Basics of Protein Phosphorylation Part II: Tools of the Trade

In the previous article in this series, we looked at the major players involved in protein phosphorylation: protein kinases, protein phosphatases, and target proteins. This time, we’ll glance over some of the tools that we can use to study various aspects of protein phosphorylation, focussing on a few I’ve personally come across.

1. Tools for Detecting the phosphorylation state of proteins

Over the course of a research project, eventually you’ll want to know what phosphorylation state a protein is in, whether it’s a result of in vivo regulation or in vitro modification. The four following techniques rely on protein electrophoresis. Once you’ve got your protein of interest in solution – whether in a crude mixture of proteins, or purified, you have the following options:

Stains to detect all phosphoproteins

This method is right for you if you want to keep yourself free and open to all potential phosphoproteins or phosphorylation states of a protein that might come your way; you can detect all phosphoproteins without restricting yourself to a single protein, or a single motif or residue. The Pro-Q Diamond Phosphoprotein Gel Stain from Invitrogen will do just that, and can be visualized with a laser scanner or UV transillumination. You can even go a step further and instead use the Blot Stain kit to detect phosphoproteins on a PVDF or nitrocellulose membrane. Then wash away the stain and multiplex with western blotting.

Pros: You get to see any and all phosphoproteins, on a gel or a membrane. Fast and easy procedure.
What to watch for: Only sensitive to the low nanogram range- better than Coomassie but sometimes not good enough for precious little protein. Also, detecting all phosphoproteins may be biting off more than you can chew if you electrophorese and stain complex and concentrated mixtures of phosphoproteins- you might get more of a “smear” than clean bands of the protein you’re looking for.

Antibodies for specific amino acids

Say you’re interested in proteins phosphorylated specifically on serine residues, not threonine, tyrosine, or anywhere else. You could use an antibody that would only bind to such phosphoserine proteins. I’ve used the PhosphoDetect Phosphoserine Detection Kit from Calbiochem, complete with four clones of antibodies to detect phosphorylated serines, as well the surrounding amino acid environment. That point is very important to keep in mind- the antibody will not bind to just any phosphoserine present in a protein, but must recognize and have affinity for adjacent “epitopes” as well.

Pros: High specificity and high sensitivity, as expected with western blotting.

What to watch for: “Epitope” requirement can be bothersome. I wanted to use casein, a protein rich in phosphoserines as a positive control/standard. Casein is highly phosphorylated at sites surrounded by acidic amino acids, and I learned that these antibodies unfortunately detect phosphoserines in environments with basic amino acids. I ultimately couldn’t get the antibodies to detect a single speck for casein! Also, with regards to this method and the next two- antibodies aren’t cheap!

Antibodies for specific proteins and specific phosphorylation sites

The first two methods had the potential to detect a multitude of phosphoproteins- what if you were interested in just one? You could use antibodies targeted to that single protein, and moreover, specific phosphorylation sites within that protein. The Phospho-PKC Antibody Sampler Kit from Cell Signaling Technology helped me when I was working on protein kinase C (PKC), to differentiate between the different isozymes and where they were phosphorylated (Ser744, Thr505, Ser916 to name a few).

Pros: Specificity for one single protein and phosphorylation site, and the sensitivity of western blots all rolled into one.

What to watch for: Limiting. For this method (and the next), the more specific and narrow you get with your detection methods… what happens if what you think you’re looking for, turns out not to be what you’re looking for?

Antibodies to detect specific targets of phosphorylation

Let’s take the previous method and flip it around- say you weren’t interested in PKC per se, but you were instead more concerned with what PKC phosphorylated and which of its targets you could find. You’d be interested in detecting the specific motif in proteins to which PKC binds while phosphorylating the serine residue within it. Surely enough, there are antibodies for those as well, like the Phospho-(Ser) PKC Substrate Antibody from Cell Signaling Technology.

Pros: Once again, specificity and sensitivity, what’s not to love?

What to watch for: As before, in the context of this example- what happens if your protein of interest turns out to be possibly phosphorylated at a threonine instead of a serine, and this antibody doesn’t detect it? What happens if your protein of interest turns out to not be phosphorylated by PKC in certain conditions… or at all?

