What area of phosphorylation research is your focus?
My background is in comparative biochemistry and physiology, with an emphasis on the molecular mechanisms involved in the adaptation to stress. I’ve looked at frogs that freeze solid and ground squirrels that hibernate over the winter, along with cancer cells that continue to grow and proliferate in deficient media. I look at enzymes that catalyze key (suspected) reactions in their survival, and whether these enzymes are regulated by post-translational modification to make them more or less active. Since phosphorylation is the most common PTM, that’s what I’ve studied.
How do you perform Kinase Assays?
Typically I rely on radioisotopes to run kinase assays, namely gamma-32P-ATP. I’ll use the ATP, a kinase- either commercial, purified from a homogenate/cell lysate, or in crude condition- and a target for that kinase like a peptide or whole protein. After the reaction is over, what I do next depends on whether I used a peptide or protein. If it’s a peptide, then it involves spotting on phosphocellulose (P81) paper, along with washes in phosphoric acid. If I used a protein as the target, I’ll boil with SDS-PAGE sample buffer, load into an acrylamide gel and run SDS-PAGE. Finally, the paper or gel is then exposed to a phosphor storage screen and scanned with a laser scanner/phosphorimager.
Have you been able to move away from doing radioactive kinase assays?
Sir Philip Cohen once referred to radioactive kinase assays as the “gold standard” and so it’s very, very difficult to move away from them. The thing about radioisotopes is that the methods for detecting them are very sensitive, so you can detect low levels of phosphorylation. Also, you can detect a hot phosphate no matter where it’ll turn up in a protein. That’ll become relevant and important in a minute.
I’ve used another method with some success- in fact it was my first publication. It involved a peptide substrate for kinases that have a conjugated fluorophore. When the peptide is phosphorylated, magnesium ions are chelated by the phosphate and fluorophore, enhancing the fluorescence. The reaction can be detected in real-time, kinetically, using a fluorometer microplate reader.
What I’ve also tried to do in the case of protein substrates and SDS-PAGE is, using nonradioactive ATP of course, use a phospho-specific stain, like the Pro-Q diamond phosphoprotein gel stain to see if any of the protein bands light up after reaction with a kinase. The problem with that is that it’s not very specific- only to the nanogram range, and so if you have very little phosphorylation going on, you’ll have a hard time detecting it.
Finally, going further along that SDS-PAGE path, I’ve transferred proteins to a PVDF membrane and then probed with phospho-specific antibodies. The problem is, phospho-specific antibodies have limitations, so you can’t be too specific. If your antibody detects phosphorylation at serine-234 and instead, your protein is being phosphorylated- albeit beautifully- at threonine-658, your antibody won’t detect a speck and you’ll wrongfully assume that phosphorylation isn’t taking place. Antibodies of wider specificity won’t necessarily help because all antibodies out there rely, to some extent or another, on an epitope: not just a single amino acid of interest but a surrounding environment of amino acids, all of which an antibody binds to.
If you’re interested in my comparing and contrasting of different methods, I’d like to remind you about a Bitesize Bio article I did awhile back.
We understand that you have used some of our bioactive small molecules in the work that you’ve done. Can you tell us which ones and how you used them?
I relied on high-quality Sigma biochemicals for stimulating (and inhibiting!) my endogenous signaling enzymes. I used compounds like phorbol myristate acetate (PMA) to stimulate protein kinase C activity; cyclic AMP and cyclic GMP to activate protein kinase A and protein kinase G, respectively; AMP to activate AMP-dependent protein kinase; calmodulin to activate calcium/calmodulin-dependent protein kinase, and so on.
Some of the inhibitors included okadaic acid, to distinguish between protein phosphatise 1 and 2A, and cypermethrin to inhibit calcineurin or PP2C. In kinase assays using tissue extracts, protein kinase A is a very active kinase and can present a lot of interference, so we used protein kinase A inhibitors. The same was true for protein kinase G inhibitors.
All of that doesn’t even begin to include the coupling enzymes and substrates that I used to assay enzymes after I had stimulated protein kinases or phosphatases. For instance, I would take a muscle extract, and stimulate protein kinases to try and phosphorylate the something like AMP deaminase within the muscle. But then, what effect did that potential phosphorylation have on AMPD enzyme activity/kinetics? To find that out, I’d need to assay AMPD enzyme activity once again, which required Sigma products like AMP as the substrate for AMPD, and then the coupling reaction involving glutamate dehydrogenase, alpha-ketoglutarate, and NADH, so that I could detect it spectrophotometrically at 340 nm.
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