Sequential phosphorylation in vivo but not in vitro - (Feb/16/2012 )

I´m working on a protein that becomes phosphorylated following DNA damage. From in vivo results using phosphorylation mutants we can say the phosphorylation is sequential at two sites.
We identified the kinases acting on both sites using kinase inhibitors and Ipp.

My question is, how is it possible that when I perfomed in vitro kinase assays using the kinase acting at the second phosphorylation residue I get a phosphorylated band although the first residue is not modificated (the phosphorylation is sequential as concluded by in vivo results).

Thank for yout time.

Did you have the first phosphorylase in the in vitro mix? If not - then you won't see phosphorylation of the first residue. The sequential nature probably isn't significant for functional purposes outside of the cell, but is critical for some function within the cell.

Thanxs for your reply!
I didnt have the first phosphorylase in the in vitro mix, so I´m looking for an explanation about that, how is it that in vivo the phosphorylation is sequential but in vitro the second phosphorylase doesnt need the first modification to act, what can be happening in vivo is my question.

bob1 on Thu Feb 16 21:49:04 2012 said:

Without permissing first phosphorylation, second phosphorylation may occur to lower extent; only second-site phosphorylation may have different result in kinase properties than first+second-site phosphorylation;

For first site phosphorylation you may need, f.i. second messenger activation; if it is blocked or missing, second-site phosphorylations increase

are the phosphorylations which you are analyzing mainly cis- or trans-phosphorylations?

Inmost sun on Fri Feb 17 14:51:59 2012 said: