Disruption of the CD4/PLSCR1 interaction by SLPI.

<p>A) In vitro inhibition of the CD4/PLSCR1 interaction by SLPI. GST or GST-PLSCR1 (left panel, coomassie blue) was incubated with equal amounts of lysates from Jurkat CD4-positive T cells in the presence of the indicated concentrations of either GST-SLPI or GST-ARF1 (left panel) used as a control. Bound proteins were analyzed by Western blot with anti-CD4 (right panel). B) Mapping of the PLSCR1 determinants required for binding to CD4 and SLPI. L40 yeast strain expressing either the cytoplasmic domain of CD4 (CD4c) or SLPI fused to LexA in combination with each of the deleted forms of the Gal4AD-PLSCR1 hybrids indicated on the left was analyzed for histidine auxotrophy and beta-gal activity. The interactions between hybrid proteins were scored as follows: (+), cell growth on medium without histidine and development of a β-gal activity; (−), no growth on medium without histidine and no β-gal activity.</p

Anti-HIV-1 activity of recombinant SLPI.

<p>A) Inhibition of virus replication. Primary T lymphocytes were isolated from peripheral blood mononuclear cells and used for infection with the HIV-1JR-CSF isolate in the presence of increasing concentrations (0–50 µg/ml) of purified GST-SLPI (see inset, coomassie blue) or GST (50 µg/ml). After washing, infected cells were cultured for 6 days and the viral production was quantified by measuring the p24 production in the cell-culture medium. B) Inhibition of virus transfer from dendritic cells to T lymphocytes. Dentritic cells were derived from purified primary monocytes, and incubated with either HIV-1Bal (R5 strain, upper panel) or HIV-1NDK (×4 strain, lower panel) for 1 h at 37°C in the presence of 50 µg/ml of purified GST-SLPI or GST. After washing, IL-2 activated-lymphocytes were added in a 1/5 ratio and maintained in co-culture for 72 h. Viral production was quantified by measuring the p24 production in the cell culture medium.</p

Mapping of the CD4 determinants required for PLSCR1 binding by co-immunoprecipitation assay.

<p>A) Schematic representation of the human CD4 mutants. The extracellular and transmembrane (TM) domains of CD4 are represented in grey and hatched, respectively. The a.a. sequences of the cytoplasmic tail of the CD4 mutants are aligned with that of the wild-type CD4 (CD4 WT). Dashes (−) indicate a.a. identities with the wild type protein and a.a. substitutions are identified. B) Co-immunoprecipitation assay. Protein extracts were prepared 48 h post transfection from 293T cells co-expressing HA-PLSCR1 together with wild type or mutated CD4 as indicated at the top. Crude extracts were then subjected to CD4 immunoprecipitation followed by Western blot analysis with anti-CD4 (upper panels) and anti-HA (lower panels).</p

Mapping of the CD4 determinants required for PLSCR1 binding by ELISA.

<p>A) Primary a.a. sequences of the CD4 long (405–433) and short (405–426) peptides used in the ELISA test. The primary sequence of the entire cytoplasmic domain of CD4 is shown at the top; the 2 Cys residues required for p56Lck binding and mutated in Ala in both peptides are indicated in red. B) ELISA interaction test. 96-well plates were coated with the CD4c long (left panel) or short (right panel) peptides at a final concentration of 0.5 µM. After washings and blocking of non-specific binding sites, GST or GST-PLSCR1 was incubated at concentrations ranging from 1 nM to 300 nM. Binding was then revealed with an anti-GST antibody and a secondary peroxidase-conjugated anti-mouse IgG. The peroxidase substrate solution was incubated for 10 min and the optical density was measured at 450 nm.</p

Co-distribution of scramblases and CD4 at the plasma membrane.