2. Tools for determining activity, affinity and specificity of protein kinases

Just as important as the proteins being phosphorylated are the protein kinases doing the phosphorylating. What are the kinetic parameters of a kinase in given conditions? Will it phosphorylate a novel protein of interest or won’t it? These are important questions you may come across over the course of your research.

Radioisotopes

Hot stuff! Take a protein kinase and give it either a protein or peptide substrate, use normal ATP spiked with a bit of radioactive gamma-32P-ATP, and watch the results light up. Anything phosphorylated will be detectable with high sensitivity to radiometric methods such as phosphor storage screens and laser scanners, and even greater sensitivity with liquid scintillation counting (LSC).

Pros: Very high sensitivity and broad detection- as long as a kinase phosphorylates a protein and transfers that terminal 32P-phosphate over, it’s detectable- regardless of which amino acid is phosphorylated, the nature of the surrounding epitope, antibody recognition, and so forth.

What to watch for: The joys of working with lucite shields, double-gloves, Geiger counters, radiation safety offices, and everything else that comes with an ionizing-radiation source that can potentially be pretty harmful!

Peptides and P81 paper

Sir Philip Cohen and his group at Dundee wrote a Nature Protocols paper referring to this method, quite correctly, as the “gold standard” of protein kinase quantification. Simply, it involves reacting a protein kinase with a peptide containing a motif known to be recognized by the kinase and, importantly, rich in basic amino acids and hot ATP. The reaction mixture is then spotted onto negatively-charged P81 phosphocellulose paper, allowing the positively-charge peptide to bind and everything else- including any autophosphorylated kinase and unreacted ATP- to wash away. The hot peptides on the paper can then be detected by phosphor storage screens or LSC.

Pros: Simple enough to get the hot ATP, and you can get the peptides virtually anywhere- potentially even at your own core facility.

What to watch for: Some peptides can be expensive depending on where they’re purchased, surprisingly even more expensive than their whole-protein counterparts. Using only those peptides rich in basic residues can also limit which kinases you can assay. Conceivably, you can use acidic peptides and DEAE (positively-charged) paper but I’ve never come across that. Further, what happens when you use neutral or somewhat hydrophobic peptides that won’t interact with ionic paper at all?

Proteins and precipitation, or electrophoresis

Limited by peptides? Take Sir Philip’s protocol above and substitute a short peptide for a whole protein. In fact, it’s actually a reaction more similar to physiological conditions. The problem, however, is that if you had issues getting certain peptides to stick to P81 paper, then a whole protein could be a nightmare. Instead, precipitate the protein chemically, such as with TCA, to filter paper, leaving the unreacted ATP to remain in solution and wash away. Or, better yet, load your reaction mixture into an acrylamide gel and electrophorese away. You’re ultimately looking for a radioactive protein band at the correct molecular weight in the gel, and can even Coomassie stain it (safely- keep in mind you’re working with a hot gel) to help you find what you’re looking for.

Pros: Greater choice of protein substrates to be phosphorylated. With electrophoresis, you’re sure to identify your target of interest, at the correct molecular weight, appearing as a radioactive band.

What to watch for: By using TCA washes or electrophoretic running buffer, and then staining and destaining a gel, you may be spreading radioisotopes into even more solutions that require careful disposal.

