by Koji Nakamura, Alexander Malykhin, and K. Mark Coggeshall The Src homology 2 domain–containing inositol 5-phosphatase negatively regulates Fcγ receptor–mediated phagocytosis through immunoreceptor tyrosine-based activation motif–bearing phagocytic receptors by Koji Nakamura, Alexander Malykhin, and K. Mark Coggeshall Blood Volume 100(9):3374-3382 November 1, 2002 ©2002 by American Society of Hematology

Phagocytosis assay using BMMs from gene-targeted mice Phagocytosis assay using BMMs from gene-targeted mice.Fluorescent IgG-opsonized RBCs were incubated with BMMs from gene-targeted mice indicated at a ratio of 20:1. Phagocytosis assay using BMMs from gene-targeted mice.Fluorescent IgG-opsonized RBCs were incubated with BMMs from gene-targeted mice indicated at a ratio of 20:1. Internalized IgG-RBCs were counted under a fluorescence microscope. The results were expressed as the number of the internalized IgG-RBCs per 100 BMMs (phagocytic index). Results shown are the average of duplication and are representative of 2 independent experiments. Koji Nakamura et al. Blood 2002;100:3374-3382 ©2002 by American Society of Hematology

Calcium mobilization upon FcγR stimulation in BMMs from gene-targeted mice.BMMs (5 × 105) from gene-targeted mice indicated were loaded with Indo-1 AM and stimulated with 40 μg/mL ΔIgG. Calcium mobilization upon FcγR stimulation in BMMs from gene-targeted mice.BMMs (5 × 105) from gene-targeted mice indicated were loaded with Indo-1 AM and stimulated with 40 μg/mL ΔIgG. The intracellular Ca++ was monitored by spectrofluorometry. The bar indicates intracellular Ca++ as a reference. The arrow indicates the time when ΔIgG was added. Koji Nakamura et al. Blood 2002;100:3374-3382 ©2002 by American Society of Hematology

Tyrosine phosphorylation of SHIP upon FcγR stimulation in BMMs from gene-targeted mice.(A) BMMs (4 × 106) from gene-targeted mice indicated were stimulated with 40 μg/mL ΔIgG for indicated minutes, lysed, and immunoprecipitated with anti–SHIP antibody. Tyrosine phosphorylation of SHIP upon FcγR stimulation in BMMs from gene-targeted mice.(A) BMMs (4 × 106) from gene-targeted mice indicated were stimulated with 40 μg/mL ΔIgG for indicated minutes, lysed, and immunoprecipitated with anti–SHIP antibody. The immunoprecipitates were blotted with antiphosphotyrosine antibody (upper panel; anti-pTyr) or anti–SHIP antibody (lower panel). (B) The amount of tyrosine-phosphorylated SHIP to total SHIP shown in panel A was quantified and expressed as fold increase of the ratio. The results were shown as relative values of the time-zero controls and as averages from 2 independent experiments. Bars represent standard errors of duplicate measurements. Koji Nakamura et al. Blood 2002;100:3374-3382 ©2002 by American Society of Hematology

Clustering of FcγRIIa or the γ-chain of FcγRs is sufficient for SHIP phosphorylation.(A) The expression of CD8 chimeras in stable RAW264.7 transfectants was examined by fluorescence-activating cell sorter (FACS) analysis. Clustering of FcγRIIa or the γ-chain of FcγRs is sufficient for SHIP phosphorylation.(A) The expression of CD8 chimeras in stable RAW264.7 transfectants was examined by fluorescence-activating cell sorter (FACS) analysis. The cells were stained with biotinylated F(ab′)2 fragments of OKT8 followed by FITC-conjugated streptavidin and analyzed by FACS. Dotted lines indicate fluorescence of unstained cells. (B,C) The RAW264.7 transfectants were stimulated with biotinylated F(ab′)2 fragments of OKT8 followed by streptavidin. Whole-cell lysates (B) or SHIP immunoprecipitates (C) were separated by SDS-PAGE and blotted with antiphosphotyrosine (anti-pTyr). The filter in C was reprobed with anti–SHIP antibody (lower panel). (D) The expression of CD8 chimeras in stable THP-1 transfectants was examined by FACS analysis using biotinylated F(ab′)2 fragments of OKT8 followed by FITC-conjugated streptavidin. (E,F) THP-1 transfectants were stimulated with biotinylated F(ab′)2fragments of OKT8 followed by streptavidin. Whole-cell lysates (E) or SHIP immunoprecipitates (F) were blotted with anti-pTyr. In panel F, untransfected cells were also stimulated with Fab fragments of IV.3 antibody followed by F(ab′)2 fragments of goat anti–mouse antibody (2 lanes on the farthest right). The membrane was reprobed with anti–SHIP antibody (lower panel). Koji Nakamura et al. Blood 2002;100:3374-3382 ©2002 by American Society of Hematology

