I have a protein with a phosphorylation site on the extracellular surface of the membrane, and I would like to figure out which kinase is phosphorylating it, and if possible which phosphatase dephosphorylates it. Any suggestions?
Hi Adrianne,
you can use some computational tools to identify kinases that phosphorylates your site. There are many free servers that can help you with that.
If you want to do this in the lab, you can use specific inhibitors for different kinases. Using them you can see which one couse decrease of transfered phosphate. Of course you have to use radiolabeled ATP.
The best way would be cloning and expressing your protein in bacteria or yeast and than you can use simple kinase assays to identify the one that phosphorylates your protein.
If you know the phosphorylation site, you can try one of the proteomic tools on expasy.org to predict the phosphorylation site. A combination of kinase inhibitors and in vitro phosphorylation assays could be used to verify that the site is indeed phosphorylated by that kinase.
If you don't what the site is, you will need to do tryptic digestion coupled with either mass spec or phosphopeptide mapping before you can really identify kinase/phosphatase of interest.
Your question should be précised to find out at which stage you are in studying a target protein at the moment. If only sequence is known check first the presence of specific motifs by informatics (tools are available but they are limited in the prediction capacity). If phosphorylation is a hypothesis based on virtual and/or experimental data and you can purify the target protein under the conditions required for phosphorylation then you can use this protein as a substrate for lambda phosphatase (it has wide specificity) from Santa CruzBiotechnolgy to be sure that the protein is indeed phosphorylated (do not forget about blanks and different controls). Next or alternatively you can perform peptide analysis of the purified protein (do not forget using phosphatase inhibitors) or the spots separated by 2DE (in this case you need not purify protein but should take into consideration whether it is reasonable to do in terms of your project) using detection approaches of phosphorylated amino acid accessible for you. The detection of modified amino acid(s) helps in prediction of kinase(s) and probably phosphatase(s) to be involved (compare with virtual prediction). So, you will have a possibility to check the only protein kinase and protein phosphatase or you will have a short list of enzymes to be tested. There are several commercial kits allowing identification of some PTK and PTP (Millipore, etc), which might include the enzyme predicted. Bon courage!
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