Papers Flashcards
Src (tyrosine kinase) SH2 domain forms a tyrosine binding pocket between what residues of the substrate?
What is the EF1 residue?
What is src’s EF1?
What is sem5’s EF1?
To what consensus sequence does WT Src SH2 bind to?
To what consensus sequence does T215W Src SH2 bind to?
pY+1 and pY+3
Residue at the p+3 binding pocket of the SH2 domain
Threonine T 215
Tryptophan W
pYEEI
pYENP
What consensus sequence does Grb2’s SH2 domain bind to?
How did T215W Src SH2 compare when binding to this peptide?
How is FAK binding mediated?
Therefore how does the difference in WT Src SH2 and T215W Src SH2 compare when it binds?
How did FAK bind to W215G Src SH2?
What is the conclusion?
pYVNV
With similar binding affinity
Through Src SH2 as they recognise pYAEI of FAK
WT bound to pY of FAK
T215W did not bind to pY of FAK
W215G bound to pY of FAK as glycine doesn’t obstruct pY
T215W decreased selectivity due to pY+2 E and pY+3 I
How do WT Src SH2 bind to phosphorylated sem5 proteins?
How does this compare with T215W?
What other protein’s SH2 domain binds sem5?
What happens when it’s mutated from W to T?
What can you conclude about T215W?
Don’t bind
Binds
Grb2
Doesn’t bind
Converted to binding specificity closely resembling Grb2
How was a mutant sem5 protein created?
When both the WT sem5 and mutant sem5 placed in a vulvaless phenotype - what was the difference in restoration?
How is a vulvaless phenotype produced?
What happened when a mutant sem5 had a T215W SH2?
What other protein binds to sem5?
What does the W amino acid do?
What does this mutation of the EF1 then resemble?
Its SH2 domain replaced with WT Src
WT ~ 75%
Mutant ~ 20%
Inactive let23 tyrosine kinase which activates ras pathway without sem 5 present
Similar restoration to the wildtype
Grb2
Protrudes into p+3
Sem5/Grb2
How were the old cAMP biosensors made up of fluorescin and rhodamine crap? 4 ways
What is the new way?
Therefore what is seen in low cAMP levels?
High cAMP levels?
Why?
How is FRET changes calculated?
Why is using a beam splitter & 2 bandpass filters better than 1 beam and 2 bandpass filters?
How is the images processed in 3 steps?
- Needed large complexes
- Needed large amount of biosensor
- Not applicable to all cell types
- Can aggregate & precipitate
RII beta subunits fused to CFP and C-alpha (catalytic) subunits fused to YFP & using FRET
Excitation CYP 430nm, emission CFP 480nm & YFP emission at 545nm
Only CYP excitation & emission
No non-radiative transfer as PKA is active
Ratio between donor fluorescence : acceptor fluorescence when PKA is active and inactive
Means that there are 2 discrete beams - introduces artefacts otherwise due to time required to shift between filters
- Align both CFP & YFP images
- Subtract background noise – reduces artefacts & increases range of measure
- Calculate ratio between CFP & YFP images such that each pixel in the superimposed picture CFP/YFP corresponds to the ratio of that pixel & then use this to measure average ratio kinetics
Which PLC gamma 1 subunit mutants had an effect when PLC gamma was BCR-induced to the membrane?
What does this say about the domain & role in translocation?
Which mutants had an effect when PLC gamma 1 interacted with BLNK adaptor protein after western blot?
Conclusion?
What happened when mutant domains had a myr sequence added for membrane translocation along with their tyrosine phosphorylation?
Conclusion?
Which domains hydrolysed PIP2? Conclusion?
What was the difference in domain activity when stimulated with BCR?
Conclusion?
SH2N = no translocation
SH2N & SH2C = no translocation
SH2N required for BCR induction of membrane translocation
SH2N mutation did not coprecipiate with phosphoprotein BLNK whereas everything else did
SH2N is required for tyrosine phosphorylation & binds to BLNK via this domain
SH2N showed no phosphorylation with or without myr whereas SH2C and SH3 were phosphorylated and translocated
SH2N required for protein interaction for PLC phosphorylation for its translocation
None - all involved in BCR coupling to PLC gamma for hydrolysis
SH2N showed basal activity - same as unstimulated WT
SH2C had higher than WT activity even when unstimulated
SH2C plays role in coupling PLC to BCR receptor as an intrinsic regulator of activity