SHIP binds directly to the ITAM of FcγRIIa in vitro and in vivo SHIP binds directly to the ITAM of FcγRIIa in vitro and in vivo.(A) Sequences of peptides used are shown and described previously.29 (B) THP-1 cells were stimulated with Fab fragments of IV.3 antibody followed by F(ab′)2 fragments of goat anti–mouse antibody. SHIP binds directly to the ITAM of FcγRIIa in vitro and in vivo.(A) Sequences of peptides used are shown and described previously.29 (B) THP-1 cells were stimulated with Fab fragments of IV.3 antibody followed by F(ab′)2 fragments of goat anti–mouse antibody. The lysates were incubated with biotinylated peptides indicated and purified by Neutravidin beads. The precipitates were resolved by SDS-PAGE, and blotted with anti–SHIP antibody. (C) The recombinant GST-SH2-SHIP fusion protein (right 5 lanes) or GST protein (left 5 lanes) was incubated with biotinylated peptides indicated, precipitated with Neutravidin beads, resolved by SDS-PAGE, and blotted with anti–GST antibody. The phosphorylated peptides were pretreated with calf intestinal alkaline phosphatase (CIAP) before the incubation with recombinant proteins as indicated. The position of GST-SH2-SHIP was indicated at right. (D) THP-1 transfectants were stimulated with biotinylated F(ab′)2 fragments of OKT8 followed by streptavidin and lysates were immunoprecipitated with anti-myc. The immunoprecipitates (ip) were separated by SDS-PAGE and probed with antibody to SHIP (upper panel) and reprobed with anti-myc (lower panel). Koji Nakamura et al. Blood 2002;100:3374-3382 ©2002 by American Society of Hematology

Measurements of affinities between GST-SH2-SHIP fusion protein and phosphopeptides by surface plasmon resonance.Biotinylated phosphopeptides indicated were captured on a streptavidin-coated sensor chip, and GST-SH2-SHIP was injected for 5 minutes at a flow ... Measurements of affinities between GST-SH2-SHIP fusion protein and phosphopeptides by surface plasmon resonance.Biotinylated phosphopeptides indicated were captured on a streptavidin-coated sensor chip, and GST-SH2-SHIP was injected for 5 minutes at a flow rate of 30 μL/min at 25°C. The chip was washed with binding buffer for a further 5 minutes to examine dissociation rates. Koji Nakamura et al. Blood 2002;100:3374-3382 ©2002 by American Society of Hematology

Both tyrosine residues in FcγRIIa ITAM are responsible for SHIP phosphorylation in vivo.(A) The expression of CD8 chimeras with substitutions of tyrosine residues with phenylalanine was examined by FACS analysis using biotinylated F(ab′)2 fragments of OKT8 ... Both tyrosine residues in FcγRIIa ITAM are responsible for SHIP phosphorylation in vivo.(A) The expression of CD8 chimeras with substitutions of tyrosine residues with phenylalanine was examined by FACS analysis using biotinylated F(ab′)2 fragments of OKT8 followed by FITC-conjugated streptavidin. Dotted lines indicate fluorescence of unstained cells. (B) The THP-1 transfectants were stimulated with biotinylated F(ab′)2 fragments of OKT8 followed by streptavidin. The lysates from the cells were immunoprecipitated with anti–SHIP antibody, separated on SDS-PAGE gels, and blotted with antiphosphotyrosine (anti-pTyr) antibody. The filter was reprobed with anti–SHIP antibody. Koji Nakamura et al. Blood 2002;100:3374-3382 ©2002 by American Society of Hematology

The introduction of SH2-SHIP enhances the phagocytic abilities in the absence of FcγRII(b).(A) THP-1 cells were transiently transfected with GFP or GFP-SH2-SHIP. The introduction of SH2-SHIP enhances the phagocytic abilities in the absence of FcγRII(b).(A) THP-1 cells were transiently transfected with GFP or GFP-SH2-SHIP. The GFP-positive cells were sorted and analyzed by FACS analysis. Dashed lines indicate untransfected THP-1 cells. (B) The sorted GFP-positive cells were incubated with RBCs coated with Fab fragments of IV.3 antibody for 20 minutes at 37°C. The results were expressed as the number of the internalized RBCs per 100 cells which phagocytosed at least one RBC (phagocytic index). Open and closed bars represent phagocytic indexes for GFP-expressing cells and GFP-SH2-SHIP–expressing cells, respectively. Results are shown as the averages of duplication and are representative of 2 independent experiments. Bars represent standard errors of duplicate samples. Koji Nakamura et al. Blood 2002;100:3374-3382 ©2002 by American Society of Hematology