(A) Sequence of BAP-135. The nucleotide sequence is shown on the upper line; the deduced amino acid sequence is shown on the lower line. Sequences of tryptic peptides used to identify BAP-135 cDNA are underlined. (B) Identification of a repetitive amino acid motif in BAP-135. The BAP-135 amino acid sequence is diagrammed (Upper). The 95-residue repeat unit (hatched boxes), sequences homologous to the Src autophosphorylation site (open boxes), and regions of unique sequence (solid boxes) are indicated. Numbers refer to amino acid residues at the boundaries of repeat units. Amino acid sequences of the individual repeats are compared (Lower). Residues are numbered at left and right; amino acid identities, with reference to the second repeat (residues 304–398), are shaded. A consensus sequence (core) was derived by comparison of the four full-length repeats (residues 304–398, 409–503, 514–608, and 676–770). (C) BAP-135 is associated with Btk in B cells. Immunoprecipitation from lysates of pervanadate-stimulated (lanes 6–10) or unstimulated (lanes 1–5) RAMOS cells was performed using anti-Btk antibodies (Ab1280, lanes 2 and 7; Ab1300, lanes 4 and 9), preimmune sera (lanes 1, 3, 6, and 8), or rabbit IgG (lanes 5 and 10). BAP-135 (Upper) was detected by immunoblotting with Ab2240; Btk (Lower) was detected with Ab1280. Epitope-tagged (lane 12) and untagged (lane 13) BAP-135 coding sequences were transferred to the expression vector pCIS-2 and expressed in 293 cells by transient transfection; BAP-135 was detected in total lysates of untransfected (lane 11) and transfected cells by immunoblotting.

Association of Btk with BAP-135 in transfected cells. (A) Cells expressing Btk (lanes 1–4), epitope-tagged BAP-135 (lanes 5–8), or both (lanes 9–12) were lysed in RIPA buffer containing protease inhibitors. Btk was immunoprecipitated in the presence of 0 μg (lanes 1, 5, and 9), 1 μg (lanes 2, 6, and 10), 2 μg (lanes 3, 7, and 11), or 5 μg (lanes 4, 8, and 12) of affinity-purified Ab1280. Precipitates were washed three times with RIPA buffer, once with a buffer containing 0.5 M NaCl and 50 mM Tris (pH 7.4) and twice with water. Proteins were separated by SDS/PAGE; epitope-tagged BAP-135 was detected by immunoblotting with antibody 9E10 (Upper, lanes 1–12) and Btk by Ab1280 (Lower, lanes 1–12). Total cell lysates were fractionated in lanes 13–15 at 1/10 the amount used for immunoprecipitation, and overexpressed BAP-135 was visualized by staining with amido black. (B) 293 cells expressing Btk (lanes 2 and 8), kinase-inactive Btk (lanes 3 and 9), epitope-tagged BAP-135 (lanes 4 and 10), Btk and epitope-tagged BAP-135 (lanes 5 and 11), kinase-inactive Btk and epitope-tagged BAP-135 (lanes 6 and 12), and cells transfected with vector alone (lanes 1 and 7) were lysed in the presence of 1% Nonidet P-40 (12). Before lysis, cells were treated with pervanadate (lanes 7–12) or untreated (lanes 1–6). Immunoprecipitation of epitope-tagged BAP-135 was carried out with antibody 9E10 and proteins were fractionated by SDS/PAGE (IP myc). In the upper three panels, Btk was detected by immunoblotting with Ab1280 (Btk, Top), tyrosine phosphorylated BAP-135 was detected by immunoblotting with antibody 4G10 (p135m [P-tyr], Middle) and BAP-135 protein was detected by amido black staining (p135m [stain], Bottom). In the lower two panels, total cell lysates were fractionated at one-tenth the amount used for immunoprecipitation (lysate); BAP-135 was detected by immunoblotting with 9E10 (p135m [myc], Upper) and Btk was detected with Ab1280 (Btk, Lower).

