L3 - GF and signalling

Question 1

What is the function of src?

Answer 1

It is a protein kinase, specifically a tyrosine kinase. It phosphorylates tyrosine residues by taking a phosphate group from ATP

Question 2

What are the principles of RTK signalling?

Answer 2

• R in ‘off’ state is often a monomer
• Ligand binding induces dimerisation
• Can have transphosphorylation
• Phos R acts as platform for signalling events

Question 3

How was src identified?

Answer 3

RSV is a retrovirus that induces transformation of cells. Retroviruses normally have 3 genes; gag,pol and env. RSV has a 4th gene - Src. This gene must be responsible for causing transformation in cells when integrated into the genome

Question 4

How was Src identified as a protein kinase?

Answer 4

Src antiserum was developed to visualise Src. In transformed cells treated with antiserum (on beads - binds to src), src immunoprecipitates with y32P-ATP (a RA phosphate). This suggests that Src is a protein kinase

Question 5

What type of protein kinase is Src and how was this discovered?

Answer 5

Src is a tyrosine kinase. PLT is a viral protein that is phosphorylated. Inc of PLT with HCL breaksdown the protein into single aa. Run the aa in 2D separation (chromatography and electrophoresis). Known spots for phos threonine and serine residues. This 2D sep showed that it was a tyrosine residue that was being phosphorylated.

Question 6

What does the identification of Src as a tyrosine kinase tell us?

Answer 6

Src must touch many different signalling pathways that induce change in the shape/function of targets - this causes transformation. This introduced the idea of signalling networks and GFRs

Question 7

How was EGFR identified?

Answer 7

HeLa (cancer cell line) needed serum to proliferate. Identification of GF in serum that allows proliferation. Attach EGF to bead, mush HeLa cells and pass through column with beads. Only 1 protein was found to interact with EGF

Question 8

How was the function of EGFR determined?

Answer 8

1. Digest protein into 3 segments - seq the segments and compare to known libraries
2. Functional map - run three segments and see that they bind to. First fragment bound to I-EGF
3. Kyte and Dolittle plot - measure hydrophobicity. Second segment was very hphobic - suggests a TMD
4. Sequence homology - end segment is similar to Src - suggests it is a tyrosine kinase

Question 9

What 2 studies backed up the suggestion that EGFR was a TK?

Answer 9

Frackleton et al - increased amount of phosphotyrosine in the presence of EGF
Yamamoto - avian erythroblastosis virus has same structure as EGFR minus the regulatory domain

Question 10

TRUE OR FALSE

Many GFR are protooncogenes?

Answer 10

True. The over-activation of these receptors activates pathways that are important in cell growth and proliferation