Dear Yang, some kinases are active when over-expressed, some others need additional activation / mutations / co-expression of upstream factors for showing detectable activity. We have seen that in some cases co-transfection of some substrates could also activate certain kinases (directly or indirectly). Some substrates could also be heavily phosphorylated just by over-expression independent of whether you co-express a kinase or not. It will be very difficult for anyone to speculate without knowing the identity of your kinase. 

Good Luck: Manoj

I agree with Manoj. Are the kinase and respective substrate expressed endogenously in the cell? If it is, you may even try to express the kinase first and monitor the endogenous substrate. 

Hi yes I agree with the above - some kinases (including trans-membrane receptor tyrosine kinases) can be activated by over-expression in the absence of their ligands (I have personally seen/done this).

When people perform an in vitro tyrosine kinase assay the kinases are activated when they are precipitated.

So there are two possibilities a) expression of substrate and kinase in same cell does not cause phosphorylation of the substrate in the absence of a ligand and b) the over-expression of the kinase causes activation which will lead to substrate phosphorylation.

I would suggest, when you make your double-transfectant you stably transfect and select clones (with the expectation that the clones expressing lower levels of the kinase won't have the kinase illegitimately activated).

However to make the double transfectant perform it step-wise (so generate kinase over-expressing first). You can, as suggested above, then IP endogenous substrate and see if this is being phosphorylated before proceeding to transfecting in the substrate.

I hope this makes sense?

Gary

There is always a baseline level of constitutive activity between a kinase and its substrate depending on the cellular environment so it is quite possible that if the conditions in the cell allow there could be phosphorylation.

Thank you so much, everyone!

The reason why I want to try this "in vivo phosphorylation" is that I'm not sure the kinase involved phosphorylates the substrate directly.

The substrate is not expressed in HEK293 cells and by overexpression of the substrate alone I can see a low level of phosphorylation in WB.

For the co-overexpression of the substrate and kinase, I would use pBudCE4.1 (which has two promotors to express two genes simultaneously, Invitrogen) so that the substrate and kinase genes can be transfected to HEK293 at the same time.

Though over expression of the kinase may have an effect of HEK293 cell growth, I'm expecting to see a phosphorylation of the substrate with overexpression of the kinase simultaneously.

Finally, I will do an in vitro phosphorylation the reaction condition of which is well controlled.

Anyway, I'm going to have a try. 

Will be good to have a bonafide substrate as control under similar settings to make sure that co-expression of your kinase is enough  to get enhanced specific-kinase activity. Good Luck.