BLNK Immunity Volume 9, Issue 1, Pages 93-103 (July 1998) Chong Fu, Christoph W. Turck, Tomohiro Kurosaki, Andrew C. Chan  Immunity  Volume 9, Issue 1, Pages 93-103 (July 1998) DOI: 10.1016/S1074-7613(00)80591-9

Figure 1 Characterization of BLNK (A) Biochemical and molecular characterization of BLNK. Top, Commassie blue staining of the purified proteins is shown on the left. The deduced amino acid sequence of human (top) and murine (bottom) BLNK is shown on the right. The seven peptide sequences obtained from protein sequencing are underlined. The 23 amino acids not present in the alternatively spliced form of human BLNK-s (amino acids 203–225) are denoted by the boxed sequence. Bottom, schematic diagrams of hBLNK, hBLNK-s, and hSLP-76. The 470 bp PCR probe is depicted at the top. The percentage of amino acid identity of the various domains between BLNK and SLP-76 is shown. (B) BLNK mRNA is predominantly expressed in the spleen. Human multi-tissue Northern blots (Clontech) were hybridized with BLNK (top) or β-actin (bottom) cDNA probes. (C) BLNK proteins are expressed only in B cell lines. Total cell lysates from different cell lines were immunoblotted with an anti-BLNK antiserum (top) or an anti-β-actin MAb (bottom). Ramos, Daudi, and Raji are human Burkitt lymphoma cells; WEHI-231, A20, and 70Z/3 are mouse B cells; Jurkat and EL4 are transformed T cell lines; K562 is a human erythroleukemia line; Thp-1 is a human monocytic cell line; RBL-1 is a rat basophil leukemia cell line; 293 is a human embryonic kidney line; and LAK represents lymphokine-activated killer cells. Immunity 1998 9, 93-103DOI: (10.1016/S1074-7613(00)80591-9)

Figure 2 Tyrosine Phosphorylation of BLNK by Syk (A) Kinetics of BLNK tyrosine phosphorylation in Daudi cells and mouse B cells. Daudi cells (lanes 1–7) or mouse splenocytes (lanes 8–15) were stimulated for the indicated times with an anti-human IgM F(ab)′2 fragment or an anti-mouse IgM F(ab)′2 fragment, respectively. BLNK was then immunoprecipitated and analyzed by immunoblotting with the indicated antibodies. (B) BLNK tyrosine phosphorylation is affected in Lyn− and in Syk− DT40 cells. Myc-hBLNK was transfected into DT40 cells (lanes 1–2) or its derivative Lyn− (lanes 3–4), Syk− (lanes 5–6), or Btk− (lanes 7–8) cells. Following transfection, the cells were either left unstimulated (lanes 1, 3, 5, and 7) or stimulated with an anti-chicken IgM MAb (M4; lanes 2, 4, 6, and 8). Myc-hBLNK was immunoprecipitated and immunoblotted with the indicated antibodies. (C) Syk phosphorylates BLNK in insect cells. Myc-hBLNK was expressed in Sf9 cells either alone (lane 1) or coexpressed with GST-Syk (lane 2), GST-Lyn (lane 3), or GST-Btk (lane 4). BLNK was immunoprecipitated and analyzed by immunoblotting with the indicated antibodies. Expression levels of the kinases were confirmed by immunoblotting with an anti-GST MAb (bottom panel). Immunity 1998 9, 93-103DOI: (10.1016/S1074-7613(00)80591-9)

Figure 3 BLNK Associates with PLCγ, Vav, Grb2, and Nck (A) Interaction of Myc-BLNK with PLCγ, Vav, Grb2, and Nck. Myc-BLNK(WT) was expressed in Daudi cells and immunoprecipitated from lysates of resting (−) or BCR-activated cells (+). Anti-Myc immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. The bottom panel demonstrates comparable levels of Myc-BLNK(WT) in all immunoprecipitates. Conversely, immunoprecipitates with anti-PLCγ1 (lanes 9–10), anti-Vav (lanes 11–12), anti-Grb2 (lanes 13–14), or anti-Nck (lanes 15–16) antiserum were immunoblotted with an anti-Myc MAb (top panel). The bottom panel demonstrates comparable levels of PLCγ1, Vav, Grb2, or Nck in each immunoprecipitate. IgL, light chain of the immunoprecipitating Ab. (B) Interactions of BLNK and BLNK-s with PLCγ, Vav, Grb2, and Nck. PLCγ1 (lanes 1–2), Vav (lanes 3–4), Grb2 (lanes 5–6), or Nck (lanes 7–8) were immunoprecipitated from resting (−) or BCR-activated Daudi cells (+) and immunoblotted with an anti-BLNK MAb (top panel). The bottom panel demonstrates comparable levels of PLCγ1, Vav, Grb2, and Nck present in resting or BCR-activated conditions. Immunity 1998 9, 93-103DOI: (10.1016/S1074-7613(00)80591-9)

