Growth factors and signalling Flashcards
Definition of what protein kinases are?
Protein kinases operate by removing the high energy phosphate group from ATP and attaching it to the hydroxyl groups in side chains of serine, threonine or tyrosine residues
How was an antibody of src made?
Src viral proteins were injected into rabbits , and the antiserum was collected from the rabbits
What was the basis of the experiment looking at whether or not src had any kinase activity?
Src was immunoprecipitated with cell lysates and radioactively labelled ATP- and then exposed to an autoradiogram
What happened when Src antiserum was immunoprecipitated with transformed chicken embryo fibroblasts cell lysates and radioactively labelled ATP? Thus what was the conclusion
There was a band present- showing the presence of the phosphorylated src antibody
Conclusion= Src is a protein kinase
What was the key experiment showing that src was a tyrosine kinase ?
When transformed mouse cells expressing src were exposed to antiphosphotyrosine antibodies- there was the presence of phosphotyrosine
Lack of bands in untransformed cells
Src is a tyrosine kinase
What did they purify from serum used for cell proliferation in cell culture?
EGF
epidermal growth factor
How did extract the protein that binds to EGF?
Attached EGF to a solid column- porous
Then they passed HeLa cells through the column
eluted what was bound to too EGF and ran it on a gel
How do you usually identify a mystery protein?
Digest the protein with a protease, use the peptide sequences in an automatic protein sequencer (sanger method)
What were the characteristics of the 3 fragments of the mystery protein?
- Large N terminal domain which was thought to bind to a growth factor
- A transmembrane domain
- A similair sequence to src- a domain which extends into the cytoplasm
What was the protein that bound to EGF?
EGFR
How does the kyle and doolittle plot help with identifying a protein?
Looks at the hydrophobic/ hydrophilic nature of amino acids a protein
Hydrophobic= most likely in the membrane or cytoplasm
Hydrophillic= most likely to be outside the membrane
What sort of protein is EGFR, and how does EGF emit a signal?
EGFR= receptor tyrosine kinase
when EGF binds to the receptor- triggers transphosphorylation of the kinase - phosphorylation of tyrosine residues
What are the 2 main conclusions concerning RTKs, growth factors and oncogenes
- Growth factors induce their cellular effects through activating receptor tyrosine kinase signalling
- Oncogenes might work by activating the same signalling pathways, but in the absence of appropriate extracellular cues
What is the evidence for the 2 main conclusions?
- The amount of phosphotyrosine goes up in the presence of EGF
- The structure of some viral oncogene proteins are almost identical to EGFR- but they lack the domain that binds to EGF- so functions like EGFR but in the absence of a growth factor
What are the main principles of receptor tyrosine kinase signalling?
The receptor is off when in a monomeric state
Ligand binding induces dimerisation or de-auto-inhibition
Increased local concentration of active receptor kinase and receptor substrate leads to trans-phosphorylation
