Lecture 3

Question 1

Structural components of viral envelope

Answer 1

Gag, pol, env

Question 2

What is Src ?

Answer 2

protein Tyrosine kinase

-enz that catalyse transfer of phosphate

Question 3

Collet and Erikson

Answer 3

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

Question 4

Transformed cells elicit what type of behaviour

Answer 4

anchorage independent growth

Question 5

Tony Hunter - Polyoma Large T experiment

Answer 5

-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

Question 6

Src

Answer 6

Tyrosine kinase

• induces functional changes in whatever it “touches”
• promiscuous- phosphorylates anything

Question 7

How did they show function of Src ?/

Answer 7

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

Question 8

phosphorylation

Answer 8

Occurs everywhere

Question 9

Stan Cohen - experiment into Growth factors

Answer 9

• Using HeLa cells

- cells in culture require a serum, purify out this serum - GF fulfil serum requirements

Question 10

Cohens questions

Answer 10

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

Question 11

Cohens Protein

Answer 11

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

Question 12

Techniques to identify Cohens protein?

Answer 12

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

Question 13

how do you work out the structure ? how do we know EGF-R goes through the membrane ??

Answer 13

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

Question 14

significant homology is shown between what structures ??

Answer 14

EGF-R similar Homology to a phosphotyrosine kinase

Question 15

What does Cohens findings suggest ?? two explanations

Answer 15

• 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

Question 16

How do growth factors elicit an effect ??

Answer 16

Growth factors induce their cellular effects by triggering a tyrosine kinase signalling pathway

Question 17

Kyte and Doolittle plot

Answer 17

The plot has amino acid sequence of a protein on its x-axis, and degree of hydrophobicity and hydrophilicity on its y-axis.

Question 18

EGF-R

Answer 18

Receptor Tyrosine kinase

Question 19

Evidence that EGF-R was working by phosphorylating things in the cell

Answer 19

Amount of Phosphotyrosine in response to growth factors in cells goes up
- amount of Phosphotyrosien goes up significantly in the presence of EGF-R

Question 20

EGF-R structure

Answer 20

tyrosine kinase with Transmembrane domain have receptor on the outside that brings together subunits of tyrosine kinases and phosphorylate themselves

Question 21

Immunofluroescene exp allows look at what

Answer 21

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

Question 22

Oncogenic mutation- How might they work ?

Answer 22

mutation can bring about a functional receptor in the absence of extracellular cues

Question 23

Principles of RTK signalling

Answer 23

• 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

Question 24

Receptor tyrosine kinase theme

Answer 24

Variations between kinases

• The kinase may not be tyrosine
• cytoplasmic domain may not be a kinase