So you think your protein might be phosphorylated? Maybe it runs as a fuzzy smear on the gel, slightly higher than predicted. Or perhaps you think it is involved in a signaling cascade. In any case, if you suspect phosphorylation, then you should check it out.
And the first step you can take is to see if you can predict protein phosphorylation by examining your protein’s sequence.
The signal needs to be there
If your protein is going to be phosphorylated, then it needs to contain a phosphorylation consensus sequence.
Some kinases, such as the epidermal growth factor receptor, have very specific consensus sequences that have been mapped. You can check your protein sequence to see if it has a particular kinase recognition site.
Some kinases though, don’t require such a specific consensus sequence or the consensus sequence hasn’t yet been mapped. For those, some general rules can be applied.
In eukaryotes, protein phosphorylation generally occurs on serine, threonine, tyrosine and histidine (in lower eukaryotes) residues.
Recognition and subsequent phosphorylation of a protein by a kinase depends on the context in which the residue presented. The amino acids surrounding the site make up the consensus site. In eukaryotes, phosphorylation on serine and threonine residues often occurs when the residue(s) lies near one or more basic amino acids. In contrast, tyrosine phosphorylation occurs when the residue is in close proximity to acidic amino acids. These general rules are complicated by the fact that some kinases rely heavily on recognition of the 3d structure of the protein, which can be determined by amino acids that are not close to the phosphorylation site. In addition, many proteins contain multiple phosphorylation sites and a phosphorylation hierarchy may occur; phosphorylation of one residue might only occur after another residue is phosphorylated.
You don’t need to go it alone
Thanks to the power of computing and ingenious programmers, you can use online programs to predict potential phosphorylation sites in your protein.
Alternatively, there is ample freeware or free online services to predict protein phosphorylation. A quick Internet search pops up multiple sites, including (in no particular order):
- GPS from The Cuckoo Workgroup
- NetPhos 2.0 Server from the Center for Biological Sequence Analysis
- PhosphositePlus from Cell Signaling Technology
I’ll leave you to find your favorite.
Verify, verify, verify
The most important thing to remember when using protein analysis software, is that it is only a prediction. Many factors, including: phorylation site opens up a whole set of experiments you will need to perform to prove it is phosphorylated.
Who knows; it might be the start of your career (or at least a great paper).