Small G proteins, GAPs and GEFs

Question 1

GTPases and ATPases are protein switches WITH/WITHOUT covalent modification

Answer 1

GTPases and ATPases are protein switches WITHOUT covalent modification

Question 2

GTPase/ATPase switches involve conformational changes driven by cycles of GTP/ATP binding and hydrolysis (Respectively)
TRUE OR FALSE

Answer 2

TRUE
Associated with motor protein complexes or transporters that move material into and out of cells and organelles.
For example, myosin and kinesin are ATPase switches

Question 3

3 conserved features of GTPase and ATPase structures:

Answer 3

• the P-loop (phosphate-binding loop)
• switch I sequence motif
• switch II sequence motif

Question 4

Growth control by Ras (Rat sarcoma)

Answer 4

Ras is a GTPase – functions as a GDP/GTP binary on-off switch.
Ras identified as the oncogenic protein of tumour virus that causes rat sarcoma.
1982, mutations in human RAS genes (H-Ras, K-Ras, N-Ras) implicated in cancer development.
Microinjecting active Ras protein induced proliferation of normal mammalian cells.
Conversely, interfering with Ras function by microinjection of anti-Ras antibody or expression of a dominant negative Ras mutant blocked growth factor-induced cell proliferation.
Thus, activated Ras induces abnormal growth of cancer cells, and is required for the response to normal cells to growth factor stimulation.
Ras proteins are guanine nucleotide-binding proteins.
Ras is smaller than G alpha AND acts as a monomer rather than in a heterotrimer with beta and gamma subunits (Simon Arthur lectures).
Ras proteins alternate between inactive GDP-bound and active GTP-bound forms.

Question 5

The conformation of Ras-GTP differs from Ras-GDP:

Answer 5

Ras-GDP=‘OFF’ with Switch I and switch II relaxed into an inactive conformation
Ras-GTP=‘ON’ in ‘spring loaded’ conformation

Question 6

How Ras is interconverted between GDP- and GTP-bound forms:

Answer 6

Ras has high affinity for GDP AND for GTP
Ras is ‘off’ with GDP bound in the site.
- To turn Ras ‘on’, tightly bound GDP is exchanged for GTP
- Ras has a low intrinsic nucleotide exchange
- Ras is turned off again by hydrolysis of GTP to GDP and Pi
- Ras has a slow intrinsic GTPase

i.e. Ras, by itself, is an incomplete enzyme

Question 7

GDP/GTP cycling is controlled by:

Answer 7

Positive (GEFs) and negative (GAPs) regulators.

Question 8

GEF proteins (guanine nucleotide exchange factors)

Answer 8

Insert close to the P loop and accelerate the process of turning the Ras switch on.
GEFs wrench open the binding site, allowing GDP to exit and allowing GTP to take its place (intracellular GTP is ~10-fold more abundant than GDP).
Sos is one of the Ras-specfic GEFs.
A single G protein may be recognised by multiple GEFs, enabling one GTPase to serve as the focal point for integrating signals from different upstream pathways.

Question 9

GAPs (GTPase-activating proteins)

Answer 9

Accelerate hydrolysis of GTP by Ras.
RasGAP stabilizes switch II and Gln61.
RasGAP inserts a catalytic Arg finger into the GTP-bound G protein, which changes its conformation to orient the GTP for better nucleophilic attack by water.
In other words, GAP proteins ‘complete the active site’ of Ras.
Ras-specific GAPs include p120GAP and NF1.
Avoid confusion: GAPs activate the GTPase of Ras, which becomes inactive in its binding to effector proteins. ‘ON’ enzyme = ‘OFF’ protein

Question 10

Ras association with the plasma membrane
– via phosphorylation –
is critical for its function in growth factor signalling
TRUE OR FALSE

Answer 10

FALSE
Ras association with the plasma membrane
– via FARNESYLATION –
is critical for its function in growth factor signalling

Question 11

Why does Ras need to be at the plasma membrane to function?

