PLD2 and, to a lesser extent, PLD1 are tyrosine phosphorylated by c-Src tyrosine kinase. (A) COS-7 cells were transiently cotransfected for 40 h with various combinations of plasmids encoding PLD1, PLD2, c-Src, Lck, and Fyn. Cell lysates were immunoprecipitated with anti-P-Tyr antibody, and precipitated proteins were subjected to immunoblot analysis with an anti-PLD antibody. Western blot analysis using PLDs and c-Src family tyrosine kinase demonstrated that the respective proteins were expressed to equal extents. (B) COS-7 cells were transiently transfected with the indicated expression plasmids, and cell lysates were subjected to immunoprecipitation with anti-P-Tyr antibody or anti-PLD antibodies. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-PLD or anti-P-Tyr antibody. Expression of PLD and c-Src was determined by using anti-PLD or anti-c-Src antibodies. (C) COS-7 cells were transiently transfected with various combinations of plasmids encoding PLD1, PLD2, wild-type (WT) c-Src, and dominant negative (DN) c-Src. Cell lysates were immunoprecipitated by using anti-P-Tyr antibody or anti-PLD antibody, and immune complexes were analyzed by immunoblotting. Immunoreactive proteins were visualized by use of horseradish peroxidase-coupled secondary antibody and chemiluminescence. Data are representative of three experiments.

EGF induces tyrosine phosphorylation of PLD2 and its association with c-Src in A431 cells. (A) Mouse monoclonal antibody specific for PLD2 was generated as described in Materials and Methods. To analyze its specificity, PLD1 or PLD2 was transfected into COS-7 cells and the lysates were immunoblotted with anti-PLD antibody which recognizes both PLD1 and PLD2 or anti-PLD2 antibody. (B) Quiescent A431 cells were pretreated with or without a selective inhibitor of Src family tyrosine kinase, PP2 (2 μM), for 30 min and stimulated with 200 ng of EGF/ml for 20 min. Cell lysates were prepared, and 1 mg of the lysates were immunoprecipitated with anti-P-Tyr antibody or anti-c-Src antibody. Immunoprecipitated proteins were separated on SDS-PAGE gel and probed with mouse monoclonal antibody specific for PLD2. The result shown is representative of three experiments.

The catalytic domain of c-Src interacts with PLD2. (A) Schematic representation of GST-fusion protein constructs. (B) c-Src was fragmented into SH3 (88-137), SH2 (144-245), and kinase (265-523) domains. The fragments were cloned as GST fusion proteins, expressed in Escherichia coli, and purified by use of glutathione-conjugated Sepharose beads. An equal amount (2 μg) of GST or GST-Src fragment was incubated with the lysates of COS-7 cells transfected with PLD2 and c-Src. The pull-down proteins were subjected to immunoblot analysis with an antibody against PLD (upper panel). The amount of the GST fusion proteins was visualized by immunoblotting with an anti-GST antibody (lower panel). Data are representative of three experiments.

c-Src does not affect EGF-induced PLD activation in COS-7 and FaO cells. COS-7 cells transiently transfected with various constructs (A) and FaO cells expressing constitutive active c-Src (Y529F) or dominant inactive c-Src mutant (K296R/Y528F) (B) were labeled with [³H]myristic acid for 18 h. The cells were untreated or treated with 200 ng of EGF/ml for 30 min in the presence of 0.3% 1-butanol. Radioactivity incorporated into PtdBut was measured as described in Materials and Methods. *, P < 0.05 compared to cells transfected with vector and treated with EGF. Results show means ± standard deviations of three independent experiments.

PLD enhances not only c-Src-induced tyrosine phosphorylation of paxillin but also c-Src-mediated ERK activation. COS-7 cells were transiently cotransfected for 40 h with various combinations of plasmids encoding an empty vector, PLD1, PLD2, c-Src, and c-Src plus either wild-type (wt) or catalytically inactive mutant (mt) PLD1 or PLD2. Cell lysates were immunoblotted with an antibody directed against tyrosine-phosphorylated paxillin (A) or an anti-phospho-ERK antibody (B). Expression of total paxillin and ERK was visualized by immunoblotting with antipaxillin antibody or anti-ERK antibody. Data are representative of three experiments. The intensity of phosphorylated paxillin or phospho-ERK immunoreactive bands quantified by densitometry of immunoblot was expressed as relative intensity of the bands. Results show means ± standard deviations of three independent experiments.

