High baseline phopshporylation - How to lower the baseline before stimuli? (Jun/10/2007 )

Hi,

We are investigating a RTK recptor which we overexpress in cells. When Western blotting for tyrosine-phopsphorylation of the receptor the signal in unstimulated condition is as high as in the ligand stimulated. This is true for both neuronal celllines and fibroblasts.

The protocols we have tied are basically starvation in serum free medium or 0.2-0.5% serum medium over night or longer. Then the ligand (or no ligand) are added to cells. Cells are washed in PBS and lysed for WB. The phopshotyrosine-blots show a similarily high signal in all conditions!

Any ideas? Have anyone tried to stimulate in PBS rather than medium?

Other ways to decrease baseline to get a clear ligand induced phoshoprylation of the receptor tyrosines?

Thanks a lot!

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there should be some more analysis in put;

overexpression of RTK does not necessarily mean that all inserted to the plasma membrane; try to demonstrate the subcellular localization by ICC;

transregulation by other RTK´s may occur; basal phosphorylation events occur even in unstimulated cells;

RTK have multiple Y-phosphorylations sites, f.i. to induce dimerization;
try to discriminate the quality of various Y-phosphorylations to get the difference between unstimulated and stimulated RTK´s

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Hi and thanks!

This is not a new RTK for us and most of the above points have been sorted out. The problem is most certainly due to overexpression related auto-dimerization. I realize that a lower expression level could be suitable for solving the problem but that is unfit for other resons (co-IP). Hence, any cultural/stravation conditions that may reduce autophosphorylation/ dimerazation despite the overexpression is most welcome.

Not easy, i know

Thanks again,

/Kallun

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the high abundance of expression increase the conc of RTK whcih also works as a substrate in cisphosphorylation

the only idea I have is to co-express the appropriate PTP to limit RTK cisphosphorylation; to gear the activity of PTP, it should have an inducible off-promoter f.i. tet-off system

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That is a very good idea! Alternatively perhaps add low concenterations of a phosphatase prior to ligand and the hope that ligand addition will overcome the phosphatase and activate the receptors whereas no-lignad obviously will not.

Thoughts?

/Kallun

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am I right? you like to add a phosphatase outside to your cells? but phosphorylations of RTK work at their endo- not at their ectodomains; as I understand you like to diminish your basal RTK (cis)phosphorylations, and you are already working with minimal medium which means offering no ligands; so you will not decrease cisphosphorylation from outside;

another possibility would be to lower overexpression RTK; you may select stable transfectants and find a clone with medium or low level-overexpressed RTK with possibly reduced basal phosphorylations...

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Well you are correct in your biology of course. Many phosphatases in peptide form however do readily cross membranes in solution. I have not thought of that idea before, just came up as an offspin to your (very good) suggestion above.

In the end I guess a titration of expression levels allowing for sucsessfull co-ip but reducing cis-dimerization will have to be the cumbersome way to go ahead.

In all cases I enjoy the discussion!

/Kallun

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what do you mean by "phosphatases in peptide form"? even truncated form reduced to the active site would be too large to cross the membrane;

you may succeed with titration with less DNA but try in parallel a selection of stable-expressing clones; in the beginning, it is very less work...

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