Figure 7.

PLD2-regulated myosin II activity is independent of Rac, PI3 kinase, FAK, and MAPK pathways. (A) Lack of effect of PLD2 overexpression on the activation of Rac1. Levels of active GTP-bound Rac1 at 15 min after plating in the indicated lines were determined by incubating cell lysates with GST fusion proteins containing the PAK1 RBD. Affinity precipitates and samples of the whole cell lysates were analyzed by SDS-PAGE and Western blotting with Rac1 antibody. (B) Failure to rescue PLD2-inhibited cell spreading by expression of RAC1-V12, a constitutively active mutant of Rac1. Rac1-V12–transfected cells were identified by immunofluorescent staining of the T7 tag at the Rac1 N-terminus. Actin was visualized using rhodamine-phalloidin staining. (C) PLD2 doe not affect the activation of PI3 kinase. (D) FAK activation is not affected by changes in PLD2 expression. (E) MAPK activation is the same in control cells and cells expressing PLD2 or PLD2 K758R. Control and PLD2-expressing cells were plated on tissue culture plates coated with 10 μg/ml fibronectin and collected at indicated times for the assays shown (C–E). Western blotting was performed using an Odyssey imaging system. All results shown are representative of at least three experiments.