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1.
Figure 2.

Figure 2. From: Phospholipase D2 acts as an essential adaptor protein in the activation of Syk in antigen-stimulated mast cells.

Association of PLD2 with Syk is dependent on phosphorylation of Syk. (A) RBL-2H3 cells were exposed to 20 μM PP2, 120 μM piceatannol (Pi), 50 mM 1-butanol (Bu), or 50 mM tertiary butanol (tBu) for 10 minutes before stimulation with 25 ng/mL DNP-BSA (Ag) for 7 minutes or left unstimulated (NS). Endogenous Syk was immunoprecipitated with anti-Syk antibody for detection of tyrosine-phosphorylated Syk (pY-Syk) with antiphosphotyrosine antibody and Syk with anti-Syk antibody by immunoblotting. (B-C) RBL-2H3 cells transiently cotransfected with HA-PLD2 and myc-Syk plasmids were stimulated with antigen in the presence or absence of inhibitors as described for panel A. HA-PLD2 (B) and myc-Syk (C) were immunoprecipitated with agarose-conjugated antibody against HA-tag or antimyc antibody for immunoblotting and detection of HA-PLD2, myc-Syk, and their phosphorylated counterparts (pY-) with the appropriate antibody. Representative immunoblots from 3 experiments are shown.

Jun Ho Lee, et al. Blood. 2006 August 1;108(3):956-964.
2.
Figure 6.

Figure 6. From: Phospholipase D2 acts as an essential adaptor protein in the activation of Syk in antigen-stimulated mast cells.

Overexpression of HA-PLD2 or Syk enhances phosphorylation of LAT activation as well as degranulation. (A-B) RBL-2H3 cells were transfected with cDNA constructs for HA-PLD2 (PLD2 or P2), catalytically inactive PLD2K758R (PLD2m or P2m), Syk (S), or vector (–). Cells were stimulated for 7 minutes with antigen (Ag) or not stimulated (NS). LAT was immunoprecipitated from cell lysates with anti-LAT antibody, and precipitated proteins were subjected to immunoblot analysis for detection of LAT and phosphorylated LAT (pY-LAT) with anti-LAT and antiphosphotyrosine antibodies. Representative blots (A) and densitometric data (B) from 3 experiments are shown. (C) Cells were also stimulated with antigen for 7 minutes for measurement of release of the granule marker, β-hexosaminidase. Values are expressed as percent of release of β-hexosaminidase in vector-transfected cells (about 31% release) and are the mean ± SEM of values from 3 experiments. Significant increase or decrease in release: *P < .05; **P < .01.

Jun Ho Lee, et al. Blood. 2006 August 1;108(3):956-964.
3.
Figure 3.

Figure 3. From: Phospholipase D2 acts as an essential adaptor protein in the activation of Syk in antigen-stimulated mast cells.

Phosphorylation of Syk is enhanced by PLD2 and by Lyn. (A-C) RBL-2H3 cells were transiently cotransfected with myc-Syk along with HA-PLD2, catalytically inactive HA-PLD2K758R cDNA plasmids, or vector (V). Transfection was performed with 25 μg plasmid per 2 × 107 cells (A) or as indicated (B-C). Cells were stimulated for 7 minutes with 25 ng/mL antigen (Ag) or not stimulated (NS). Myc-Syk was immunoprecipitated (IP) from cell lystates with antimyc antibody, and precipitated proteins were subjected to immunoblot analysis for detection of tyrosine-phosphorylated Syk (pY-Syk) and myc-Syk with antibodies against phosphotyrosine and myc, respectively. (D-E) RBL-2H3 cells were cotransfected with cDNA constructs for myc-Syk along with vector (V), flag-Lyn, HA-PLD2, or the combination of flag-Lyn and HA-PLD2. Transfection was performed with 5 μg flag-Lyn and PLD2 DNA per 2 × 107 cells (E) or as indicated (D). Cells were stimulated with antigen or not for 7 minutes, and immunoblotting was performed for detection of phosphorylated Syk and myc-Syk. Representative immunoblots from 3 experiments are shown for all 5 panels, and relative densities are indicated in italics in panel E.

Jun Ho Lee, et al. Blood. 2006 August 1;108(3):956-964.
4.
Figure 1.

Figure 1. From: Phospholipase D2 acts as an essential adaptor protein in the activation of Syk in antigen-stimulated mast cells.

Colocalization of PLD2 with LAT and Syk on the plasma membrane. (A) RBL-2H3 cells were transiently transfected with EGFP-PLD2 plasmid and then left unstimulated (NS) or stimulated with 25 ng/mL DNP-BSA (Ag) for 7 minutes for examination by confocal microscopy. Cells were counterstained with rhodamine-labeled antibodies against LAT or Syk. Representative photomicrographs are shown. (B-C) RBL-2H3 cells transiently cotransfected with HA-PLD2 and myc-Syk cDNA plasmids or mock transfected (MOCK) were stimulated or not with antigen as in panel A. Proteins were immunoprecipitated (IP) with anti-HA (B) or antimyc (C) antibodies and then subjected to immunoblot analysis with anti-Syk or anti-HA antibodies. (D) The period of stimulation of the cotransfected RBL-2H3 cells was also varied as indicated, and immunoprecipitates obtained with anti-HA antibody were subjected to immunoblot analysis with anti-Syk or anti-HA antibodies. (E) Cells were also stimulated with antigen to calculate the rate of degranulation at the given times as determined by the release of the granule marker, β-hexosaminidase. Values are expressed as percent of cellular β-hexosaminidase that was released into the medium per minute and are the mean ± SEM of values from 3 experiments.

