I have a protein that is active when expressed in SF9 cells and I am curious whether phosphorylation is important for enzyme activity. For a very blunt test, I would like to dephosphorylate it (ie. with NEB calf intestinal alkaline phosphatase) and then test enzyme activity. If it is lower, though, I would like to see if re-phosphorylating it rescues activity. So what I am looking for is a very general set of kinases (preferably readily available, not something I would have to clone and express) that would phosphorylate a protein. Any suggestions?
Tyrosine kinases, serine/threonine kinases or histidine kinases
You have first to determine if there are consensus phosphorylation sites for a given kinase. Then some kinase are commercially availbale and you can perform in vitro phosphorylation assays
Many active protein kinases are commercially available. You need to determine what kinase phosphorylatiobn motifs are in your protein and order an active kinase that will phosphorylate those sites. Sometimes there is overlap in their consensus phosphorylation sites so you might be able to identify a single kinase that can phosphorylate multiple sites depending on what is actually present.
You have to analyse your amino acid sequence to identify if there are phosphorylation site(s). Therefore, basing on the type of residues in the consensus site you can choose the right enzyme: Serine kinases, tyrosine kinases.... like PKC which control phosphorylation of hydroxyl groups of serine and threonine residues.
Many tools are available in SWISSPROT data base.
Thanks all. These are helpful suggestions. Also, recombinant human kinases (ie PKC) seem to be quite pricey. Does anyone have luck using animal / yeast kinases, or are they pretty much human only?
Not sure in vitro phosphorylation of your protein will tell you very much. I can pretty much guarantee you that X or Y kinase will incorporate some phosphate but is it meaningful? Best to determine what the sites are in vivo (i.e. in immunoprecipitated protein from cells/tissues) and then try to identify the kinases based on consensus and on the impact of cell stimulation/inhibitors. Kinases are fairly promiscuous in vitro (some a lot more than others) and you could easily be mislead.
By the way, there are essentially *no* specificity differences between mammalian kinases. No need to worry about using human on human unless you are also interested in Florida swampland (companies will sell you anything). Yeast kinases are more distant.
As James said, I am not sure how much in vitro phosphorylation would tell you about functional significance of specific phosphorylation sites. In my opinion it depends on what you are really looking for. If you are looking for the specific kinase that phosphorylates your protein of interest, there are elegant methods -- e.g. http://onlinelibrary.wiley.com/doi/10.1002/anie.200905244/abstract -- that give you really specific answers. If you just want to phosphorylate the protein in vitro, I am sure you already got good suggestions. One thing to keep in mind is to have appropriate controls to ensure that the site you are interested in, is the one that is indeed phosphorylated in vitro and also the one that imparts functional significance to the protein in vivo. Good luck :)
Some years back I used a cheap protein kinase A from bovine heart (Sigma)
to phosphorylate a protein expressed with the kinase recognition sequence ARRASV.
http://www.sigmaaldrich.com/catalog/product/sigma/p2645?lang=en®ion=US
This kinase is not very specific and I found that it can phosphorylate other sequences that look similar.
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