Figure 5.
Figure 5. Interaction of MIST with Fgr is essential for the attenuation of NK-cell receptor-mediated activation. (A) Expression of Fgr mRNA in NK but not NKT cells. The cDNAs were synthesized from total RNA obtained from IL-2-expanded FACS-sorted spleen NK (NK1.1+TCRαβ-) and NKT (NK1.1+CD4+) cells. PCR was carried out for Fgr, MIST, and β-actin mRNA using the specific primer pairs described in “Materials and methods.” (B) Constitutive association of MIST with Fgr in NK cells. Cell lysates from wild-type NK cells were immunoprecipitated (IP) with anti-MIST or control antibody and immunoblotted with anti-Fgr or anti-MIST antibody. Whole-cell lysates (WCL) were also blotted with the same antibodies. (C) Association of Fgr with the C-terminal proline-rich region of MIST. COS-7 cells were transfected with expression plasmids encoding Fgr (left) in combination with that encoding T7 epitope-tagged wild-type (WT) or mutant forms of MIST lacking either single proline-rich domains (ΔPR1 and ΔPR2) or both (ΔPR1/2) or transfected with MIST plasmid (right) in combination with Fgr (WT) or the nonfunctional SH3 domain mutant (W102A/W103A). Cell lysates were immunoprecipitated with anti-T7 antibody and then blotted with anti-Fgr or anti-MIST antibody. WCLs were also blotted with anti-Fgr antibody, which showed equal expression levels of Fgr protein. (D) Ectopic expression of Fgr attenuated NK1.1-mediated IFN-γ production in MIST-sufficient but not in MIST-deficient NKT cells. IL-2-expanded NKT (NK1.1+CD4+) cells were transduced with a bicistronic retrovirus expressing Fgr with EGFP, stimulated either with anti-NK1.1, anti-CD3, or PMA plus ionomycin (P+I), and analyzed for intracellular IFN-γ. The numbers represent percentages of IFN-γ-producing and nonproducing cells in GFP-positive populations. The percentages of IFN-γ-producing cells in the noninfected cell population are also shown in the top left quadrant. Representative data from 3 independent experiments are shown. (E) IFN-γ release from wild-type (□) and Fgr-deficient NK cells (▪) stimulated with the anti-NK1.1 antibody or IL-12. *P < .05. (F) α-GalCer-induced IFN-γ release from freshly isolated spleen cells from Fgr-deficient (▪) and wild-type mice (□). (G) IFN-γ production by Fgr-deficient NK cells reconstituted with wild-type Fgr, its nonfunctional SH3 domain, or kinase-inactive (KD) mutants. The percentages of IFN-γ-producing and nonproducing cells in the GFP-positive- and -negative populations are shown as in Figure 6D. Data are representative of 2 independent experiments. The retrovirus-mediated expression of wild-type and mutant forms of Fgr protein was confirmed by Western blotting as described in Figure 4A.

Interaction of MIST with Fgr is essential for the attenuation of NK-cell receptor-mediated activation. (A) Expression of Fgr mRNA in NK but not NKT cells. The cDNAs were synthesized from total RNA obtained from IL-2-expanded FACS-sorted spleen NK (NK1.1+TCRαβ-) and NKT (NK1.1+CD4+) cells. PCR was carried out for Fgr, MIST, and β-actin mRNA using the specific primer pairs described in “Materials and methods.” (B) Constitutive association of MIST with Fgr in NK cells. Cell lysates from wild-type NK cells were immunoprecipitated (IP) with anti-MIST or control antibody and immunoblotted with anti-Fgr or anti-MIST antibody. Whole-cell lysates (WCL) were also blotted with the same antibodies. (C) Association of Fgr with the C-terminal proline-rich region of MIST. COS-7 cells were transfected with expression plasmids encoding Fgr (left) in combination with that encoding T7 epitope-tagged wild-type (WT) or mutant forms of MIST lacking either single proline-rich domains (ΔPR1 and ΔPR2) or both (ΔPR1/2) or transfected with MIST plasmid (right) in combination with Fgr (WT) or the nonfunctional SH3 domain mutant (W102A/W103A). Cell lysates were immunoprecipitated with anti-T7 antibody and then blotted with anti-Fgr or anti-MIST antibody. WCLs were also blotted with anti-Fgr antibody, which showed equal expression levels of Fgr protein. (D) Ectopic expression of Fgr attenuated NK1.1-mediated IFN-γ production in MIST-sufficient but not in MIST-deficient NKT cells. IL-2-expanded NKT (NK1.1+CD4+) cells were transduced with a bicistronic retrovirus expressing Fgr with EGFP, stimulated either with anti-NK1.1, anti-CD3, or PMA plus ionomycin (P+I), and analyzed for intracellular IFN-γ. The numbers represent percentages of IFN-γ-producing and nonproducing cells in GFP-positive populations. The percentages of IFN-γ-producing cells in the noninfected cell population are also shown in the top left quadrant. Representative data from 3 independent experiments are shown. (E) IFN-γ release from wild-type (□) and Fgr-deficient NK cells (▪) stimulated with the anti-NK1.1 antibody or IL-12. *P < .05. (F) α-GalCer-induced IFN-γ release from freshly isolated spleen cells from Fgr-deficient (▪) and wild-type mice (□). (G) IFN-γ production by Fgr-deficient NK cells reconstituted with wild-type Fgr, its nonfunctional SH3 domain, or kinase-inactive (KD) mutants. The percentages of IFN-γ-producing and nonproducing cells in the GFP-positive- and -negative populations are shown as in Figure 6D. Data are representative of 2 independent experiments. The retrovirus-mediated expression of wild-type and mutant forms of Fgr protein was confirmed by Western blotting as described in Figure 4A.

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