Fig. 5. Effect of the catalytic activity and autophosphorylation of PYK2 on
cytoskeletal reorganization. (A) Swiss 3T3 cells were microinjected with
plasmids encoding Myc-tagged PYK2 (PYK2-WT), catalytically inactive PYK2
(PYK2-KD) or the autophosphorylation mutant (PYK2-Y402F) and stained with
antibodies against PYK2 (polyclonal, UBI) and vinculin (monoclonal, Sigma).
White arrows indicate the microinjected cells. Bar, 50 µm. (B) PYK2-WT,
PYK2-KD and PYK2-Y402F protein were immunoprecipitated from transfected HEK293
cells and subjected to SDS-PAGE or to an in vitro kinase assay using
GST-paxillin N-terminus as a substrate. (C) Increased tyrosine phosphorylation
of several proteins including p130Cas was observed in
PYK2-expressing cells. HEK 293 cells lysates expressing Myc-tagged
PYK2-WT and PYK2-KD were either directly immunoblotted with antibodies against
Myc and phosphotyrosine (RC20, Transductions Labs) or immunoprecipitated with
antibodies against p130Cas (Transduction Labs) and immunoblotted
with antibodies against p130Cas and phosphotyrosine. Arrows
indicate proteins with increased tyrosine phosphorylation in PYK2-WT
expressing cells. (D) Histograms summarizing results in (A). These show the
percentages of means ± s.d. of three or more different samples (total
of 200 microinjected cells). The asterisk indicates a P value of
<0.05 compared with expression of (PYK2-WT) (Mann-Whitney u
test).
