The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2

Abstract

The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2, and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase.

Figure 1. Detection of PLD2 as constitutively phosphorylated on tyrosine

(A) COS-7 cells were co-transfected with pcDNA3.1-mycPLD2, with the empty vector (pcDNA3 alone) or with plain COS-7 cells (no lipofectamine) for 36 hours. After this, cells were harvested and whole sonicates were immunoprecipitated with anti-Myc (9E-10) monoclonal antibody and immunocomplexes were analyzed by SDS/PAGE and immuoblotting developed with anti-PLD2 antibodies. (B) Cells treated as in the previous panel were also immunprecipitated with anti-myc antibodies and Western blots were also probed with anti-myc. (C) anti-Myc immunoprecipitates were used for measuring enzymatic activity in PC8 liposomes and [³H]n-butanol. (D, E) Cellular co-localization of myc and PLD2 by immunofluorescence micrographs: COS-7 cells transfected with pcDNA3-mycPLD2 were immunolabeling of slides with PLD2-TRITC (D) and myc-FITC (E) antibodies. (F) Same blot as shown in panel B, derived from anti-Myc immunoprecipitates and Western blots, were stripped and developed with anti-phosphotyrosine antibodies. Results are representative of 4 experiments in duplicate and, for panel C, data represent mean ± SEM of 4 independent experiments.

Figure 2. Effect of tyrosine kinase inhibitors on PLD2

Transfected COS-7 cells were cultured continuously with genistein for 48 hours. Cells were harvested and cell lysates (~0.8 mg protein/ml) were immunoprecipitates with anti-myc antibodies that were used for measuring enzymatic activity (A) or immunoblotting (B). In (C) the effect of the EGF-R inhibitor AG-18 was assayed n vitro in the presence or assensu of PTP1B. Both activity and immunoblotting (inset) were measured.

Figure 3. Action of PTP1B on PLD2 tyrosine phosphorylation and activity

Transfected COS-7 cells were harvested and cell lysates were immunoprecipitates with anti-myc antibodies. Immunocomplex beads were treated with the indicated concentrations of PTP1B and then processed for by SD/PAGE and Western blot with anti-PY antibodies or with anti-myc for confirmation of equal protein loading (A) (shown is the region corresponding to PLD2, 103 kDa) or for PLD2 activity measurement also in immunocomplexes (B). Data represent mean ± SEM of 3 independent experiments in duplicate.

Figure 4. Confirmation of the presence of phosphatase activity

(A) Increase in the detection of the phosphatase reaction product, PNP, over a range of exogenously added PTP1B. Insert: Absorption maxima for PNPP and PNP. (B) Effect of exogenously added PTP1B on the cleavage of PNPP and production of PNP in the same experimental conditions as those used in Figure 2. Also indicated is the effect of DTT, ZnCl₂ and EDTA on phosphatase activity in. Data represent mean ± SEM of 3 independent experiments in duplicate.

Figure 5. A stable cell line overexpressing PLD2 also shows constitutive tyrosyl phosphorylation of PLD2

(A) Several clones of transformed COS-7 cells were grown on 6-well tissue culture plates (1×10⁶ cells/well) in the continuous presence of 400 μg/ml geneticin. Shown in the figure is clone #3, out of 4 valid ones, that was harvested and sonicated briefly in lysis buffer, followed by immunoprecipitation with anti-Myc (9E-10) monoclonal antibodies. Immunocomplex beads were used for SDS/PAGE and subsequent Western blotting with anti-Myc antibodies. (B) Detection of in vitro PLD2 activity in anti-myc immunoprecipitates and its comparison to cells transiently transfected, and the effect of PIP₂. (C, D) Confirmation of exchangeability of myc and PLD2 antibodies in the detection of overexpressed PLD2 in two similar clones of stable cells (clones #3 and #4). Shown are representative blots among a total of 4.

Figure 6. PTP1B increases tyrosine phosphorylation of PLD2 in a stable cell line

(A) Stable COS-7 cells were harvested and cell lysates (~0.8 mg protein/ml) were treated with the indicated concentrations of recombinant PTP1B, immunoprecipitated with anti-myc antibodies and used for SDS/Western blotting developed with anti-phosphotyrosine antibodies (clone PY100). (B) COS-7 cell lysates treated with the indicated concentrations of PTP1B were immunoprecipitated with anti-myc antibodies. Immunocomplex beads were run in SDS/PAGE gels and resulting blots were developed with anti-PY antibodies. The arrow marks the position a heavily Tyr-phosphorylated protein with an apparent Mw of 29 kDa.

Figure 7. Induction of a hyperphosphorylated PLD2 band by recombinant Grb2

(A) COS-7 lysates were treated with the indicated concentrations of Grb2 and immunoprecipitated with anti-myc antibodies followed by Western blotting, probed with anti-phosphotyrosine antibodies. (B) Confirmation of Grb2 tyrosyl phosphorylation and of the presence of the Grb2 protein itself.

Figure 8. Co-immunoprecipitation of PLD2 and Grb2

(A) Immunoprecipitation with anti-Grb2 antibodies and immunoblotting with anti-PLD2. Shown is the region of the immunoblots where PLD2 localizes (103 kDa). (B) Immunoprecipitation with anti-myc antibodies and immunoblotting with anti-Grb2. Shown is the region of the immunoblots where Grb2 localizes (29 kDa). The COS-7 “Control” in (A) refers to non-transfected cells. The “Grb2 positive control” in (B) refers to an extract of cells that are overexpressing Grb2. Shown are typical experiments among three total.

Figure 9. Co-immunoprecipitation of PTP1B and Grb2 and of PLD2 and PTP1B

(A) Upper panel: Immunoprecipitation with anti-Grb2 antibodies and immunoblotting with anti-PTP1B. Shown in the is the region of the immunoblots where PTP1B localizes (37 kDa). “Control COS-7” refers to plain COS-7 cells. Lower panel: Immunoprecipitation with anti-PTP1B antibodies and immunoblotting with anti-Grb2. Shown is the region of the immunoblots where Grb2 localizes (29 kDa). (B) Upper panel: Immunoprecipitation with anti-myc antibodies and immunoblotting with anti-PTP1B. Shown is the region of the immunoblots where PTP1B localizes (37 kDa). Lower panel: Immunoprecipitation with anti-PTP1B antibodies and immunoblotting with anti-PLD2. Shown is the region of the immunoblots where PLD2 localizes (103 kDa) of an experiment typical among a total of three.