Figure 4 Interaction of BLNK and Grb2 (A) Coimmunoprecipitation of BLNK, Grb2, and SoS. Anti-Myc immunoprecipitates of Daudi cells overexpressing Myc-BLNK(WT) from lysates of resting (lane 1) or BCR-activated cells (lane 2) were immunoblotted with the indicated antibodies. IgL, light chain of the immunoprecipitating Ab. (B) BLNK is translocated into the membrane fraction following BCR stimulation. Cytosolic and membrane fractions were prepared from resting (−), BCR stimulated (+), or pervanadate stimulated (PV) cells as described in Experimental Procedures. Top panel, BLNK was immunoprecipitated from cytosolic (lanes 1–3) or membrane (lanes 4–6) fractions and analyzed by immunoblotting. Four times as many cells were used in the membrane fractions. Bottom three panels, cytosolic and membrane fractions were immunoblotted with the indicated antibodies. Equal numbers of cells were used in each fraction. Analysis of the nonmembrane localized c-Jun kinase 1 (JNK1) and the transmembrane protein CD45 (bottom two panels) confirmed the integrity of the cytosol and membrane fractions, respectively. (C) BLNK/Grb2 complexes are independent of Shc/Grb2 complexes. Shc (lanes 1–2), BLNK (lanes 3–4), or Grb2 (lanes 5–6) were immunoprecipitated from resting (−) or BCR-activated Daudi B cells (+) and immunoblotted for BLNK (top), Shc (middle), and Grb2 (bottom). Immunity 1998 9, 93-103DOI: (10.1016/S1074-7613(00)80591-9)

Figure 5 BLNK Regulates PLCγ Tyrosine Phosphorylation (A) Tyrosine phosphorylation of stably transfected BLNK proteins. Myc-BLNK(WT) or Myc-BLNK(4F) was stably expressed in Daudi cells. BLNK proteins were immunoprecipitated and analyzed by immunoblotting with an anti-PTyr MAb (top panel) or an anti-BLNK MAb (bottom panel). WT, Myc-BLNK(WT); C, vector control; 4F, Myc-BLNK(4F). The data shown here are representative of at least two clones of each transfected cDNA. (B) Regulation of PLCγ1/γ2 tyrosine phosphorylation by BLNK. PLCγ1/γ2 (lanes 1–6) were immunoprecipitated from either resting (−) or BCR-stimulated (+) Daudi clones that overexpress BLNK(WT) (lanes 1–2), vector control (lanes 3–4), or BLNK(4F) (lanes 5–6), and immunoblotted with the indicated antibodies. The antiserum used for immunoprecipitation recognizes both PLCγ1 and PLCγ2. Lanes 7–12 represent Shc immunoprecipitates from either resting (−) or BCR-stimulated (+) Daudi clones that overexpress wild-type BLNK (lanes 7–8), vector control (lanes 9–10), or BLNK(4F) (lanes 11–12) immunoblotted with the indicated antibodies. Similar data were observed from a minimum of two independent clones for each cDNA. (C) BLNK facilitates PLCγ1 tyrosine phosphorylation by Syk. FLAG-tagged PLCγ1 was coexpressed in insect Sf9 cells with Myc-BLNK (lane 1), coexpressed with GST-Syk (lane 2), or coexpressed with GST-Syk and Myc-BLNK (lane 3). Forty hours following infection, FLAG-PLCγ1 was immunoprecipitated and immunoblotted with an anti-PTyr MAb (PY20, top panel). Expression levels of FLAG-PLCγ1, GST-Syk, and Myc-BLNK were confirmed by immunoblotting analysis as demonstrated in the bottom three panels. Immunity 1998 9, 93-103DOI: (10.1016/S1074-7613(00)80591-9)

Figure 6 Regulation of PLCγ-Mediated Responses by BLNK (A) Regulation of [Ca2+]i by BLNK. [Ca2+]i was measured by spectrofluorimetry following stimulation with an anti-hIgM F(ab)′2 Ab (3 μg/ml). The left column depicts two representative clones overexpressing BLNK(WT) or parental control cells. The right column depicts two representative clones expressing BLNK(4F) or parental control cells. This data is representative of greater than ten independent experiments of multiple clones. (B) Increased NF-AT transcriptional activity by BLNK(WT). A control plasmid vector or a vector encoding Myc-BLNK(WT) at the indicated concentrations was transiently cotransfected into 10 A20 cells with a NF-AT reporter plasmid as described in Experimental Procedures. Induction of NF-AT activity was analyzed in resting cells, cells stimulated with an anti-mouse IgG F(ab)′2 fragment (4.0 μg/ml), or cells stimulated with 0.5 μg/ml PDBu and 0.5 μM ionomycin for 6 hr. Data are graphed as percentage of luciferase activity compared to cells stimulated with PDBu plus ionomycin. Cells (2 × 105) were lysed and analyzed for Myc-BLNK overexpression, as compared to endogenous BLNK in the right panel. Data are representative of greater than five independent experiments. (C) Attenuation of NF-AT transcriptional activity by BLNK(4F). A control plasmid vector or the indicated concentrations of a vector encoding Myc-BLNK(4F) was transiently cotransfected into A20 cells with a NF-AT reporter plasmid as described in Figure 6B. The level of expression of Myc-BLNK(4F) as compared to endogenous BLNK was determined by immunoblotting with an anti-BLNK antiserum (right panel). Data are representative of greater than five independent experiments. Immunity 1998 9, 93-103DOI: (10.1016/S1074-7613(00)80591-9)

Figure 7 BLNK, a Central Linker Protein in BCR Activation Studies in T cells suggest that the TCR-associated PTKs ZAP-70 and Syk phosphorylate two linker proteins, SLP-76 and LAT. Neither SLP-76 nor LAT are expressed in B cells. Our data here demonstrate that BLNK associates with Vav, Grb2, PLCγ, and Nck, suggesting that B cells utilize a single linker protein in linking the Syk PTK with these downstream effector proteins. Immunity 1998 9, 93-103DOI: (10.1016/S1074-7613(00)80591-9)