Kinase activity of the mutant receptors. (
A) Tyrosine
phosphorylation of the mutant PDGFRs. NIH 3T3 cells expressing an empty
vector (EMP) or the indicated mutant receptors were serum-starved and
exposed to either buffer (−) or 50 ng/ml of PDGF-AA (+) for 5 minutes.
F cells expressing the human αPDGFR (Fα)
11 were
processed in parallel and served as a positive control. Cells were
lysed and immunoprecipitated with the 292 antibody, which is
primate-specific, and recognizes an extracellular epitope. This
antibody selectively recovers the mutant receptor. Immunoprecipitates
representing approximately 1 × 10
6 cells
were first subjected to anti-phosphotyrosine (P-Y) Western blot
analysis (
top panel), the blots were stripped and then
reprobed with an anti-αPDGFR antibody (
bottom panel). The
data presented are representative of three independent experiments. IP,
immunoprecipitation; IB, immunoblot (Western); the abbreviations for
the receptor mutants are detailed in the legend of
Figure 1 .
(
B) Kinase activity of the mutant receptors. The mutant
receptor, immunoprecipitated with the 292 antibody, was incubated with
2 μg of GST–PLCγ, an exogenous substrate, and[
32P]-γ ATP in an in vitro kinase assay, as
described in the Methods section. The proteins were resolved by 7.5%
SDS–PAGE, and the gel was dried and exposed to film. A portion of the
resultant autoradiogram is shown, and the
arrowhead indicates the position of GST–PLCγ. In at least three independent
experiments, the T665M and V858M mutants showed substantially elevated
kinase activity, compared with either the other mutants, or the WT
receptor.