Jun Ho Lee, et al. Blood. 2006 August 1;108(3):956-964.
5.
Figure 4.

Figure 4. From: Phospholipase D2 acts as an essential adaptor protein in the activation of Syk in antigen-stimulated mast cells.

Association of PLD2 and Syk, although dependent on initial phosphorylation of Syk, results in additional phosphorylation and the activation of Syk. (A) Immunoprecipitated (IP) Syk from RBL-2H3 cells was incubated initially in the presence or absence of 20 μM ATP before addition of lysates of cells that had been transfected with HA-PLD2 cDNA or not (MOCK). The precipitated proteins were subjected to immunoblot analysis for detection of HA-PLD2 and phosphorylated Syk (pY-Syk) as described in “Materials and methods.” (B) Free recombinant Syk was phosphorylated with or without ATP and HA-PLD2 that had isolated by the Catch and Release system as described in “Materials and methods.” An equal amount of BSA was used as the negative control for HA-PLD2. The mixtures were subjected to immunoblot analysis for detection of phosphorylated Syk, HA-PLD2, and Syk. Representative immunoblots from 3 experiments are shown.(C) Free recombinant Syk was assayed for kinase activity in the absence or presence of the indicated amounts of HA-PLD2 as described for panel B by use of an in vitro kinase assay kit. An equal amount of BSA (B) was used as the negative control for each HA-PLD2 sample. Values are the mean ± SEM of 3 separate experiments.

Jun Ho Lee, et al. Blood. 2006 August 1;108(3):956-964.
6.
Figure 5.

Figure 5. From: Phospholipase D2 acts as an essential adaptor protein in the activation of Syk in antigen-stimulated mast cells.

PLD2 interacts via its PX domain with Syk in vitro, an interaction that is dependent on Syk tyrosine 317. (A) Following the incubation of recombinant hexahistidine (His6)–PLD2 with recombinant Syk, Syk was immunoprecipitated with anti-Syk antibody, and immunoblots were prepared for detection of His6-PLD2 and Syk as shown. (B) Structures of the individual fragments as well as the PX and PH domains of human PLD2 that were prepared as GST fusion proteins for studies shown in panels C-F. Numbers indicate the terminal amino acid of each nonoverlapping fragment (F) as described in “Materials and methods.” The initial and terminal amino acids of the PH and PX are also indicated (C-F). The GST fusion proteins were incubated with recombinant Syk (C-D), endogenous Syk (E), and the indicated mutants (tyrosine to phenylalanine) of myc-Syk (F). Lysates of RBL-2H3 cells and of cells made to overexpress the myc-Syk mutants were used as sources of endogenous Syk and mutated myc-Syk, respectively (see “Materials and methods”). Immunoblots of the protein precipitates were probed with antibodies against Syk, Myc, and GST as shown. (G) Cells were made to overexpress wild-type or the Y317F mutant of myc-Syk along with HA-PLD2. Cells were stimulated or not with antigen (Ag). HA-PLD2 was immunoprecipitated with anti-HA antibody, and HA-PLD2 and coimmunoprecipitated myc-Syk were detected by immunoblotting and use of antimyc and HA antibodies.

Jun Ho Lee, et al. Blood. 2006 August 1;108(3):956-964.
7.
Figure 7.

Figure 7. From: Phospholipase D2 acts as an essential adaptor protein in the activation of Syk in antigen-stimulated mast cells.

Suppression of PLD2 expression with siRNA impairs tyrosine phosphorylation of Syk, LAT, and SLP as well as degranulation. (A) RBL-2H3 cells were made to transiently express HA-PLD2 and siRNAs (RNAi) directed against PLD2 or as a control green fluorescent protein (GFP). Expression of HA-PLD2 and actin was determined by immunoblotting, and expression of PLD2 mRNA was determined by RT-PCR. (B) Cells made to express the PLD2 siRNA were also stimulated with 25 ng/mL antigen (Ag) or not (NS) for 5 minutes for detection of Syk, LAT, and SLP and their tyrosine phosphorylated (pY-) counterparts by immunoblotting after immunoprecipitation. (C) Cells were also stimulated with antigen for 15 minutes to measure release of the granule marker, β-hexosaminidase. Values are expressed as percent of cellular β-hexosaminidase that was released into the medium and are the mean ± SEM of values from 3 experiments. Significant decrease in release: *P < .01. (D) Model for the dual actions of PLD2. The data suggest that PLD2 acts in a catalytically independent and dependent manner to associate directly with Syk to enhance tyrosine phosphorylation and activation of Syk and downstream targets such as LAT and SLP-76 and form phosphatidic acid (PA), which may facilitate activation of PKC and other, as yet poorly defined, mechanisms that are essential for degranulation.

Jun Ho Lee, et al. Blood. 2006 August 1;108(3):956-964.

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