FIGURE 6.

Pak1 catalytic activity is synergistically enhanced by Cdc42 and PIP₂. A, [γ-³²P]ATP kinase assays were conducted with Pak1 in the presence of the indicated activators and Pak1 catalytic activity was measured by either Pak1 autophosphorylation or Pak1 phosphorylation of a peptide substrate (GST-Paktide). Activators were either PIP₂-containing liposomes (PC:PI:PIP₂:DGS-NTA(Ni), 43.5:43.5:6:7 mole ratio) or control liposomes (PC:PI:DGS-NTA(Ni), 46.5:46.5:7 molar ratio) with 200 nm Cdc42-His or their combination. These activator concentrations were selected on the basis of their ability to maximally activate Pak1 when combined (supplemental Fig. S3A). Results are normalized to phosphorylation observed in the reaction containing both activators and are shown for two independent replicates ± S.D. B, activation for 8T-Pak1 was measured as in A. Note that higher concentrations of PIP₂ (500 μm total lipid and 15 μm Cdc42-His) were required to achieve full activation of 8T-Pak1 (supplemental Fig. S3B). C, 50% activation contour for Pak1 autophosphorylation. The concentration of Cdc42-His required to achieve 50% activation of Pak1 was determined at each indicated PIP₂ concentration. Horizontal error bars (smaller than the data symbol in most cases) represent the S.D. calculated from two replicates. The curve that can be drawn through these points represents all combinations of PIP₂ and Cdc42-His that lead to 50% activation of Pak1. Table 1 presents the data used to generate the plots in C–F. D, Pak1 catalytic activity in the reactions in C was also measured by phosphorylation of a Pak1 substrate peptide included in the reaction and the results are presented as a 50% activation contour. The significant departure from linearity of the contour is indicative of synergistic activation. 8T-Pak1 autophosphorylation (E) and 8T-Pak1 substrate phosphorylation (F) is not enhanced by PIP₂. The 50% activation contours were determined as in C and D and the data are summarized in Table 1. The vertical nature of the 50% activation contour demonstrates a lack of sensitivity to PIP₂.