You are studying a receptor tyrosine kinase and want to investigate the nature of the
autophosphorylation of this protein. To do this, you construct three forms of the receptor in an
expression vector: 1) a normal form with an active kinase domain and one tyrosine that is
phosphorylated upon activation, 2) a “kinase-dead” mutant that is larger than wildtype and carries an
inactivating mutation in the kinase domain, and 3) a truncated form that has an active kinase domain
but is missing the tyrosine residue (see Figure below). You express these forms in a cell line that
lacks expression of this receptor (that is, there is no endogenous receptor at the plasma membrane in
these cells). To test whether cis or trans phosphorylation occurs, you express the forms singly and in
pairs (see lanes on gel below). You then treat these cells with ligand and radioactive ATP (32P on the
γ-P) and then immunoprecipitate the receptor proteins from a cell extract. You analyze expression of
the receptors by immunoblotting with an antibody that recognizes all three forms of the receptor and
you detect phosphorylation by autoradiography. Your expression results are shown in the first blow on the attached document.
What would you expect of the autoradiography results in the receptor autophosphorylates in trans (cross-phosphorylation) and then in cis (phosphorylation of same molecule)?
Essentially, I'm not sure how to go about solving this problem. I couldn't find a lot in my book about it so any clarification would be really appreciated.
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