lecture 10

Question 1

what are GTPases and ATPases

Answer 1

protein switched without covalent modifications

Question 2

how do ATPase switches work

Answer 2

atpase switches involve conformational changes driven by cycles of atp binding with hydrolysis
associated with motor protein complexes or transporters that move material into and out of cells and organelles
myosin and kinesin are atpase switches that move along actin filaments and microtubules respectively
conformational changes in atpase parts of these proteins are ultimately propagated into large movements of attached proteins and domains

Question 3

how to gtpase switches work

Answer 3

gtpase switches are g proteins which involve conformational changes driven by cycle of gtp binding and hydrolysis

Question 4

growth control by Ras (Rat Sacroma)

Answer 4

ras is identified as the oncogenic protein of tumour virus that causes rat sarcoma
mutations in human Ras genes is implicated in cancer development
microinjecting active ras protein induced proliferation of normal mammalian cells
interfering with ras function by microinjection of anti-ras antibody or expression of a dominant negative ras mutant blocks growth factor induced cell proliferation
so, activated ras induces abnormal growth of cancer cells and is required for the response of normal cells to growth factor stimulation

Question 5

what are ras proteins

Answer 5

they are guanine nucleotide binding proteins
smaller than a g alpha and act as a monomer rather than a hetero-trimer with beta and gamma subunits
they alternate between active GTP bound forms and inactive GDP bound forms

Question 6

conformation of Ras-GDP

Answer 6

off conformation, switch 1 and 2 both off
TH35 within switch one binds magnesium but makes no contact with gdp
Gln1 within switch 2 is positioned where it has activated a catalytic water for hydrolysis of gtp to gdp

Question 7

conformation of Ras-GTP

Answer 7

on, spring loaded conformation
Thr35 within switch 1 binds to magnesium, together the Th35 and magnesium help position the terminal gamma-phosphate of gtp
Gln61 within switch 2 is positioned where it cannot activate a catalytic water for hydrolysis of GTP to GDP

Question 8

conversion of ras between gdp and gtp bound forms

Answer 8

ras has high affinity for both gdp and gtp
to turn ras on, tightly bound gdp is exchanged for gdp
ras has a low intrinsic nucleotide exchange
ras is turned off again by hydrolysis of gtp to gdp and pi
ras has a sloe intrinsic gtpase
ras itself is an incomplete enzyme

Question 9

GDP/GTP cycling controlled positively by GEFs

Answer 9

gef proteins insert close to the p loop and accelerate the process of turning ras switch on
they wrench open the binding site allowing gdp to exit and gtp to take its place
son of sevenless is one of ras specific 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 10

GDP/GTP cycling controlled negatively by GAPs

Answer 10

gaps accelerate hydrolysis of gtp by ras
rasGAP stabilises switch 2 and gln61
rasGAP inserts a catalytic arginine finger into gtp bound g protien which changes its confromation to orient the gtp for better nucleophillic attack by water
gap proteins complete the active sites of ras
ras specific gaps include p120GAP and NF1

Question 11

what is Ras association with the plasma membrane via farnesylation critical got

Answer 11

its function in growth factor signalling

Question 12

how are Ras proteins tethered to the inside of the plasma membrane for lipid modification

Answer 12

c terminal CAAX motif is necessary and sufficient to dignal for modifation of the cystine by a 15 carbon farneyl isoprenoid lipid by farnesyl transferase
AAX is removed by prenyl protein specific endoprotease
then a new c terminus is methylated by a methyl transferase
N-Ras and H-Ras are further processed by palmitoylation of 1 or 2 cystine 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 factors causes dimerization of the receptor
juxtaposition of intracellular domains causes trans-phosphorylation
SH2 domain in Grb2 binds to tyrosine phosphorylated receptor
Grb2 has 2 sh3 domains and one binds to proline containing motif on son of sevenless, so brings sos in proximity to the membrane tethered to an inactive ras gdp
sos is a guanine nucleotide exchange factor for ras that causes dissociation of gdp and its replacement by gtp
ras gtp complex is active and can activate effector proteins

Question 14

what does the active GTP bound ras bind to

Answer 14

down stream effectors

it binds and regulated effectors with roles in growth factor signalling and human oncogenes

Question 15

what does Ras GTP activate to activate the MAPK kinase cascade

Answer 15

RasGTP activates Raf to activate the MAPK kinase cascade
key effectors for activated ras are protein serine/threonine kinases of raf family
they are 3 conserved regions - CR1, CR3 and kinase domain
activated ras GTP binds to the conserved region 1 domain in the n terminus part of raf serine/threonine kinase
binding of ras GTP recruits raf kinase to the mebrane and relieves their auto-inhibition
raf kinases are then phosphorylated at 2 residues in the kinase domain causing activation
once activated ras kinases switch on MAP kinase cascade
raf binds to and phosphorylates MEK, a dual specificity prtoein kinase
MEK phosphorylates both a tyrosine and threonine residue on mitogen activated protein kinase
MAP kinase phosphorylates many different proeins including nuclear trancription factors that mediate cellular responses
some of the switched on genes stimulate cell growth and proliferation

Question 16

what do mutations of Ras in cancers and developmental disease cause

Answer 16

the switch is jammed in the on position

Question 17

Ras in cancer

Answer 17

mutational activation in ras occurs in 30% of human cancers
certain single amino acid mutations stabilise Ras in constitutively active gtp bound conformation
discovery: mutant ras is insensitive to GAP protein
oncogenic mutations in the switch region of ras cause cancer because they inactive the gtpase activity of ras - the gap complex leaving ras chronically gtp bound and active
so growth promoting signal is constantly on
most common mutations are in switch 1 G12 and G13 and switch 2 Gln61

Question 18

gln61 mutations and gly12 mutations

Answer 18

gln61 muts - miss the catalytic residue

gly12 muts are sterically compromised, they are unable to stabilise transition state in gtpase reaction

Question 19

how is tumour derived Ras activated

Answer 19

by single amino acid substitutions

Question 20

neurofibromatosis - loss of rasGAP

Answer 20

a set of genetic disorders which can casue tumours to grow along various nerves and also affects development of bones and skin
mutation of essential arginine in the GAP causes the diease

Question 21

germline rasGEF mutations in human developmental syndromes

Answer 21

noonan syndrome is a clinically variable development disorder defined by short stature, facial dysmorphism and congenital heart disease
mutations that slightly enhance GEF action of SOs1 or increase sos1 recruitment to the membrane have been identified

Question 22

MAP kinase and cancer

Answer 22

AV599E mutation in B-raf possibly by exposure to uv radiation in sunlight is often found in lesions of malignant melanoma
this mutation replaces an uncharged valine side chain with a negatively charged glutamate, mimicking phosphorylation at nearby thr598 and ser601
mutant b raf stimulates cell division even in absence of growth factor
PLX4032 inhibits V600-B-Raf and has dramatic beneficial effect in melanomas driven by V600-B-Raf
drug resistant mechanisms include N-ras mutations that reactivate raf pathway

Question 23

Ras is the prototype of a eukaryotic superfamily of small gtpases

Answer 23

ras - growth factor signal cascades
rab - vesicle targeting and fusion
arf - forming vesicle coatamer coats
ran - transport of protein into and out of nucleus
rho - regulation of cytoskeleton