Fig. 2.
Fig. 2. SAP interacts with the phosphorylated cytoplasmic tail of Ly-9. / A human cDNA library made from poly A+ RNA of the human T-cell line KT3 in pGAD424 was screened with the altered yeast 2-hybrid system. Thus, 5 cDNA clones encoding the cytoplasmic tail of Ly-9 were isolated; an example is shown in panel A. To map the binding sites in the cytoplasmic tail of human Ly-9, 3 mutations of Ly-9 were analyzed in panel B. SAP binding to phospho-Ly-9 was shown in murine thymocytes in panel C. (A) Interactions of SAP and the cytoplasmic tail of Ly-9 in yeast are dependent on the presence of Fyn 420, 531 Y-F. The interaction of SAP with the cytoplasmic tail of Ly-9 in the presence or absence of Fyn 420, 531 Y-F took place in the yeast cell and was measured in a β-galactosidase assay. For each construct, at least 3 independent colonies were tested in the galactosidase assay. Open bars: cells transfected with empty pBRIDGE vector and pGAD424 encoding the cytoplasmic tail of Ly-9; solid bars: cells transfected with pBRIDGE-SAP and pGAD424 encoding the cytoplasmic tail of Ly-9; hatched bars: cells transfected with pBRIDGE-SAP and Fyn420, 531 Y-F and with pGAD424 encoding the cytoplasmic tail of Ly-9. Control, empty pGAD424 with pBRIDGE encoding the indicated DNA sequences. (B) SAP interacts with 2 phosphotyrosine motifs in the cytoplasmic tail of Ly-9. The interaction of SAP with 3 Ly-9 cytoplasmic tail mutations in the presence or absence of Fyn420, 531 Y-F took place in the yeast cell and was measured in a β-galactosidase assay. For each construct, at least 3 independent colonies were tested in the galactosidase assay. The cytoplasmic tail of Ly-9 was mutated in tyrosine residue 558 (558-YF), tyrosine 581 (581-YF), or in both tyrosine 558 and tyrosine 581 (558-581-YF). Open bars: cells transfected with empty pBRIDGE vector and pGAD424 encoding the cytoplasmic tail of Ly-9. Solid bars: cells transfected with pBRIDGE-SAP and pGAD424 encoding the cytoplasmic tail of Ly-9 or one of the Ly-9 mutants. Hatched bars: cells transfected with pBRIDGE-SAP and Fyn420, 531 Y-F and with pGAD424 encoding the cytoplasmic tail of Ly-9 or one of the Ly-9 mutants. CONTROL, empty pGAD424 with pBRIDGE encoding the indicated DNA sequences. (C) Association of SAP and Ly-9 in mouse thymocytes. Mouse thymocytes (50 × 106 cells/mL) were biotinylated and then incubated in the presence or absence of 1 mM pervanadate. Cells were lysed, and Ly-9 was immunoprecipitated with 1 μg antimouse Ly-9 monoclonal antibody (IP aLy9). Proteins were transferred to PVDF, and Western blot analysis (WB) identified specific proteins with streptavidin, antiphosphotyrosine (WB α-PY) and a rabbit antimouse SAP antibody (WB α-SAP). CONTROL, immunoprecipitation with a monoclonal hamster antibody.

SAP interacts with the phosphorylated cytoplasmic tail of Ly-9.

A human cDNA library made from poly A+ RNA of the human T-cell line KT3 in pGAD424 was screened with the altered yeast 2-hybrid system. Thus, 5 cDNA clones encoding the cytoplasmic tail of Ly-9 were isolated; an example is shown in panel A. To map the binding sites in the cytoplasmic tail of human Ly-9, 3 mutations of Ly-9 were analyzed in panel B. SAP binding to phospho-Ly-9 was shown in murine thymocytes in panel C. (A) Interactions of SAP and the cytoplasmic tail of Ly-9 in yeast are dependent on the presence of Fyn 420, 531 Y-F. The interaction of SAP with the cytoplasmic tail of Ly-9 in the presence or absence of Fyn 420, 531 Y-F took place in the yeast cell and was measured in a β-galactosidase assay. For each construct, at least 3 independent colonies were tested in the galactosidase assay. Open bars: cells transfected with empty pBRIDGE vector and pGAD424 encoding the cytoplasmic tail of Ly-9; solid bars: cells transfected with pBRIDGE-SAP and pGAD424 encoding the cytoplasmic tail of Ly-9; hatched bars: cells transfected with pBRIDGE-SAP and Fyn420, 531 Y-F and with pGAD424 encoding the cytoplasmic tail of Ly-9. Control, empty pGAD424 with pBRIDGE encoding the indicated DNA sequences. (B) SAP interacts with 2 phosphotyrosine motifs in the cytoplasmic tail of Ly-9. The interaction of SAP with 3 Ly-9 cytoplasmic tail mutations in the presence or absence of Fyn420, 531 Y-F took place in the yeast cell and was measured in a β-galactosidase assay. For each construct, at least 3 independent colonies were tested in the galactosidase assay. The cytoplasmic tail of Ly-9 was mutated in tyrosine residue 558 (558-YF), tyrosine 581 (581-YF), or in both tyrosine 558 and tyrosine 581 (558-581-YF). Open bars: cells transfected with empty pBRIDGE vector and pGAD424 encoding the cytoplasmic tail of Ly-9. Solid bars: cells transfected with pBRIDGE-SAP and pGAD424 encoding the cytoplasmic tail of Ly-9 or one of the Ly-9 mutants. Hatched bars: cells transfected with pBRIDGE-SAP and Fyn420, 531 Y-F and with pGAD424 encoding the cytoplasmic tail of Ly-9 or one of the Ly-9 mutants. CONTROL, empty pGAD424 with pBRIDGE encoding the indicated DNA sequences. (C) Association of SAP and Ly-9 in mouse thymocytes. Mouse thymocytes (50 × 106 cells/mL) were biotinylated and then incubated in the presence or absence of 1 mM pervanadate. Cells were lysed, and Ly-9 was immunoprecipitated with 1 μg antimouse Ly-9 monoclonal antibody (IP aLy9). Proteins were transferred to PVDF, and Western blot analysis (WB) identified specific proteins with streptavidin, antiphosphotyrosine (WB α-PY) and a rabbit antimouse SAP antibody (WB α-SAP). CONTROL, immunoprecipitation with a monoclonal hamster antibody.

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