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.