Lecture 3 - Growth factors and signalling Flashcards
What was the 4th gene discovered in Avian sarcoma virus?
Src
How was it discovered that Src is a tyrosine kinase?
An antibody was produced for Src. The antibody was found to become phosphorylated when they were incubated with radioactive ATP and src. Phosphorylation was known to involve the covalent attachment of phosphate groups to the side chains of specific amino acid residues. Hence it was discovered that Src was a protein kinase.
Src doesn’t normally phosphorylate antibody molecules but the fact that it had suggested that its usual mode of action was to transfer a high energy phosphate onto a target protein.
What was the result of a western blot experiment of Src transformed compared to non-transformed cells.
Antibody raised to recognise phosphotyrosine bound to multiple proteins in the Src transformed cell and none in the non-transformed cell. This shows that lots of things are changing in cells which have the capacity to become tumour cells.
How was it shown that src is a tyrosine kinase?
Electrophoresis towards two different catchodes - one at pH 1.9 and one at pH 3.5.
Phosphoserine, phosphothreonine and phosphotyrosine have expected locations when this is applied.
In non transformed cells - there was an abundance of phosphothreonine and phosphoserine
In src transformed cells there was an abundance of phosphotyrosine compared to the other two.
How was the EGFR discovered
EGF was taken and attached to a solid support in a glass column which is filled with permeable beads to allow liquid flow. Growing cells (EGF responsive) were lysed and pored down the column, washed and then eluted out whatever was attached to EGF. After this was done the protein gel revealed that only one protein comes off after elution in the presence of EGF - this must be the molecule that EGF binds so tightly. (very uncommon protein gel)
How was the protein gel with EGFR present used to sequence EGFR.
Cloned the gene by getting a small section of the protein sequence and use a chemical sequencer tosequence it and design a DNA probe that corresponds to those amino acids. After finding out the sequence they called it the EGF receptor.
How was it discovered that the EGF binding portion existed in the first 60kD of the EGFR
They analysed the sequence of the protein by cutting it into 3 pieces. These pieces were run on a gel and probed for with radioactive EGF
How did bioinformatics help to find the TMD of the EGFR
Plot how hydrophobic an amino acid sequence is. Isoleucine and valine are hydrophobic and can be measured and quantified. The area where they are most concentrated is the most hydrophobic and usually implicates the TMD.
What did the fact that EGFR and Src had similar structures suggest about oncogenes
- Growth factors induce their cellular effects by triggering a tyrosine kinase signalling pathway.
- Oncogenes might work by triggering signalling pathways in the absence of appropriate extracellular cues.
What did a radioactive 32P labelled phosphate experiment show about EGFR signalling
When EGF was added to EGFR, the phosphotyrosine signal goes up massively. This is due to the cross-phosphorylation of the receptor when it dimerises upon growth factor binding.
What was the result of introducing an artificial phospho-tyrosine domain binding protein into cells.
Using immunofluoresence microscopy to see where it is in the cell. They saw that EGFR was found at the plasma membrane and in the absence of EGF the phosphotyrosine binding protein was cytoplasmic. Addition of EGF the binding protein colocalises with the receptor.
The phosphotyrosine binding protein is probing the autophosphorylation of the EGF receptor, proving that this is what is happening in the presence of EGF.
What are the principles of RTK signalling
Receptor is in an off (monomeric) state
Ligand binding induces dimerisation or de-autoinhibition
Increased local concentration of active receptor kinase and receptor substrate results in trans-phosphorylation (the tyrosine kinase domain of each recepter monomer is able to phosphorylate the C terminal cytoplasmic tail of the other monomer)
Phosphoreceptor acts as a platform for signalling events
Which is the most prevelant mutation which activates the catalytic activity of the EGFR
The most prevalent of these mutations in tumours was found to be EGFRvIII, an EGFR deletion mutant that lacks exons 2–7, which can arise from gene rearrangement or alternative mRNA splicing
What are examples of RTK based cancer therapies
Neutralising antibodies - Block the bioactivity of RTK ligands
RTK targeted antibodies, which either target overexpressed receptors or heterodimerised ones.
Small molecule inhibitors - Inhibit RTK activity by interfering with RTK signal transduction.
What is the role of Trastuzumab
Trastuzumab is a HER2 overexpressing metastatic breast cancer target. It binds HER2 on the surface of tumour cells and induces receptor internalisation, inhibition of cell cycle progression and recruitment of immune effector cells.