PLD isozymes, most notably PLD2, bind to wild-type c-Src but not to the kinase-inactive c-Src mutant. COS-7 cells were transiently transfected for 40 h with various combinations of plasmids encoding PLD1, PLD2, wild-type (WT) c-Src, and dominant negative (DN) c-Src. Cell lysates were immunoprecipitated with anti-PLD or anti-c-Src antibodies, and immune complexes were subjected to immunoblotting analysis using anti-c-Src or anti-PLD antibodies, respectively. Expressions of PLDs and c-Src were determined by immunoprecipitation and immunoblotting. Data are representative of three experiments.

The N-terminal PH domain of PLD2 is required for interaction with c-Src. (A) Schematic representation of PLD2. The boxes indicate highly conserved sequences of PLD; their possible functions have been proposed or demonstrated in reference 52. CR, conserved region. (B) COS-7 cells were transiently cotransfected with plasmids encoding the empty vector (Vec) and c-Src, or PLD2 and c-Src, or N-terminal 183-amino-acid-truncated PLD2 Δ183(184-933) and c-Src, or N-terminal 313-amino-acid-truncated PLD2 Δ313(314-933) and c-Src. Cell lysates were subjected to immunoprecipitation by using anti-PLD or anti-c-Src antibodies, and the amount of coimmunoprecipitated c-Src or PLD was determined by immunoblotting with anti-c-Src or anti-PLD antibodies. Expression of PLD2 and c-Src was determined by using anti-PLD or anti-c-Src antibodies. (C) For the GST pull-down assay, GST-PH fusion protein was used as described in reference 29. The lysates transfected with c-Src were incubated with 2 μg of GST or GST-PH fragment and immunoblotted with antibody to c-Src. The amount of the GST fusion proteins was visualized by immunoblotting with an anti-GST antibody. Data are representative of three experiments.

Modulation between PLD2 and c-Src significantly enhances cellular proliferation. Mouse fibroblasts overexpressing PLD isozymes were plated at a density of 5 × 10⁴ cells in 12-well plates and transfected with empty vector (Vec), wild-type (WT) c-Src, or dominant negative (DN) c-Src mutant. After 40 h of incubation, viable cells were counted by a trypan blue exclusion method. Results show means ± standard deviations of three independent experiments.

Overexpression of PLD stimulates c-Src kinase activity. (A) COS-7 cells were transiently cotransfected with various combinations of plasmids encoding empty vector (Vec) and c-Src, or PLD2 and c-Src, or N-terminal 183-amino-acid-truncated PLD2 Δ183(184-933) and c-Src, or N-terminal 313-amino-acid-truncated PLD2 Δ313(314-933) and c-Src. Cell lysates were subjected to immunoblot analysis using a phospho Tyr⁴¹⁶ c-Src antibody. Expression of c-Src was determined by using an anti-c-Src antibody. (B) COS-7 cells were transiently transfected for 40 h with various combinations of plasmids encoding an empty vector, PLD1, PLD2, c-Src, and c-Src plus either wild-type (wt) or catalytically inactive mutant (mt) PLD1 or PLD2. Data are representative of three experiments. (C) COS-7 cells were transiently cotransfected with a catalytically inactive PLD2 mutant and wild-type c-Src, and cell lysates were subjected to immunoprecipitation with anti-c-Src antibody or anti-PLD antibodies. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-PLD or anti-c-Src antibody. Expression of PLD and c-Src was determined by using anti-PLD or anti-c-Src antibodies. Data are representative of three experiments.

Proposed model of PLD-stimulated c-Src downstream signaling. EGF binds to its receptor, thereby stimulating both PLD and c-Src activation. It is likely that the resulting PLD activity is involved in the stimulation of Src-mediated signaling pathways such as ERK and then in the stimulation of cell proliferation. EGF induces tyrosine phosphorylation of PLD2 via c-Src and its interaction with c-Src. At present, it is not clear, however, how tyrosine phosphorylation of PLD is involved in PLD-mediated signaling mechanism.