Differential Phosphorylation in response to a wild-type and mutant protein kinase

Andrew aji23 at drexel.edu
Thu Jan 8 13:49:39 EST 2004


EK, thanks for you inpute - it gave me some things to think about.


> Differentially phosphorylated meaning either phosphorylated or not
> phospharylated, or something else? I am asking because later in the text you
> say you did not see any difference on your 2D maps, which I might understand
> like either you see you protein phosphorylated by both wt and mutated
> kinase, or you see it not phosphorylated under either conditions, or you see
> it phosphorylated at different resudues. Please provide some details.

You were correct in that I do see a phosphorylated protein in the
presence of both wt and mutant kinase, and I am trying to demonstrate
a difference between these two phosphospecies.  To date, my 2D maps
are almost completely superimposible except for some modest
differences which I doubt would convince reviewers.

> There might be a chance that your mutation causes misfolding of the kinase
> that might in its turn cause fast sequestration of the misfolded protein by
> proteolysis. 

This is something I have thought about.  The GST fusion protein of the
mutant expresses poorly out of bacteria compared to wild-type as well.
 Would it be worth trying another tag for stability, you think?  The
GST version of the wild-type is still a kinase, indicating we can add
a tag to it without a problem.

> Could you do SDS-PAGE of cell extracts from cells with wt and mutant kinase
> (once you find optimal conditions for transfection and see if fusions
> approach could work for you) and then do westerns and probe for
> phosphoresidue and reprobe for the protein itself? There are antibodies
> against phosphotyrosine (don't forget to block with BSA and not dry milk).
> This way you could see disappearance of phosphotyrosine marker in the
> extracts containing mutant kinase (if that is what you are looking for).

The problem with this approach is the kinase in question seems to be a
S/T kinase, not a Y.  And again, this would only show rough
differences.  I need to show a specific difference between two
phosphorylated species.  manufacturing phospho-specific antibodies is
not feasible since the phosphorylated residues are not mapped (and
mapping these as you probably know is even more tedious than showing a
simple difference).

Any other thoughts on the matter?



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