FIG. 4.
Tie2-associated p85 confers association of PI3 kinase activity in vitro. (A) Representative thin-layer chromatogram demonstrating GST-Tie2 kinase-associated PI3-kinase activity. Autophosphorylated wild-type or mutant GST-kinase fusion proteins were immobilized on glutathione-Sepharose and incubated with endothelial cell lysates as described for Fig. 2 except that, after the final wash, protein complexes were incubated with PI and [γ-32P]ATP in kinase buffer. Radiolabeled PI3P was separated by thin-layer chromatography and visualized by autoradiography. As a control, glutathione-Sepharose without recombinant kinase was incubated with endothelial cell lysates (beads). The positions of the origin (O) and PI3P (PIP) are shown. (B) Quantitation of Tie2-associated PI3-kinase activity. PI3-kinase activity associated with wild-type or mutant GST-Tie2 from the chromatogram in panel A was quantitated with a Molecular Dynamics PhosphorImager. Activity was corrected for that associated with beads alone and expressed as a percentage of activity associated with the wild-type (wt) kinase.