Module 1- NGF Signalling

Question 1

Features of NGF ligand

Answer 1

120aa
Undergoes cleavage by proteases (furin) and dimerisation to be functional- allows regulation
Forms disulfide bonds with 6 cysteine residues to dimerise

Question 2

Features of receptor tyrosine kinases

Answer 2

All have conserved intracellular tyrosine kinase domain
Different extracellular domains allowing unique receptors and different ligand recognition on different extracellular surfaces= specificity for different responses

Question 3

Induction of TRK dimerisation and activation

Answer 3

Need 2 receptors and dimerisation can occur differently depending on the ligand type
L binding= conformational change of R, one monomer P the other, making the dimer active so it then autoP itself on specific Tyr residues recognised by different motifs

Question 4

Features of TrkA

Answer 4

Type of receptor tyrosine kinase
High affinity for NGF receptor
GoF mutations in colon cancer as is an oncogene
LoF mutations in congenital insensitivity to pain with anridosis
Homodimerisation (most common) or heterodimerisation required for activation

Question 5

Features of phospho-tyrosine binding domains

Answer 5

P of intracellular region of RTK creates a docking site for other proteins containing modular domains
Domains structurally dependent- can use things like alphafold to determine
Recognise P-Tyr in specific sequence context- not other residues eg Ser and Thr due to aa structure differences

Question 6

SHC- what is it and what does it contain

Answer 6

Adapter modular domains for P-Tyr binding
Contains SH2 and PTB
SH2 has antiparallel B sheets flanked by two a-helices, binds pY-E-E-I
PTB is a B-barrel with a-helix closing one end, binds N-P-X-pY

Question 7

Features of Grb2- what it has and what it is

Answer 7

Modular interaction domain with SH3 domains (2)
Also has SH2 domains
SH3 and WW bind to proline-rich domains
SH3 is a twisted B-barrel (P-X-X-P or P-P-L-P-X-R)
WW is a triple stranded B-sheet (P-X-P-X)

Question 8

Overall, how does the NGF pathway work (MAPK activation)

Answer 8

Adapter Shc (PTB) binds phospho-TrkA
TrkA P-Shc
Grb2 SH2 domain binds to P-Shc which induces conformational change in Grb2
SOS proline rich sequences binds Grb2 SH3 domains (x2)
SOS is bound to membrane by plextrin homology domains and is activated, which converts Ras-GDP to Ras-GTP
Ras-GTP activates MAPK pathway= amplification

Question 9

How does the phosphorylation cascade from Ras work for MAPK

Answer 9

Ras-GTP activates Raf (MAP3K)
MAP3K phosphorylates and activates MEK (MAP2K)
MAP2K phosphorylates and activates ERK (MAPK)- ERK into nucleus for response

Question 10

What are Raf and ERK
What is MEK
What does this mean

Answer 10

Raf/ERK are intracellular serine-threonine kinases
MEK is a dual specificity kinase- P Tyr and Thr
Therefore, slightly different domains as they recognise different sites

Question 11

What holds the MAPK activation together

Answer 11

Scaffolds
MEK constitutively bound to KSR scaffold

Question 12

How did Dikie et al find whether it was Shc PTB or SH2 that bound to TrkA

Answer 12

GST pulldown using bait (PTB or SH2) and prey (Trk)
Used GST tag to bind to the protein which binds to sepharose beads
Used for PTB and SH2 separately- added NGF to both to activate the pathway
Looking at size- knew Trk size, looked for where it was on gel- is on the PTB lane, not SH2 so therefore, Trk binds to PTB

Question 13

What does ERK move through to get into nucleus

Answer 13

Nuclear pore complex- over 3000/cell
~40kDa= passive transport
Larger proteins require active transport via nuclear transport receptors

Question 14

Structure of nuclear pore complexes

Answer 14

Made of nucleoporins- 30 protein subunits, lots of copies
110 megaDa= 1 million residues
Nucleoporins contain FG (hydrophobic and unstructured) motifs with phenylalanine and glycine
FG repeats ~200-700 aa in length and form a hydrogel sieve to control what goes through
Recognition by nuclear transport receptors (NTRs) which break sieve and allow proteins through

Question 15

Proteins mediating import of proteins into the nucleus

Answer 15

Imp-a and Imp-B
Imp-a binds to NLS in protein cargo (typically at start and K/R basic residues common, mono or bi-partite)
NLS can be predicted

Question 16

Proteins mediating export of proteins from the nucleus

Answer 16

CRM1/ exportin-1
Binds to NES seq in protein cargo (short peptide enriched with leucines)
NES can be predicted

Question 17

Ran-dependent trafficking for Imp and CRM1

Answer 17

Ran (ras-related nuclear protein) existing as GTP and GDP (small G protein)
RAN-GAP (GTPase activating protein)= causes RanGTP->RanGDP in cytoplasm, allows importins to go into nucleus
RAN-GEF (guanine exchange factor)= RanGDP->RanGTP in nucleus, allows importins to be exported back into the cytoplasm, Ran-GTP bound to importins

