is the role of Rac1 in actin-based motility? Which GEF is specific to this GTPase? 

pushes lamellipodia forward through protrusion

Tiam1 

Which structures in a cell are crucial for actin-based motility?

• actin

• stress fibres

• lamellipodia

• filopodia

Give the downstream effects of GTP-RhoA activation

activates Rho Kinase - phosphorylates myosin light chain = actomyosin contraction - mvt of myosin along actin fibre 

Why do Cdc42/Rac and RhoA signals need to be coordinated ?

antagonistic signals so would compete if not coordinated

What types of signals are Cdc42 and Rac in cell motility vs RhoA?

Cdc42 and Rac are protrusive signals at front of cell, pushing processes out // RhoA is a contractile signal at back, dragging body forward

How is cell movement given direction? How does this affect the speed of cell mvt // to random mvt?

through guidance queues like the string in the church lecture - gives cell directional push, specifically the restricted freedom that GAPs provide GTPases

slower but directional mvt

How would increasing Rac1 activity impact its migrational speed?

speed not proportional to Rac1 activity, specific amount of signalling needed for correct cell motility speed 

What would occur if a cell didn’t have localised signals like in a WT ie effector and GTPases at the extremity of the cell pushing towards the right direction?

leads to protrusions of cell in all directions, Rac super activated cell would be alive but not translocating to the correct target location