Answer 11

The upstream regulators of Ras are located at the plasma membrane
Activated Ras recruits effectors to the plasma membrane for downstream signalling

Question 12

Ras proteins are tethered to the inside of the plasma membrane by lipid modification:

Answer 12

The C-terminal CAAX motif is necessary and sufficient to signal for modification of the Cys by a 15-carbon farnesyl isoprenoid lipid by farnesyltransferase.
The AAX is removed by a prenyl-protein specific endoprotease.
Then, the new C-terminus is methylated by a methyltransferase.
N-Ras and H-Ras are further processed by palmitoylation of one or two Cys residues, respectively, adjacent to the CAAX box.

Question 13

Ras is activated by growth factors that act via tyrosine kinase receptors:

Answer 13

Binding of growth factor causes dimerization of the receptor
juxtaposition of the intracellular domains causes trans-phosphorylation
SH2 domain in Grb2 (growth factor receptor binding protein-2) binds to tyrosine-phosphorylated receptor (first identified for EGF receptor)
the Grb2 has two SH3 domains and one binds to a proline-containing motif on Sos; this brings Sos in proximity to the membrane-tethered inactive Ras-GDP
Sos (son of sevenless) is a guanine nucleotide exchange factor (GEF) for Ras that causes dissociation of GDP and its replacement by GTP
(Sos was first identified genetically as acting downstream of sevenless, a tyrosine kinase-linked receptor in the fruit fly Drosophila)
the Ras-GTP complex is active and can activate effector proteins

Question 14

GDP-bound Ras is the active form that binds to
‘downstream’ effectors
TRUE OR FALSE

Answer 14

FALSE
GTP-bound Ras is the active form that binds to
‘downstream’ effectors

Question 15

GTP-Ras binds to and regulates effectors with roles in growth factor signaling and human oncogenesis
TRUE OR FALSE

Answer 15

TRUE

Question 16

Ras-GTP activates Raf, which activates the MAP kinase cascade

Answer 16

key effectors for activated Ras are protein serine/threonine kinases of the Raf family, i.e. A-Raf, B-Raf and C-Raf (also known as Raf-1)
they have three conserved regions (CR1-3, CR3 is the kinase domain):

activated Ras-GTP (NOT Ras-GDP) binds to the conserved region 1 (CR1) domain in the N-terminus part of Raf serine/threonine kinase
binding of Ras-GTP recruits Raf kinases to the membrane and relieves their auto-inhibition
Raf kinases are then phosphorylated at two residues in the kinase domain (Thr-598 and Ser-601 on B-Raf), causing activation
once activated, Raf kinases switch on the MAP kinase (Erk) cascade
Raf binds to and phosphorylates MEK, a dual-specificity protein kinase
MEK phosphorylates both a tyrosine and a threonine residue on mitogen-activated protein kinase (MAP kinase, Erk)
MAP kinase phosphorylates many different proteins, including nuclear transcription factors, that mediate cellular responses
Some of the switched on genes stimulate cell growth and proliferation

Question 17

Summary of growth factor activation of the MAP kinase cascade

Answer 17

Growth factors (also known as mitogens) convert Ras to its active, GTP-bound form
Ras binds to and activates the Raf family of protein kinases, e.g. B-Raf
This in turn activates a cascade of protein kinases in which each kinase phosphorylates and activates the next
A central component of this cascade is MAP kinase (mitogen-activated protein kinase) also known as Erk
In response to certain stimuli, active MAP kinase (and MAP kinase-activated protein kinases, downstream of it) enter the nucleus, where they phosphorylate transcription factors that contribute to the response to the growth factor
Some of the genes switched on stimulate cell growth and proliferation

Question 18

Ras is mutated in cancers and developmental disease
– the switch is jammed in the ‘on’ position
TRUE OR FALSE

Answer 18

TRUE

Mutant Ras is insensitive to a GTPase Activating Protein (GAP)