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

EK someone at microsoft.net
Thu Jan 8 01:30:08 EST 2004


"Andrew" <aji23 at drexel.edu> wrote in message
news:958c94ef.0401071338.22bbffb2 at posting.google.com...
> I'm trying to establish the most efficient and convincing way of
> demonstrating a protein of interest is differentially phosphorylated
> by a mutant form of a protein kinase our laboratory investigates.

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.

>
> My current approach is 2D-phosphopeptide mapping using the Hunter TLC
> protocol.  I do not have MALDI-TOF MS at my disposal, although I do
> have an electospray MS available (with no experts to use it).  I have
> everything else a standard molecular biology laboratory has to offer
> (including plenty of radiation!)
>
> The endogenous protein is not expressed at reasonable levels, so I
> utilize an adenovirus expression system to produce exogenous protein.
> I also have transfection vectors available, although I am currently
> unable to produce sufficient levels of overexpression.
>
> As a source of kinase, I utilize adenovirus vectors to express both
> the wild-type and mutant forms of the protein.  The mutant does not
> expess to levels comparable to the wild-type (the wt protein is a
> transcription factor and unfortunately upregulates both adenovirus
> vector's promoters)

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. In such a case you will never be able to produce the protein.
In such a case you sould try C-terminal fusions of the kinase with various
partners (GFP, luciferase, etc.). Such fusion partners could promote a
certain stable conformation of the mutant protein, and will alow you to
check for expression levels by appropriate assays. You should first check of
course if fusions with WT protein still have kinase activity.

>
>
> My results thus far have been disappointing.  I have not seen a single
> difference in my 2D maps.  My current pursuits are going to be
> attempting to overexpress the protein(s) via transfection and attempt
> to get high efficiency.
>
> Does anyone have any input or suggestions on this?  Or better
> experimental ideas?  I can happily provide more information if needed.

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).

>
> Thank you very much.
>
> Andrew Ippolito





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