Figure 1.
FcεRI-mediated PLSCR1 tyrosine phosphorylation depends on the FcRγ chain.
(A) Non-transfected, FcαRI-transfected or FcαRIR209L-transfected RBL-2H3 cells were sensitized for 1 hr with anti-DNP IgE or F(ab’)2 fragment of the anti-FcαRI monoclonal antibody A77, as indicated. After washes, IgE-sensitized cells were stimulated with specific antigen (Ag), whereas A77-F(ab’)2-sensitized cells were stimulated with F(ab’)2 fragment of rabbit anti-mouse IgG (RAM) for the indicated time. PLSCR1 in cell lysates was immunoprecipitated with anti-rat PLSCR1 monoclonal antibody 129.2 (IP PLSCR1). Eluates were analyzed by immunoblotting with anti-phosphotyrosine monoclonal antibody to detect PY-PLSCR1 and, after stripping of the membranes, with 129.2 to detect total PLSCR1, as indicated. (B) Quantification of PLSCR1 tyrosine phosphorylation relative to immunoprecipitated PLSCR1. Shown are the fold increases relative to basal PLSCR1 phosphorylation. Two-way ANOVA was used to compare the two kinetics observed for each cell line (bracket); and two-tailed unpaired student t test was used to compare stimulated conditions to unstimulated condition. Data are presented as mean ± s.e.m. of at least three independent experiments. ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001.
Figure 2.
Time- and Lyn-dependency of FcεRI-mediated PLSCR1 tyrosine phosphorylation.
(A) Time-dependent FcεRI-mediated PLSCR1 tyrosine phosphorylation in WT mouse BMMC. IgE-sensitized cells were stimulated for the indicated length of time with antigen. PLSCR1 was immunoprecipitated from cell lysates and tyrosine phosphorylated PLSCR1 (PY-PLSCR1) was analyzed by immunoblotting with anti-phosphotyrosine antibody (4G10). After stripping of the membrane, total PLSCR1 was analyzed by immunoblotting with anti-mouse PLSCR1 1A8 antibody. Lower panel: quantification. Statistical analysis was done by a one-way ANOVA followed by a Tukey’s multiple comparison test. Data are presented as mean ± s.e.m. of six independent experiments. **: p<0.01; ***: p<0.001. (B) FcεRI-mediated PLSCR1 tyrosine phosphorylation is dependent on Lyn. Wild-type or Lyn−/− BMMC were transduced either with empty vector (LacZ), LynA containing vector (LynA) or dead-kinase LynA (LynAKN). After reconstitution, IgE-sensitized cells were stimulated for 30 minutes with antigen. Upper panels: PLSCR1 was immunoprecipitated from cell lysates and tyrosine phosphorylated PLSCR1 (PY-PLSCR1) was analyzed by immunoblotting with anti-phosphotyrosine antibody. After stripping of the membrane, total PLSCR1 was analyzed by immunoblotting with anti-mouse PLSCR1 1A8 antibody. Lower panels: Controls for the presence of Lyn in the reconstituted cells were performed by immunoblotting cell lysates with anti-Lyn antibody and with anti-actin antibody for loading control.
Figure 3.
FcεRI-mediated PLSCR1 tyrosine phosphorylation is negatively regulated by Fyn.
(A) Five million IgE-sensitized BMMC from wild-type (WT) or Fyn knock-out (Fyn−/−) mice were stimulated or not with antigen for 10 min. BMMC cell lysates were subjected to immunoprecipitation with anti-mouse PLSCR1 mAb 1A8 or protein G beads alone (pG), and eluates were analyzed by immunoblotting with anti-phosphotyrosine (upper panel) and, after stripping, anti-muPLSCR1 (lower panel) monoclonal antibodies. Fold increase in phosphorylation corresponds to the ratio of the value of phospho-PLSCR1 obtained for stimulated and non-stimulated cells for each condition normalized with the corresponding value of recovered total PLSCR1. (B) Quantification. Statistical analysis was done by a one-way ANOVA followed by a two tailed paired student t test. Data are presented as mean + s.e.m. of four independent experiments. **: p<0.01.
Figure 4.
The Fyn-dependent negative regulation of FcεRI-mediated PLSCR1 tyrosine phosphorylation targets Lyn-dependent PLSCR1 phosphorylation.
(A) Five million IgE-sensitized BMMC from wild-type (WT) or Lyn/Fyn-double deficient (Lyn−/−Fyn−/−) mice were stimulated or not with antigen for 10 min and PLSCR1 was immunoprecipitated from the lysates with anti-muPLSCR1 antibody 1A8. Tyrosine phosphorylated PLSCR1 (PY-PLSCR1) recovered from the lysates was detected by immunoblotting with anti-phosphotyrosine antibody and, after stripping of the membrane, the total amount of recovered muPLSCR1 was analyzed. (B) Quantification. Statistical analysis was done by a one-way ANOVA followed by a Tukey’s multiple comparison test. Data are presented as mean + s.e.m. of three independent experiments. ns: not significant; ***: p<0.001.
Figure 5.
Lyn: PLSCR1 association is not modulated by Fyn in mast cells.
