The 2 hybrid system?
toneil1 at gl.umbc.edu
Thu May 15 15:40:13 EST 1997
On 15 May 1997, Mr. G. Morley wrote:
> Hi.. I was thinking about using the two hybrid
> system to help me to identify a protein that assosciates/binds
> a putative binding site of a tyrosine kinase receptor.
> The problem is I have heard that yeast do not utilise
> phosphorylated tyrosines as part of their signaling pathways
> (except during mating?).
> I was wondering if:
> a) this was true.
> b) if this would affect the 2 hybrid system (presumably it would
> as if the expressed protein is not phosphorylated it will not bind)
> c) Does anyone know where this is mentioned/reviewed?
> Thanks in advance
> Gary Morley
> gmorley at rpms.ac.uk
Some receptor tyrosine kinases become phosphorylated on tyrosines when
using the two-hybrid system. We have used the cytoplasmic domains of
both the insulin and IGF1 receptors and have gotten great phosphorylation.
Moreover, we have been able to make site directed mutants of various
tyrosines and these have interacted with substrates as would have been
predicted from the literature. We believe that the multiple lexA operators
in the lacZ reporter plasmid (Brent system) allow for the fusion proteins
to trans-autophosphorylate each other as if they were in the cell
membrane. For a reference I suggest:
O'Neill, TJ , Craparo A, and TA Gustafson.1994. Characterization of an
interaction between IRS-1 and the insulin receptor using the two-hybrid
system. Mol. Cell. Biol. 14:6433-6442.
Hope this helps, Thomas J. O'Neill
More information about the Methods