FIGURE 5.
Time course of PtdIns(4,5)P2 hydrolysis by purified PLC-ε in the combined presence of RhoA and K-Ras. The time-dependent stimulation of purified, wild-type PLC-ε-EF-RA2 (1 ng) was tested in phospholipid vesicles reconstituted with 300 nm RhoA (closed upward triangles) or K-Ras (closed downward triangles), or vesicles containing 300 nm of both RhoA and K-Ras (closed circles). GTPases reconstituted in phospholipid vesicles were preincubated in the presence of 10 μm GTPγS for 30 min at 30 °C to allow nucleotide exchange. The activity of PLC-ε-EF-RA2 in the absence of G proteins also is shown (closed squares). The ability of GDP-bound RhoA (open upward triangles), K-Ras (open downward triangles), or RhoA and K-Ras (open circles) to regulate PLC-ε-EF-RA2 activity are indicated at the 6 min time point. These results are representative of results from five individual experiments and are presented as the mean ± S.E.