Hi,

I know the initial event of a pathway (i.e. inactivation of a kinase by dephosphorylation) and the end result (i.e. inhibition of apoptosis). However, I would like to figure out which proteins are involved in between the initial and the final events. The initial kinase has several targets and I would like to know exactly which proteins and pathway is modulated.

 

Thanks!

This is not a simple question and there is no perfect way to do this. Its actually very difficult. In the past I have worked on signaling pathways and used numerous approaches with varying degrees of success.

 

For instance, you can try to do an immunoprecipitation of your kinase. If it forms any stable complexes or if you can successfully crosslink it with interacting proteins, then you can pull those down with your kinase and identify them by mass spec. You could use a 2D gel to try and identify phosphorylated proteins in the presence or absence of your kinase and try to identify these by mass spec. If you work in a system that is easily mutable, you can always do mutant screens like suppressor screens, enhancer screens, etc.

Yeah, a suppressor mutant screen would be a pretty good way to start depending on your model organism! I also agree with chadn737 that a pull-down of your kinase is something else you can start with.

 

But if you have a good idea of what type of protein you are looking for, you could try to use bioinformatics to predict a set of proteins that are putatively involved, and then follow up with knock-outs or knock-downs to see if they are actually involved in your pathway. You might also consider a yeast-2-hybrid screen for interaction partners. It may prove useful to take a look at this article: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0065011

 

Additionally it sounds like you have some predicted targets for the initial kinase, so if you know anything about how these proteins are specifically modified by the kinase, you can try to analyze phospho-negative mutants (mutate the phosphorylation target serine/threonine to alanine for s/t kinase or tyrosine to phenylalanine if the kinase is a tyrosine kinase) or phospho-mimic mutants (by mutating serine/threonine to aspartate or glutamate, if your kinase is an s/t kinase).

 

Hope that helps! It would be nice to know what your model organism is, too. That might help determine what your plan of attack should be.