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

Tom Anderson ucgatan at socrates-a.ucl.ac.uk
Fri Jan 9 08:00:21 EST 2004

On 7 Jan 2004, Andrew wrote:

> 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.
> 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

ES is fine - i don't know why everyone's obsessed with MALDI-TOF. A former
lab of mine did a side-by-side comparison of the two (for proteomics
work), and found that while they didn't give identical results, ES was as
good as MALDI-TOF (ie, each missed some peptides that the other picked
up). I think they had a pretty good ES system, though. Anyway, ES would
be able to tell you quite a lot about what's happening in your protein. Do
you have tandem MS? That would give you fantastic data, right down to the
individual residues.

> (with no experts to use it).

Okay, well forget what i just said! Unless, of course, you're willing to
master it yourself - quite possible, but MS scares the dickens out of me.

> 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)

Are you then doing the reaction _in vitro_? Do you have controls to make
sure that the substrate and kinase you're producing are properly folded
and functional etc? I suppose that you can't really assay the mutant
kinase for proper activity without knowing what its activity is, which is
what you're trying to find out!

> My results thus far have been disappointing.  I have not seen a single
> difference in my 2D maps.

So, if i may ask, what makes you think the mutant kinase does have
different behaviour?

You mention you're using the Hunter protocol, which i'm afraid i don't
know; how are you detecting the peptide spots? If it's by a method which
reveals all peptides, you might try radiolabelling the phosphate and
detecting by autoradiography; you ought to be more likely to detect faint
differentially-phosphorylated spots.

One possibility might be that the mutant kinase is switching from one
phosphorylation site to another that is in the same peptide, which i don't
think you'd be able to detect by TLC (or single MS) - pSXXXS looks the
same as SXXXpS. If that's the case, cutting the protein with a different
protease might do the trick.

> Does anyone have any input or suggestions on this?  Or better
> experimental ideas?  I can happily provide more information if needed.

If you know which residues the WT kinase phosphorylates, you could make a
panel of mutant subtrates, in each of which all but one of the target
resides are mutated to alanines; you could then run _in vitro_
phosphorylation reactions with the WT and mutant kinases, and check for
phosphorylation by WB or radiophosphate labelling. However, you might find
that the proteins don't fold, and it would be a lot of work, and i think
you don't know what the phosphorylation sites are anyway.

How about making constructs which express fragments (eg individual
domains) of the substrate and doing the assay with each one? If the
differentially phosphorylated residue is the only phosphorylated residue
in its fragment, you should get a clear difference between the WT and
mutant kinases (which you could detect by anti-pS/T WB, or radiophosphate
labelling plus SDS-PAGE and autoradiography or scintillation counting).
You could combine this with the point-mutation approach to suppress
individual sites in each fragment; that would make the job quite a bit

How about isoelectric focusing? If the WT-phosphorylated and
mutant-phosphorylated forms of the substrate have different total numbers
of phosphate groups attatched, they will have different pIs, and so will
go to different places on an IEF gel. This doesn't really help you more
than doing a quantitative anti-pS/T WB or a quantitative radiolabelling,
as Emir suggested, though.


Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL

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