A) Quantification of PLSCR1 and Lyn relative to actine in wild-type (WT), Lyn−/− and Fyn−/− BMMC. Left: representative immunoblots for the indicated proteins from one analysis. Right: relative quantifications of three independent experiments. The relative amount of the indicated protein was standardized as 1 in WT BMMC, and served as reference for all other relative quantifications. N.D., not detected. Data are presented as mean + s.e.m. One-way ANOVA analysis showed no significant difference. B) Sensitized BMMC from wild-type (WT), from Fyn-deficient (Fyn−/−) and from Lyn-deficient (Lyn−/−) mice were stimulated or not stimulated with antigen for 10 min. BMMC cell lysates were immunoprecipitated with anti-mouse PLSCR1 mAb 1A8-coupled beads (IP PLSCR1). Eluates were analyzed by immunoblotting with anti-Lyn antibodies (Lyn) and, after stripping, with anti-muPLSCR1 monoclonal antibody (PLSCR1). Left: one experiment representative of four. Bracket: Lyn doublet. The upper band is the heavy chain of the immunoprecipitating antibody. Right: Quantification of the amounts of Lyn co-immunoprecipitated with PLSCR1. Data were normalized to values obtained with WT unstimulated BMMC. Data are presented as mean + s.e.m. of four independent experiments. One-way ANOVA analysis showed no significant difference.
Figure 6.
FcεRI-mediated PLSCR1 tyrosine phosphorylation is dependent on Syk.
(A) One million IgE-sensitized RBL-2H3 cells (RBL) and Syk-deficient RBL-2H3 cells before (Syk–) or after (Syk+) reconstitution with Syk, were stimulated for 30 minutes with antigen. PLSCR1 was immunoprecipitated from cell lysates with anti-rat PLSCR1 monoclonal antibody 129.2. Tyrosine phosphorylated PLSCR1 (PY-PLSCR1) in eluates was detected by immunoblotting with phosphotyrosine-specific antibodies and, after stripping of the membrane, PLSCR1 was detected by immunoblotting with 129.2. Lower panel: the presence and absence of Syk was confirmed in cell lysates by immunoblotting. (B) Quantification of the increase in PLSCR1 tyrosine phosphorylation after cell stimulation relative to PLSCR1 basal phosphorylation. Statistical analysis was done by a one-way ANOVA followed by a Tukey’s multiple comparison test. Data are presented as mean + s.e.m. of four independent experiments. ns: not significant; *: p<0.05; **: p<0.01.
Figure 7.
FcεRI-mediated PLSCR1 tyrosine phosphorylation is partially dependent on calcium.
(A) Phosphorylation of PLSCR1 in the absence of intra- and extra-cellular calcium. RBL-2H3 cells were stimulated or not stimulated with IgE and antigen for 30 min in the presence or absence of calcium and of the intracellular calcium chelator BAPTA-AM as indicated and as described in the Materials and Methods Section. RBL-2H3 cell lysates were immunoprecipitated with anti-rat PLSCR1 mAb 129.2, and tyrosine phosphorylated PLSCR1 (PY-PLSCR1) in the eluates was analyzed in immunoblotting with anti-phosphotyrosine antibodies. After stripping of the membrane, the presence of total PLSCR1 was analyzed by immunoblotting with anti-rat PLSCR1 monoclonal antibodiy 129.2. Quantification of the PLSCR1 phosphorylation was performed using the NIH Image J software. Fold phosphorylation corresponds to the ratio of the value of phospho-PLSCR1 obtained for stimulated and non-stimulated cells (Fold, see Materials and Methods section) for each condition. One experiment representative of three is shown. IgH: Heavy chain of immunoprecipitating antibody. (B) Quantification. Statistical analysis was done by a one-way ANOVA followed by a two tailed paired student t test. Data are presented as mean + s.e.m. of three independent experiments. *: p<0.05; **: p<0.01. (C) Degranulation. The supernatants of the cells shown in the panel (A) were tested for the percent of β-hexosaminidase released. (D) Fluorescence measurement of intracellular calcium of sensitized RBL-2H3 cells loaded with Fura-2-AM and stimulated as in (A) with antigen (Ag) in the presence (+Ca++) and absence (−Ca++ + BAPTA-AM) of intra- and extra-cellular calcium. This experiment is representative of three.
Figure 8.
Complex regulation of FcεRI-dependent tyrosine phosphorylation of PLSCR1.
Based on the data presented herein, we propose that tyrosine phosphorylation of PLSCR1 following FcεRI engagement is regulated on at least three levels in mast cells. Positive regulation is mediated by the Lyn-dependent pathway, whereas negative regulation is mediated by the Fyn-dependent pathway. After FcεRI aggregation, PLSCR1 can be phosphorylated on tyrosine directly either by Lyn or by Syk and indirectly as a result of Lyn/Syk-dependent activation of subsequent calcium mobilization. The other activation pathway initiated by FcεRI, that is dependent on Fyn, negatively regulates tyrosine phosphorylation of PLSCR1. Whether it acts by directly modulating Lyn-mediated or calcium-dependent tyrosine phosphorylation of PLSCR1, by controlling the PLSCR1 cellular localization required for its optimal phosphorylation by the Lyn pathway or by promoting its dephosphorylation is still unresolved.