To rule out GSK-3, you can treat the cells with lithium (10 mM) for an hour or one of the small molecule inhibitors such as kenpaullone, BIO or TDZD (uM concs). All inhibitors block both isoforms similarly (there are no isoform-selective GSK-3 inhibitors). Unfortunately (for you), predicted GSK-3 sites are the most common in proteins (see supplementary figure 4 in http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692296/ due to the relatively poor selectivity of the preferred "motif" which is basically Ser/Thr-Pro although many substrates require serial phosphorylation Ser/Thr-X-X-X-Ser-P/Thr-P.

The requirement for another "priming" kinase to phosphorylate a substrate before GSK-3 can target it can complicate analysis. For example, CIP will likely dephosphorylate all sites and therefore adding back GSK-3 + ATP in vitro will result in no phosphorylation. This is also true for proteins expressed in non-mammalian cells where the priming kinase may be absent.

Is it phosphorylated for sure....or an isoform.

A first issue will be for you to nail down the conclusion that the protein is indeed phosphorylated. Besides deducing this from size differences on an SDS gel, it may be good to test for the phosphorylation itself via metabolic labeling, using a phospho-specific protein stain on membranes with IP purified protein or simialr technique. Better yet, you could develop a phospho-specific antibody against this site although usually this is adviseable once you have confirmed the phosphorylation via other techniques. As far as identifying the phosphorylating kinase using kinase inhibitors, this is probably a good approach. In this type of screening, the readout is very important (will you use your 2 band readout, or will you go for a phospho-antibody?) as well as the selection of compounds. If your limited in the amount of test conditions (probably it's the case if you use western), then you would have to select a panel of inhibitors with sufficient coverage of the kinome (thus rather broad spectrum inhibitors) then go into inhibitors targeting more specific classes of kinases. From there, it would then be adviseable to also use molecular techniques (kinase knockdown, overepxression) to confirm the findings from pharmacological approaches. I hope this helps.

The company I am working for offers a screening service called KinaseFinder which should be exactly what you are searching for. The panel comprises currently 229 human recombinant serine/threonine kinases (94 Tyr-Kinases are also available but most probably not of interest for you at the moment). If you are interested please have a look at http://www.proqinase.com/content/view/90 (there are still some lower numbers for the kinases mentioned, we are just in the process of upgrading that service).

Daniel

I am not sure that an array (RNA??) is apporpriate for screening for the kinase you are looking for. The conclusions from your predictions either point out the wrong type of Kinase or your inhibitors are not working,

You might try different type of coktail inhibitors and zoom down.

Good luck

Have you tried assessing cell conditions that alter the phosphorylation (as judged by the shift on SDS-PAGE)? However, this would be difficult if the protein migrates largely as the slower band - that is, is largely phosphorylated. There are many cellular stimulants that can help narrow your putative kinase focus. Wheatgerm in vitro translation of substrates usually produces apo/unphosphorylated proteins which can then be tested with kinases to assess for shifts (including screening extracts). Lastly, you may have complex phosphorylation occurring in which one site which doesn't cause a gel-shift is required for phosphorylation of another site that does. There are examples of this such as casein kinase-2 and GSK-3. This vastly complicates testing in vitro with purified kinases or kinase arrays. Also, remember that many arrays do not necessarily present the kinases in an optimal state such that negative data doesn't necessarily exclude a particular kinase.

Excellent comment by James - to follow this line of thought - even a positive result from an in-vitro screen with recombinant kinases does not necessarily mean that the respective kinase is the actual physiological kinase targeting your substrate in-vivo. Such biochemical in-vitro assays are always highly artificial and results have to be verified by in-vivo data! Nevertheless they have the potential to give you a good starting point, if more direct methods like co-IP are not successful.

Thought of an additional avenue which would be to knockdown expression of putative kinases in a suitable cell line and test for reduction of the mobility of your target. An important caveat is that there is a lot of redundancy among kinases (they are expertly designed to foil our efforts and to extend PhD studies!) so intelligent grouping of RNAi/shRNAs is recommended. However, no one technique is foolproof and since most interesting phosphorylation events are equilibria, perturbing the on-rate by reducing even a fraction of the kinases responsible may be detectable. RNAi/shRNA screens work best when looking at collections and several companies have kinase-focused libraries available (you "only" need the 390-odd serene/threonine kinases).

To follow-up on Jean-Marc's point, if you don't want to go to the trouble of making a phospho-specific antibody, you can take the old school approach of doing the phosphorylation in the presence of 32P-ATP. Then you can easily confirm the transfer of the radiolabel to your band -- and the lack of it in the serine mutant.

As for which kinase, it is possible that more than one kinase can phosphorylate the site. First, I would do some datamining on the site itself and look for candidates. Second, I would see who currently carries a pack of screening compounds for kinases. I used to use a lot from Calbiochem, so I would look there and places like Tocris first.

Hope that helps a little.

Have you considered doing a mass spec instead for identification....as unknown kinase identification would be much more easier that way....and kinase arrays are expensive and you sure will have to revalidate your finding if you do an array for identification

you can try Ambion Silencer kinase siRNA library

good luck!

Thank you for all the reply...I am attaching an image ...it will show why I predict the band shift as phosphorylation.

I found two kinases which are predicted to phosphorylate the serine at that site and used those inhibitor...but no change in the upper band observed.....thats why I am looking for a screening method to screen the possible kinase.

@Rikhia Chakraborty ...........Thank you for your reply...I haven't used mass spec for the identification......so can u tell me how to use it to find kinase ??

@James ... GSK3 is one of the predicted kinases....can u suggest me how do I narrow down the kinases??

another point regarding the inhibitor experiment: Did you do in-vitro experiments or did you treat cells with the inhibitor and isolated the substrate protein later? If you did it in cells, two things should be considered: Make sure the inhibitor actually works in cells at the concentration you applied, because there are often large discrepancies between in-vitro and in-vivo IC50 values and it is not always trivial to find out which model system was used to determine the values.

Another point is the possibility , that even while you succeeded in inhibiting GSK3 (alpha or beta?) another kinase could also target the same site stepping in for the reduced or absent GSK3 activity.

Hi, Satadru,

You may also want to consider expressing a kinase-inactive mutant version of your protein, just to exclude autophosphorylation

To rule out GSK-3, you can treat the cells with lithium (10 mM) for an hour or one of the small molecule inhibitors such as kenpaullone, BIO or TDZD (uM concs). All inhibitors block both isoforms similarly (there are no isoform-selective GSK-3 inhibitors). Unfortunately (for you), predicted GSK-3 sites are the most common in proteins (see supplementary figure 4 in http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692296/ due to the relatively poor selectivity of the preferred "motif" which is basically Ser/Thr-Pro although many substrates require serial phosphorylation Ser/Thr-X-X-X-Ser-P/Thr-P.

The requirement for another "priming" kinase to phosphorylate a substrate before GSK-3 can target it can complicate analysis. For example, CIP will likely dephosphorylate all sites and therefore adding back GSK-3 + ATP in vitro will result in no phosphorylation. This is also true for proteins expressed in non-mammalian cells where the priming kinase may be absent.

Hi Satadru, have you done the reverse expt - i.e. taking your dephosphorylated substrate and phosphorylating it with physiological amounts of the two kinases you think might be candidates, to see if you recover the upper band again?