No need to FRET

August 30, 2010 § Leave a comment

I just found this very pretty story about a new kind of kinase sensor from Barbara Imperiali’s lab (Luković et al. 2009 Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters. Angew Chem Int Ed Engl. 48 6828-31).  I don’t have to tell you why kinases are important; phosphorylation is one of the most significant things you can do to a protein (short of cleavage) and it’s used to create new binding sites and change conformations, causing profound changes in protein activity, in an enormous variety of settings in the cell.  There are over 500 kinases that phosphorylate proteins in the human genome, and the fraction of eukaryotic proteins that are phosphorylated in eukaryotes is huge — at least 30%, and the number goes up as methods get better. Misregulation of protein kinases is common in cancer, and anti-cancer drugs like Gleevec and Iressa have their effect by inhibiting the activity of specific kinases.  Because kinases are so common, it can be hard to see the activity of an individual kinase; you often don’t know whether the activity you’re watching is the kinase you care about, or a different one with overlapping specificity.

FRET can be helpful as a kinase monitor, but it’s not a simple technology — a bit fretful (ha!) in application [“marked by worry and distress; troublesome”] — and while it’s useful in a live-cell setting, it’s not a general solution.  In particular FRET is not ideal for high-throughput screening.  One promising alternative is a chromophore called Sox, for sulphonamido-oxine, that only fluoresces if it’s interacting with magnesium ions.  But it can’t effectively chelate the magnesium on its own; instead, it’s dependent on the presence of a nearby phosphate group that provides two of the four ligands needed for tetrahedral coordination of the magnesium.  So when the peptide is not phosphorylated, there’s no magnesium chelation and no fluorescence.  When the peptide is phosphorylated, fluorescence goes up significantly; the phosphate essentially becomes part of the chromophore.  This is the kind of design where, when you look at it, you go “oh, that’s clever”.  It’s reminiscent of the FlAsH system, which is also very clever.  In that system, a tag added to your protein of interest, containing four cysteines, coordinates a metal (in this case arsenic), causing a non-fluorescent chromophore to become fluorescent.  But in the case of Sox you can see the activity of an enzyme, not just its presence.

The Sox system is only selective if the kinase selectively phosphorylates a particular peptide sequence.  And that can be a problem.  Some kinases selectively phosphorylate motifs that are 4-8 amino acids long; others only need the right 2 amino acids in a row, which can hardly be called a motif.  For example, it’s not easy to isolate the residues responsible for selective phosphorylation of the important Ets family of transcription factors by ERK1/2. Short peptides containing the Ets-1 phosphorylation site bind to ERK1/2 quite poorly. To get specific binding of ERK1/2 you need pretty much all of the “pointed” (PNT) domain of Ets-1, connected to the unstructured region where phosphorylation happens.

So, to make a specific sensor for ERK1/2 activity, Luković et al. set out to combine the PNT domain with a peptide containing both an optimized phosphorylation sequence and the Sox conditional chromophore.  They expressed and purified the PNT domain and made the peptide by peptide synthesis; then they stuck the two together using native chemical ligation.  Then they asked — does ERK1/2 recognize the result?  And is the sensor specific enough to be useful in complex mixtures like the cell cytoplasm?

Obviously I wouldn’t be telling you this story if the answer were no [well, unless the reason for the failure was somehow interesting]. The Sox-PNT sensor is phosphorylated nearly as well as wild-type PNT by both ERK1 and ERK2, and a panel of kinases that shouldn’t phosphorylate the sensor — including Jnk, p38 and cyclin-dependent kinases — don’t.  And phosphorylation does indeed make the sensor light up.

The key test, though, is what happens in unfractionated cell lysates. Is the sensor still selective for ERK1/2 activity?  To test this, Luković et al. made extracts from four different cell lines with ERK1/2 stimulated or not.  This was done by treating the cells with EGF before lysing them; ERK1/2 are activated by phosphorylation by MEK1/2 as part of the EGFR pathway.  To switch ERK1/2 off again, they used an inhibitor of MEK1/2, again before lysis. All four cell lines showed the same pattern: no EGF, no fluorescence; add EGF, strong fluorescence; add MEK1/2 inhibitor, go back to almost control levels.  The level of phosphorylated ERK1/2 showed the same pattern, as measured by Western blot.  The control Sox-peptide showed no such selectivity, giving high levels of fluorescence whether the cells were treated with EGF or not.  And if ERK1/2 activity is directly removed, either by adding a competitive inhibitor or by immunodepletion, the response to EGF goes away.

Luković et al. argue that this could be a general strategy for testing kinase activities in cell and tissue lysates; there are several other kinases whose activity has been hard to measure for similar reasons of complex substrate recognition.  The authors say that they can easily produce enough Sox-PNT using this semi-synthesis method to do >5,000 assays at a time.  Now, remember that this sensor isn’t GFP: you can’t make it inside a living cell (though I guess you could inject it). But still, a selective, easy to use activity sensor for hard-to-assay kinases will find many uses.

Luković E, Vogel Taylor E, & Imperiali B (2009). Monitoring protein kinases in cellular media with highly selective chimeric reporters. Angewandte Chemie (International ed. in English), 48 (37), 6828-31 PMID: 19681083

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