I would try specific kinase inhibitors. Synkinase is one company that sells many kinase inhibitors. I would set up an assay using your protein, and add the specific kinase inhibitors to your specific cell extract. Or you can do whole cell assays, and treat the yeast with specific inhibitors and look for inhibition of phosphorylation. Then for either assay, use a Western to look at phosphorylation of your protein.

You might want to try to search literature for some specific kinase inhibitors inhibiting one or more of these kinases. When you find an inhibitor blocking the phosphorylation in your specific domain (confirmed by western blot) you might make some conclusions.

You could get all 8 candidate kinases and test them one by one with the purified protein.

You could try an coprecipitation/proteomic experiment with your protein if you can purify it or if you have a specific antibody to it.

There is really no easy and fool-proof way to do this. Inhibitors have the problem that they often inhibit multiple kinases and you can never be sure if you are actually inhibiting the kinase that is directly phosphorylating your protein of interest or something upstream of a signaling cascade. In vitro work has the problem that purified kinases and targets very commonly lead to both false positives and false negatives.

My suggestion would be to try Adam's second approach: Run a co-iP under conditions where the protein is being phosphorylated and where it is not being phosphorylated. Identify in both cases they interacting proteins by MS and then follow up if there are any kinases that are more readily identified in the former than in the latter case.

Good luck

Hi Mario and Adam,

I am unable to purify the full length protein. I can purify the domain that gets phosphorylated but it is disordered. When I tried co-IPs before with cell lysates, the disordered domain was degraded even though I used protease inhibitors. Would co-IPs with purified kinases have the caveat of false positives and false negatives as well?

That is too bad. Rather than using purified kinases I would try some ways to reduce the protein degradation. You can increase or change the protease inhibitors you use. Make sure that you are working on ice at all times. And finally, the IP can be done very fast if necessary (less than an hour) instead of the overnight incubation that many people chose to do.

Deleting PEP4 gene may reduce the degradation of your protein, but there's no gurantee.

I really don't have any additional comments to make to what was suggested above.  I assumed that your inhibitor concentrations were effective and that you did all the steps on ice or in cold room to prevent protein degradation.  I would follow Mario's advice.  You may want to do a "titration" for the effective  amount of protease inhibitor required for your experiments.  It is a difficult situation and you may have to twick conditions as you go through suggested steps above.  Good luck.

You can try using a siRNA library targeting most of the protein kinases if you have a cell reporter system for your interested protein. But, this can be expensive.

Thank you all! I will try to tweak my IP experiment to reduce protein degradation.

You mentioned that you know already 8 candidate kinases for your substrate - in case your protein is Y-phosphorylated (or in case there is a site specific p-antibody) you could just overexpress each of the candidate kinases and check via pan-PY (or site specific) western which one induces a strong phosphorylation of your protein. As overexpression experiments should only be interpreted cautiously the next step would be to knock down the identified kinase via siRNA and check whether this reduces/abolishes the phosphorylation of your protein of interest.

Thank you Daniel, this was something I considered doing but I am under the impression that kinases are somewhat promiscuous and if you knock down one another might pick up the slack, so I am not sure high informative this would be. I am new to the kinase field though so please correct me if I am wrong. Also, is there a reason why you suggest using siRNA instead of just getting a yeast knock out strain (since my protein is from yeast)?

Thanks so much for the help!

Natasha, you are basically right, it is not uncommon that other kinases might step in for another one being inhibited or absent. But in a lot of cases one can see a significant difference especially in a specific phospho-Western even while the phosphorylation may not be completely abolished. And in combination (increased signal when kinase overexpressed, decreased signal when absent) such results may be quite conclusive. I am not sure about a ko. strain - compared to siRNA it has more time to compensate the loss of your kinase of interest (i.e. upregulate alternative pathways) but this is really dependent on the regulatory mechanism involved and their respective timing. In itself the suggested experiment may not be sufficient to identify one kinase undoubtably but it could give a significant piece of information.

As a preliminary experiment, you could use a kinase inhibitor library (targeting ~100 kinases or more). Even though inhibitors are not the best approach regarding specificity, you could find a few candidates that you can start with and rule out some others. Obviously you would need to confirm any result with inhibitors by using single/double knockout approach. The co-IP followed by MS would give you a list of candidate kinases that interact with your protein of interest in the physiological conditions that you previously determined. And the MS result would have to be confirmed by co-IP/Western Blot. If applicable, you can also perform in vitro phosphorylation assays. Best of luck!

 Hi Daniel, this is very informative for me, thank you. One more question: in order to undoubtedly identify a particular kinase, is it just a matter of consistently showing by various in vitro and in vivo methods that it is able to phosphorylate a target under pertinent conditions but unable to phosphorylate the target otherwise?

Hi Natasha, indeed if you can present data showing your candidate kinase phosphorylates your target in-vitro and also in-vivo (and even in different experimental settings) this would demonstrate that your target is a substrate for your kinase. The next step would be to demonstrate that whatever phenotype should be connected to that phosphorylation can be influenced by the modulation of your kinase - either by a specific inhibitor (if available) or knock-down/out approaches. You could also mutate the target-phosphorylation site (if identified) - if this reproduces the kinase-ko phenotype you would have another evidence for your proposed pathway.