<p>A) Schematic representation of PLSCR1. PLSCR1 is a type-II transmembrane protein with a long N-terminal cytoplasmic domain (a.a. 1–290, grey box) containing 3 Cys-rich motifs (red boxes), and a transmembrane domain (a.a. 291–310, black box). Amino acids are numbered according to Zhou et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005006#pone.0005006-Zhou1" target="_blank">[23]</a>. B) Localization of wild type and deleted GFP-PLSCR1 forms. Jurkat CD4-positive T cells expressing the full length (left panel) or deleted forms (1–310 and 1–290, central and right panels, respectively) of GFP-PLSCR1 were fixed and directly examined. Cells were analyzed by epifluorescence microscopy, and images were acquired using a CCD camera. C) Localization of GFP-PLSCR3 and GFP-PLSCR4. Jurkat cells expressing GFP-PLSCR1 (left panel), GFP-PLSCR3 (central panel) or GFP-PLSCR4 (right panel) were fixed and directly examined as in (B). D) Subcellular distribution of CD4 and PLSCR1. Jurkat cells expressing wild type GFP-PLSCR1 (middle panel) were fixed, permeabilized and subsequently stained with an anti-CD4 (left panel). Scale bars, 10 µm.</p

Interaction of PLSCR1 and PLSCR4 with the CD4 receptor.

<p>A) In vitro interaction. Lysates from Jurkat CD4-positive T cells were incubated with equal amounts of GST or GST-PLSCR1 (upper panel, coomassie blue) immobilized on GSH-sepharose beads. Bound proteins were then analyzed by immunoblotting with anti-CD4 (lower panel). B) Co-immunoprecipitation of endogenous proteins from T lymphocytes. Jurkat T cells were lyzed and CD4 was precipitated with either anti-CD4 (OKT4) or a control isotypic antibody. Precipitates were analyzed by Western blot with anti-CD4 (upper panel) or anti-PLSCR1 (lower panel). C) Co-precipitation of overexpressed CD4 and PLSCR1 proteins. 293T cells expressing HA-tagged PLSCR1 (lower panel, Cell lysate) in combination with wild-type CD4 (WT) or a mutant of CD4 deleted of its cytoplasmic domain (ΔCT) were lyzed and the CD4 forms were precipitated with anti-CD4. Precipitates were then analyzed by Western blot with anti-CD4 (upper panel) or anti-HA (middle panel). D) Co-precipitation of overexpressed CD4 and PLSCR4 proteins. 293T cells expressing GFP- PLSCR1, GFP-PLSCR3 or GFP-PLSCR4 (lower panels, Cell lysate) in combination with CD4 WT (right panels) or CD4 ΔCT (left panels) were lyzed and the CD4 forms were precipitated with anti-CD4. Precipitates were then analyzed as in (C). Of note, the differences in migration observed between PLSCR1 (318 a.a.), PLSCR3 (295 a.a.) and PLSCR4 (329 a.a.) is likely related to the respective amino acid lengths of the GFP fusion proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005006#pone.0005006-Zhou1" target="_blank">[23]</a>. E) Interactions in the two-hybrid system. L40 yeast strain expressing the LexA-CD4c hybrid in combination with the indicated Gal4AD hybrids was analyzed for histidine auxotrophy and β-gal activity. Transformants were patched on medium with histidine (upper panels) and then replica-plated on medium without histidine (middle panels) and on Whatman filter for β-gal assay (lower panels).</p

Complex regulation of FcεRI-dependent tyrosine phosphorylation of PLSCR1.

<p>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.</p

FcεRI-mediated PLSCR1 tyrosine phosphorylation is negatively regulated by Fyn.

<p>(A) Five million IgE-sensitized BMMC from wild-type (WT) or Fyn knock-out (<i>Fyn</i>^−/−) 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.</p

FcεRI-mediated PLSCR1 tyrosine phosphorylation depends on the FcRγ chain.

<p>(A) Non-transfected, FcαRI-transfected or FcαRI<i>_R209L</i>-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 (<i>Ag</i>), whereas A77-F(ab’)2-sensitized cells were stimulated with F(ab’)2 fragment of rabbit anti-mouse IgG (<i>RAM</i>) for the indicated time. PLSCR1 in cell lysates was immunoprecipitated with anti-rat PLSCR1 monoclonal antibody 129.2 (<i>IP PLSCR1</i>). 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.</p

Time- and Lyn-dependency of FcεRI-mediated PLSCR1 tyrosine phosphorylation.

<p>(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 (<i>LacZ</i>), LynA containing vector (<i>LynA</i>) or dead-kinase LynA (<i>LynAKN</i>). 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.</p