cGMP activates PKG. What attributes of "activated" PKG can be detected by IHC? For example, the attached paper mentions that binding of 2 cGMPs to PKG, releases the autoinhibitory domain of the R subunit domain from its interaction with the catalytic cleft of the C subunit domain. Thus, such a binding promotes autophosphorylation. Indeed, "activated" PKG is autophosphorylated. On the other hand, autophosphorylation can also lead to activation. A Similar mechanism has been suggested for PKA activation.
Does this mean that phosphorylated PKG (ser79 phospho-PKG) is actually an "activated" PKG? Is their any antibody available that detects this phosho-protein?
Alternative approach: If I use cGMP antibody for immunostaining and find that cGMP is abundantly expressed in experimental condition when compared to the control condition, can I infer that PKG is more likely to be activated in experimental condition? Can cGMP abundance be used as a surrogate marker for PKG activation?
as an alternative approach, you could try an immunohistochemical approach using an antibody against the phosphorylated form of a known, specific substrate for PKG.
Thank you, Bruce. Indeed, we do observe phosphorylation on a target protein. This protein and its phosphorylated site is also a target of some other kinases (shared target). That is why we wanted to see if PKG is in its activated form.
You should have a specific antibody for PKG itself. The target protein or the specific substrate may be tissue-specific.
You will not likely be successful detecting activated PKG by IHC or even western blot of proteins . Unlike PKA, PKG does not disassociate with its regulatory subunit. Additionally, the consensus site of PKG is very close to and able able to phosphorylate PKA target sequences, so antibodies are not terribly useful looking at phospho-peptides. You might want to look at the activation of a G. Cyclase upstream and choose a known PKG target downstream. The combination of this data would strongly infer PKG activity. The problem that wil remain will be PKA and cAMP. cAMP can activate PKG andcGMP can activate PKA. Furthermore the consensus sequence can be phosphorylated by either kinase
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