Lecture 3 Flashcards
Structural components of viral envelope
Gag, pol, env
What is Src ?
protein Tyrosine kinase
-enz that catalyse transfer of phosphate
Collet and Erikson
Developed a Src antiserum
- incubated src imminopreicpitates with gamma32P- ATP
- looked at transformed and untransformed
- expose it to film sensitive to energy range of particles emitted by radioactive substance s- darkness where radioactivity present
Transformed cells elicit what type of behaviour
anchorage independent growth
Tony Hunter - Polyoma Large T experiment
-Take polyoma large t radioactively phosphorylated
-Incubate in HCL and hydrolyses everything to single amino acids
-Chromatography and electrophoresis in 2D
Differential solubility – separated out
-Polyoma was phosphorylated on another a.a
But made buffer incorrectly – couldn’t repeat
Found phosphotyrosine
Phosphotyrosine kinase phenomenon
- PH buffer change
Src
Tyrosine kinase
- induces functional changes in whatever it “touches”
- promiscuous- phosphorylates anything
How did they show function of Src ?/
Developed an antibody specifically binds to phosphotyrosine in a western blot
-Recognises phosphorylated proteins just phosphorylated on the phsophotyrosine
-Shows Src doing a lot phosphorylating things on tyrosines
phosphorylation
Occurs everywhere
Stan Cohen - experiment into Growth factors
- Using HeLa cells
- cells in culture require a serum, purify out this serum - GF fulfil serum requirements
Cohens questions
What does bind to the growth factors in cell s?
- attached an epidermal growth factor EGF to a solid support
- mush up HeLa cells and pass extract through column where EGF is bound
- Eluted the contents – what ws the nature of stuff in tube and was it homogeneous
- Only one protein – huge degree of purification
Cohens Protein
EGF-R - found using sanger sequence and proteolysis -
what bits do what ?
- chopped it into three bits and ran on a gel ( unknown functional protein)
incubated with Radioactive EGF
- part that bound
Techniques to identify Cohens protein?
Take the material and chop it up
Digest it with a protease and isolate various peptides
Automated protein sequence – sanger sequencing
Screened gene libraries to identify the gene
Chopped it up again using a proteolytic mechanism
Specific proteolysis – generates 3 fragments
Then ran them on a gel
And incubated with radioactive egf
how do you work out the structure ? how do we know EGF-R goes through the membrane ??
Plot a graph of how hydrophilic/phobic amino acids are then can move along 3 at a time
Hydrophobic inside of molecule
EGF-R bit that goes through them membrane – hydrophilic
significant homology is shown between what structures ??
EGF-R similar Homology to a phosphotyrosine kinase
What does Cohens findings suggest ?? two explanations
• 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
How do growth factors elicit an effect ??
Growth factors induce their cellular effects by triggering a tyrosine kinase signalling pathway
Kyte and Doolittle plot
The plot has amino acid sequence of a protein on its x-axis, and degree of hydrophobicity and hydrophilicity on its y-axis.
EGF-R
Receptor Tyrosine kinase
Evidence that EGF-R was working by phosphorylating things in the cell
Amount of Phosphotyrosine in response to growth factors in cells goes up
- amount of Phosphotyrosien goes up significantly in the presence of EGF-R
EGF-R structure
tyrosine kinase with Transmembrane domain have receptor on the outside that brings together subunits of tyrosine kinases and phosphorylate themselves
Immunofluroescene exp allows look at what
Immunofluroescene exp allows look at what happened at the pm when trigger EGF-R – is there phosphorylation occurring
- Know EGF- R is phosphorylating things at the PM
- signalling goes up
Oncogenic mutation- How might they work ?
mutation can bring about a functional receptor in the absence of extracellular cues
Principles of RTK signalling
- Receptor in off state often monomeric
- ligand binding incuces dimerisation or de-auto-inhibition
- increased local concentration of active receptor kinase and receptor substrate results in trans- phoshporylate
- Phospho- receptor act as a platform for signalling events
Receptor tyrosine kinase theme
Variations between kinases
- The kinase may not be tyrosine
- cytoplasmic domain may not be a kinase