Question 18

Experimental determination of ERK translocation

Answer 18

Used ERK-GFP in plasmid and transfected into mammalian cells
Used FGF to activate ERK- see increase in fluorescence- get a little bit of false positive due to way looking at cell on slide under microscope, get some cytoplasmic
Used UO126 to then inhibit and decrease in ERK seen- no fresh P ERK going back into nucleus so overall decrease
Is always export of ERK but is replenished by import of ERK when active

Question 19

PI3K and phospholipids in the pathway

Answer 19

P of Y751 allows PI3K binding
PI3K catalyses P of phospholipids in response to extracellular stimuli like NGF- causes PIP2 to PIP3 by P
PIP3 recruits adaptors with PH domains like SOS
Signals through AKT kinase pathway

Question 20

PI3K structure

Answer 20

Heterodimer
P85 regulator domain- binds P TrkA via SH2 domains and contains SH3 to bind proline motif of p110
P110 catalytic domain has the kinase part with a proline motif to bind to p85 SH3 domain

Question 21

how does PI3K signal through the AKT kinase pathway

Answer 21

PI3K causes PIP2->PIP3- encourages more things to bind through its SH3 domain
PDK1 binds to PIP3 via PH domains
PIP3 binds AKT PH domain (AKT is a protein kinase B) therefore, PIP3 brings AKT and PDK1 close together
Induces conformational change of AKT
Allows PDK1-mediated P of AKT-T308
Causes activation of growth pathways and inhibition of apoptosis pathways through P events

Question 22

Testing NGF signalling dependence on PI3K

Answer 22

Using PC12 cells- they differentiate from neuronal-like precursors to neurons, and at differentiation, filopodia outgrowths occur
Added NGF to all to stimulate
Then added DMSO (allows solubilisation- acts as control) and/ or wortmannin and stained with phallodin (see actin)
Wortmannin caused decreased proliferation and stopped cell growth at decreased efficiency
DMSO only, saw the filopodia

Question 23

Switching off PI3K signalling

Answer 23

SHIP1/2- SH2 containing phosphates- converts PIP3 to PIP2
PTEN- converts PIP3 to PIP2- lost in many cancers causing over-activity and increased signalling

Question 24

phospholipase C in the pathway

Answer 24

Phospholipase C recognises P Y785 and converts PIP2 phospholipid into DAG and IP3
IP3 is a ligand for calcium channels

Question 25

IP3 and calcium channels

Answer 25

Causes calcium release- out of ER to cytoplasm or extracellularly to have effects on other cells (amplification)
Ca causes activation of PKC which is free in cytoplasm or membrane-bound via DAG
PKC phosphorylates things such as ERK for activation= another way to activate ERK and drive a response

Question 26

TrkA mutations

Answer 26

Can be oncogene with GOF mutations causing ligand-independent activation
Can be LOF with complete inactivation, change in protein processing or reduction in receptor activity. Can cauwe CIPA
Occurs from fusions and translocations- genes fused in N-terminus or regulation at C-terminal end are important for determining the outcome
Occur in many cancer types

Question 27

Melanoma features

Answer 27

Melanoma is a malignant tumour of melanocytes (cells which produce melanin)
They can be found throughout the body, not just on the skin epidermis (eye, inner ear, meninges, bones and heart)
Melanin can be beneficial- UV absorption and scattering, free radical scavenging, REDOX reactions and ion storage

Question 28

Melanoma and mutations

Answer 28

Many mutations lead to melanoma- usually in different pathways and onocgene and tumour suppressive
Melanocytes use MAPK cascade: mutations in Raf, PTEN, p110 of PI3K, RTKs, Ras

Question 29

Melanoma Raf and Ras mutations

Answer 29

Raf- 66% of primary and 68% of metastatic melanomas, causes constitutive activation from phosphomimetic (like always P) from glutamic acid neg charge
Ras- impair GTP hydrolysis so always on
Raf and Ras rarely occur in same mutation- already over-active with one mutation

Question 30

Melanoma and PTEN mutations

Answer 30

From epigenetic regulation, altered subcellular location, ubiquitination= reduction in function
Results in activation of Akt as PTEN isnt there to convert PIP3 to PIP2

Question 31

Melanoma and p110 mutation

Answer 31

Makes PI3K become overactive

Question 32

What is CIPA

Answer 32

Mendelian disorder
Congenital insensitivity to pain with anhidrosis
Loss of pain/ temp sensation, no sweating, mild-moderate ID, immune abnormalities
Autosomal recessive= 1:125 million
TrkA variants (all LOF) and also NGF variants (shown with PC12 cells)
Many die within 3 years- prone to febrile seizures- cant control internal temp when have a fever

Question 33

How does NGF normally signal with brain and relation to CIPA

Answer 33

NGF normally signals as pain signal to the brain from the periphery
In CIPA the NGF-dependent dorsal root ganglions are lost so this signalling doesnt occur