Module 1- NGF Signalling Flashcards
Features of NGF ligand
120aa
Undergoes cleavage by proteases (furin) and dimerisation to be functional- allows regulation
Forms disulfide bonds with 6 cysteine residues to dimerise
Features of receptor tyrosine kinases
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
Induction of TRK dimerisation and activation
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
Features of TrkA
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
Features of phospho-tyrosine binding domains
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
SHC- what is it and what does it contain
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
Features of Grb2- what it has and what it is
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)
Overall, how does the NGF pathway work (MAPK activation)
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
How does the phosphorylation cascade from Ras work for MAPK
Ras-GTP activates Raf (MAP3K)
MAP3K phosphorylates and activates MEK (MAP2K)
MAP2K phosphorylates and activates ERK (MAPK)- ERK into nucleus for response
What are Raf and ERK
What is MEK
What does this mean
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
What holds the MAPK activation together
Scaffolds
MEK constitutively bound to KSR scaffold
How did Dikie et al find whether it was Shc PTB or SH2 that bound to TrkA
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
What does ERK move through to get into nucleus
Nuclear pore complex- over 3000/cell
~40kDa= passive transport
Larger proteins require active transport via nuclear transport receptors
Structure of nuclear pore complexes
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
Proteins mediating import of proteins into the nucleus
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