We have tried IP for total protein detection using the p-Tyr antibody, but we can only occasionally detect a signal. Does the fact that the proportion of PDK1 that gets tyrosine-phosphorylated extremely small, mean that you have to use cells that overexpress PDK1? There are WB antibodies that detect PDK1 only when it is phosphorylated at Tyr9, but none that specifically detect PDK only when it is phosphorylated at Tyr373/Tyr376. Any suggestions?
First off I would question the fundamental hypothesis that PDK1 is tyrosine phosphorylated. However, if you are convinced that previously published data is convincing and that the pY on PDK1 is turning over rapidly, you could pre-treat the cells for 30 minutes with Sodium Orthovanadate (make sure you get the correct ionization state!) in a range from 100microM to ~1mM and then add your stimulus. Over-expression of PDK1 might help you explore the importance of pY-PDK1 in pathway regulation but I would suggest that you obtain PDK1 null cells to do those experiments. Bear in mind that over-expression can sometimes allow for events that do not occur on the native protein so assessing this on endogenous PDK1 is essential. Hope that helps.
Hi:
You may need to follow your own data. Your data showed that pTyr phosphorylation stoichiometry of PDK1 is very low (consistent with everything that I know about PDK1).
You need to decide how it is possible for a pTyr site with very low stoichiometry (say 1/100) to affect the activities of PDK1 (known to be constitutively phosphorylated at Ser241 at high stoichiometry, say 1/2).
PDK1 from recombinant and endogenous sources were characterized by Alessi's group in great detail. They essentially found that PDK1 is a constitutively active kinase due to constitutive auto-phosphorylation at T241 (Mora, A., Komander, D., van Aalten, D.M. & Alessi, D.R. PDK1, the master regulator of AGC kinase signal transduction. Semin Cell Dev Biol 15, 161-70 (2004)). I have not seen many believable cellular modifiers that affect PDK1 catalytic activity without affecting T241.
If PDK1 is catalytically constitutively active, the only mode of regulating PDK1 enzyme in cells would be through selective localization or protein binding partners. It may be functional relevant to find a sub-population of PDK1 that had increased pTyr signal stoichiometry.
So far, your own findings suggest that pTyr is not relevant to PDK1 function.
tung
Just an additional comment. There is evidence to suggest that phosphorylation of C-RAF on either tyrosine 340 or 341 (in the SSYY motif upstream of the core kinase domain) is important for regulation of C-RAF kinase activity downstream of either RTK, SRC or JAK family PTKs. However, as you propose for PDK1, the stoichiometry of pY on those sites on endogenous C-RAF after growth factor stimulation is very low indeed (see papers by Debbie Morrison, Richard Marais, Catrin Pritchard and others on this topic). Hence, it remains somewhat controversial as to whether pY on those sites on endogenous C-RAF is or is not important for C-RAF regulation despite an abundance of strong circumstantial evidence that they are. Hence, you may find yourself in the same situation with PDK1.
if there's no pdk1-pTyr antibody you can IP with anti-PDK1 and then do western with pan pTyr antibody.
Dear Thomas,
If I understand you correctly you are performing an IP with PY20 and then looking for PDK1. If so, and your suspicion that PDK1 phosporylation at Tyr373/6 is low, then I would IP for PDK1 and western for PY, simply because the fraction of PDK1 in a PY20 IP would be miniscule. If you are looking for phosphorylation at TYR373/6 alone, then this strategy might work, but you also pick up phospho TYR9. Over expression of PDK1 might help you perform the experiment, but might mess with the normal stoicheometry of endogenous PDK1 containing complexes meaning the results might not reflect normal function, as Martin suggested.
You could always (I say "you could always"-this is a fair amount of work!) mutate the Tyr sites in PDK1 to unphosphorylatable or phospho mimetic sites, knock down endogenous PDK1 and transfect in your constructs one by one and use a functional assay to determine the effect. This would be a lot of work and I would think long and hard about how important the answer is to you, and even if the strategy works it still might not give you the answer.
Hej Thomas,
I suggest that you immunoprecipitate PDK1 with pan- PDK1 and then run p- PDK1 WB. many Phospho antibody fails to pulldown / Ip total protein due to too little phosphorylated protein or simply the ab does not work for IP. If there is too little phospho-protein in the cell then I recommend you to run insitu detection using PLA. choose two different antibodies from different speices where one recognizes phosphorylated PDK-1 while the second total PDK. you can contact me so we can discuss this.
Goodluck
Echoing Tung and Martin, you should question the relevance of tyrosine phosphorylation of PDK1. It can certainly be detected if you treat cells with tyrosine phosphatase inhibitors such as pervanadate but the usual stoichiometry is very low. Just because you can detect something, doesn't mean its important - and this is particularly relevant to phosphorylation. Much of what can be detected, I suspect, is the equivalent of collateral damage of kinase (lack of) specificity vs the need for nature to ramp up effective signal transmission within seconds of an agonist engagement. Kinases are akin to Bren machine guns. Loud, pump out a lot of lead in a short time, but often spray/miss their targets.
Hi Jim
I suspect that one has to be of a sufficient vintage (or read a lot of Commando books) to appreciate the analogy of protein kinases to Bren guns. Personally, I rather liked it -- but I read lots of Commando books as a boy......
Hi Martin (fellow closet commando),
Maybe a super-soaker is a more apt analogy :)
Jim
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