Non-radiometric assays

As the safety demands and distaste for using radioisotopes grow, many enterprising biotech companies are coming up with ways to eliminate that dependency. New kits are continually being developed that rely on fluorometric or colourimetric detection of protein kinase reactions. My grad lab was one of the first that tested the Omnia Kinase Assays by Invitrogen on non-recombinant and non-human kinases. The assays use a peptide with an unnatural amino acid fluorophore- upon phosphorylation of the peptide, magnesium is chelated between the phosphosite and the fluorophore, enhancing fluorescence. What was especially nice about this assay as opposed to others was that, instead of merely detecting the endpoint of phosphorylation, the Omnia Kinase assay principle allows phosphorylation progress to be tracked in real-time. I also tried out the IMAP FP kinase assay by Molecular Devices, which showed promise but ultimately didn’t suit my needs. That assay uses peptides which once again contain a fluorophore, but rely upon fluorescence polarization. Upon phosphorylation, the phosphopeptide binds to a trivalent metal (M3+) nanoparticle, and fluorescence polarization increases. Unfortunately, what I also discovered was that virtually anything with phosphates can bind to these metal nanoparticles, occupying binding sites and quenching the fluorescence polarization of the peptide: unreacted ATP, ADP, AMP, cofactors, phosphoproteins- all of which you can easily find when using crude lysate or using high concentrations of ATP.

Pros: Sensitive, convenient kits, eliminating the need to use radioisotopes.

What to watch for: “Dime a dozen” may refer to the abundance of non-radiometric kinase assays being developed by companies these days, but certainly not the price. These can easily cost orders of magnitude above what is required for a simple assay using hot ATP.

3. Tools for determining activity, affinity and specificity of protein phosphatases

So far, we’ve looked at proteins that have phosphates on them, and the kinases that put them on there. Just as important and certainly not to be forgotten, are the protein phosphatases which dephosphorylate them.

Go Green!

Ekman and Jager presented, back in 1993, a method now commonly used to assay subnanomolar inorganic phosphate hydrolyzed from phosphoproteins by phosphatases. The principle is based upon the strong green colourimetric complex formed between the malachite green dye, molybdate, and free orthophosphate. As with protein kinase assays, a protein or peptide substrate can be used for a protein phosphatase, but in this case, the substrate is phosphorylated to begin with, and presumably loses phosphate as the incubation continues. At the end of the desired incubation time, the reaction is quenched with the dye reagent, and absorbance at 600 nm is read in a simple spectrophotometer.

Pros: Sensitive (hey, it’s subnanomolar!), easy mix-and-read method. Conceivably one type of assay for any protein phosphatase with any phosphopeptide or phosphoprotein substrate.

What to watch for: If you thought peptides were expensive, phosphopeptides can be even more so! Try to find cheap, bulk proteins to use as substrates, like casein. Also, that dye can definitely make a staining mess- which can actually be less trivial and more serious than it sounds if you spill it in your spectrophotometer.

The yellow glow of para-nitrophenyl phosphate

Remember 2nd-year undergraduate biochemistry lab? If so, then you’ll recall the classic lab that involves, in one way or another, alkaline phosphatase reacting with a molecule to produce an increasing yellow colour over time. That molecule is actually para-nitrophenyl phosphate (pNPP), and its hydrolysis product, para-nitrophenol (pNP) is the yellow-coloured molecule which absorbs at 400 nm. You can use a simple spectrophotometer to detect hydrolysis in real-time.

Pros: Cheap, simple, real-time method.

What to watch for: Not the highest sensitivity. Moreover, pNPP is a rather small molecule and not a physiological peptide or protein with recognition motifs- not all phosphatases will bind and hydrolyze it, and so it may not be usable for all phosphatases.

Radiolabeled protein and peptide

Earlier, we saw how peptides and proteins can be radiolabeled by reacting them with a protein kinase and hot ATP. With a bit of ambition, skill, and luck, those peptides or proteins can be purified and used as substrates for phosphatases. The assay principle is the same, but in reverse- instead of detecting radioactivity being incorporated into a protein by a kinase, the aim is to detect radioactivity being removed from a protein by a phosphatase. In addition to making your own radiolabeled protein and peptide, you can also purchase commercially-available ones.

Pros: Sensitive, universal, all the advantages of assay protein kinases with radioisotopes now applied to protein phosphatases.

What to watch for: The use of radioisotopes and everything that goes with them, again.

I hope you found this useful, although it doesn’t even scratch the surface of available tools! Use this as a foundation and go see for yourself what best suits your needs! If you have anything to add, or any questions about the tools I have described, please be sure to leave a comment.

Next, we’ll take a closer look at protein kinases, beyond what we covered in their introduction in Part I.