I am doing some work on type 1 transmembrane receptors and I have been using some bioinformatic tools to analyse possible protein domains present in them and one of the things that has come from these searches is the high probability of an ERK D binding domain being present in the extracellular part of the protein. I know in silico analysis does not provide proof of a domain being present but I was wondering if anybody had any thoughts or research papers which looked at a protein kinase being bound to the extracellular region of a transmembrane protein?
I don't have any experience with the subject, but was curious about the question. These might serve as a starting point.
http://stke.sciencemag.org/cgi/content/abstract/5/255/re7
http://www.sciencemag.org/content/336/6085/1150.full
Did you try to find out whether this extracellular domain is really extracellular; as I am facing similar issue with my protein but I am willing to tag this extracellular loop and try to determine whether it is really extracellular!!!
Uniport database postulates the domains of the membrane proteins, you might find through it that this binding domain in intracellular!
A kinase called Four Jointed phosphorylates the extracellular domains of the Fat and Dachsous cadherins. See paper in Science from Ken Irvine's lab. But your kinase needs to be able to enter the secretory pathway.
If you receptor is internalized for trafficking or other purposes it may be phosphorylated in the intracellular compartment.
Although the idea of kinases working in extracellular spaces has never received a lot of traction, kinases that are normally intracellular have been detected in the extracellular milieu. PKA is one such example (see PNAS 2005, 102:4718-4723). Likewise, the existence of proteins phosphorylated in their extracellular domains is a well-established fact (see review in TIPS 1999, 20:453-459). Four-Jointed is in a different class: Fj is present in the lumen of the Golgi, just like glycosyl transferases. Thus, it phosphorylates proteins as they traffic through the secretory pathway.
As to the question posed here: the presence of potential ERK docking domain by itself is not good enough, not even to predict that this domain binds ERK. So, the answer in short is: first you must decide what you want to do with this information. If you are willing to pursue it, start by showing that there is a phosphorylation site in the extracellular domain of your receptor doing some wet lab work. To me, that is one of the issues with bioinformatics-only approaches: too much information oft leads to too much confusion :)
The question you raise is intriguing and potentially of high interest.
Do you think that any of the elements of your extracellular interaction may be tagged in live cultured cells with a specific antibody and then rapidly fixed to detect the antibody itself (which certainly does not enter the membrane) ?
In summary:
1) on the extra-cellular side of the membrane has been found a putative site for phosphorylase on a protein.
2) the extra-cellular phosphorylases are well documented
3) outside the cell exists ATP or GTP in sufficient quantity to phosphorylate a protein, so there is a great probability that the putative site has a real biological significance..
4) in general, the phosphorylated site is not always plentiful so you have to do some enrichment
5) because often transmembrane receptors have extracellular protein segments not so long (up to 40 - 50 aa), you can try to synthesize the peptide and use it to see if it is phosphorylatable. It could be an intermediate solution but useful to go deeper(nothing says that this phosphorylation also happens in vivo).
I know that this is an old question - but wondering if you were able to identify any examples of such an interaction. We are studying a transmembrane protein that we think may traffic to the cytosol and interact with ERK - so this question is of great interest to us as well. Thanks!
This in fact is a very old question and still controversially discussed. The first ever reported secreted phosphorylated protein casein for example was found around 130 years ago and the kinase responsible for it only 4 years ago. I have been working on this question for the last few years. We have summarized the topic in the following articles and some unpublished manuscripts will hopefully follow soon:
http://stke.sciencemag.org/content/5/255/re7
http://onlinelibrary.wiley.com/doi/10.1002/cam4.368/full
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427915/
While the topic is interesting and may be a game changer, if proven true in a physiological setting, key data sets are missing to exactly show this. It is a tough question to unconditionally show the significance in vivo as there are many challenges and hurdles to overcome.
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