Do you have Kd data for either or both states? Phosphorylation could have just slowed the rates of interaction but not the binding affinity. It is going to take more than pull downs to suggest this mechanism.

Indeed as Andrew said you should probably try out something fancier like surface plasmon resonance. At least it should be successful now that you know from pull downs that you have an interaction.

Have you checked if this phenomenon varies as a function of pH? Might be relevant as phosphorylation most often introduces a new charged group to the protein.

Thank you for your answers.

We really think that phosphorylations promotes interaction because more than 10 sites are involved in the interaction and when we mutate them one by one we clearly see a decrease of the avidity. Moreover, we have strong in vivo data that reinforce this model. Unfortunatly, we do not have better experiements that pull down, so we do not have acces to such values as kd ect.. We are working in the pH of our animal model (C. elegans embryo).

I think you can support that phosphorylation promotes interaction but that isn't the same as saying that they aren't needed to maintain it. You should either work to do the in vitro experiments or figure out a way to monitor the modification state and its interaction in vivo in real time (difficult).

The only other thing I can think of you could try is to to measure the rate of elution off of the pull down bead with and without out phosphatase. This could take days and if you protein isn't stable that long, it won't work. If the rates are the same you could suggest phosphorylation is possibly increasing the on rate and that is driving the complex formation.

good luck.

What kind of phosphorylation is this. The usual one is an ATP dependant reaction which requires Mg2+. If so you could try with EDTA or minus Mg to kill the reaction and also titerate with phosphate ions seperately. This will also give you infromation on whether you need a covalent phosphate or whether a non-covalent phosphate is sufficient. Removing any ATP,, phosphate or Mg will tell you if there is strong protein-protein affinity alone. I know that for autophosphorylation in histidine kinase the interacting surfaces are large and in your case you have 10 aa that affect binding so you also have large surface areas which can result in large gibbs free anergy changes which can also result in strong binding. The mechanosm may therefore require protein-protein interactions first before phosphorylation.

If it is just a phosphotranspher between proteins then you need to also try phosphatase treatment on the seperate proteins then try binding them.

Try this line of thought: Prior to phosphorylation, one, or both of the proteins are sequestered or bound by other regulatory proteins. Upon phosphorylation, they are released from the regulatory complex, or translocated, or something that brings them into contact with each other. The interaction may not require phosphorylation, but their mutual availability to start interacting may require phosporylation.

Alternative line of thought: Have you ruled out the possibility that other proteins are involved in the binding. Perhaps other proteins are in the complex and maintain the interaction after phosphatase treatment.

Dear all,

Thank you so much for your feeback.

I have to mention that, we performed pull down assay using higly purified proteins, phophorylated by cdk1 or not. In this reconstitued system, we show that other proteins are not involved in the interaction and that the interaction is totally phosphorylation dependent.

Nico

Maybe you have enough to go on already. But consider designing an experiment in which all sites are phosphorylated (measured stoichiometry of 10 sites) or psuedo-phosphorylated (10 sites mutated to D/E) and compare the binding to when all sites are dephosphorylated or not able to be phosphorylated (10 sites mutated to A). Maybe you have done this and I didn't understand, but your information supplied is limited. Are all your sites cdk1 substrates? If not, you don't have the full picture and you need to mutate. Rather than mutate all 10 we prefer to order the DNA mutated. If you do this then I think might find that binding requires dephosphorylation. Often the simplest explanation is correct. A time cource of binding is also sometimes required to see subtle effects (e.g 10 min, 1 hr, 3 hrs, 12 hr).