BAP-135 is phosphorylated on tyrosine in response to surface immunoglobulin crosslinking. (A) Serum-starved RAMOS cells were stimulated with goat anti-human IgM F(ab′)₂ (μ heavy chain-specific; lanes 4–10) or goat anti-mouse IgG F(ab′)₂ (lanes 1–3) for the indicated times at 37°C. Cells were lysed in RIPA buffer and BAP-135 was immunoprecipitated under conditions described in Fig. 3C. Following fractionation by SDS/PAGE, phosphotyrosine was detected by immunoblotting (Upper); BAP-135 was subsequently detected by probing with Ab2240 (Lower). (B) BCR-induced tyrosine phosphorylation of Btk and BAP-135 occurs within a preformed complex. RAMOS cells were stimulated with goat anti-human IgM F(ab′)₂ (lane 2) or goat anti-mouse IgG F(ab′)₂ (lane 1) on ice for 10 min, followed by incubation with rabbit anti-goat IgG at 37°C for 10 min. Cells were lysed in the presence of 1% Nonidet P-40 and Btk was immunoprecipitated. Phosphotyrosine-containing proteins (P-tyr, Top), BAP-135 (p135, Middle), and Btk (Btk, Bottom) were detected by sequential immunoblotting.

Btk is associated with proteins of 135 and 140 kDa in B lymphoblastoid cells. (A) Coimmunoprecipitation assays. RAMOS cells were treated for 10 min with 1 mM pervanadate (lanes 1–4) or they were left untreated (lanes 5–8), and they were all lysed in the presence of 1% Nonidet P-40 (12). Immunoprecipitation was performed using 10 μl of preimmune sera (lanes 2, 4, 6, and 8) or anti-Btk antisera (lanes 1, 3, 5, and 7) as described (12). Phosphotyrosine-containing proteins present in immune complexes were detected by immunoblotting with anti-phosphotyrosine antibody 4G10 (Upper). Btk was detected using anti-Btk antibody Ab1280 (Lower). Btk is indicated by the open arrow; p135 and p140 by filled arrows. (B) Immune complex kinase assays. Btk was immunoprecipitated from lysates of pervanadate-stimulated (lanes 1 and 2) or unstimulated (lanes 3 and 4) RAMOS cells using affinity-purified antibody Ab1300 in the presence (lanes 2 and 4) or absence (lanes 1 and 2) of competitor peptide. Immune complex kinase assays were carried out as described (15). Protein was fractionated by 7.5% SDS-PAGE and detected by silver staining (Lower); after alkalai treatment to preferentially dephosphorylate phosphoserine and phosphothreonine residues, ³²P was detected by autoradiography (Upper). Positions and masses of standards (in kDa) are indicated at left.

Specific binding of BAP-135 to the combined PH and TH domains of Btk in vitro. (A) A lysate of 293 cells expressing BAP-135 was diluted 3-fold serially and incubated with agarose beads bearing GST (lanes 1–4), or GST fused to residues 1–218 (PH/TH, lanes 5–8), 219–268 (SH3, lanes 9–12), or 269–382 (SH2, lanes 13–16) of Btk. Retained protein was fractionated by SDS/PAGE and tagged BAP-135 was detected with anti-c-myc antibody 9E10. An amount of total lysate corresponding to the highest dilution used in binding assays was run in lane 17. (B) Binding assays were performed as in A, using GST fusions to residues 1–80 (PH, lanes 5–7), 104–218 (TH, lanes 8–10), or 1–218 (PH/TH, lanes 11–13) of Btk.

Btk-dependent tyrosine phosphorylation of BAP-135. 293 cells were transfected with vector alone (lane 1), Btk alone (lane 2), epitope-tagged BAP-135 alone (lane 3), epitope-tagged BAP-135 with wild-type Btk (lane 4), or epitope-tagged BAP-135 with the following Btk mutants: R28C (lane 5), E41K (lane 6), P189A (lane 7), Y223F (lane 8), W251L (lane 9), R307K (lane 10), K430E (lane 11), and Y551F (lane 12). Total cell lysates were fractionated by SDS/PAGE, and phosphotyrosine-containing proteins were detected by immunoblotting with antibody 4G10 (P-tyr, Top). The filter was subsequently probed for Btk using Ab1280 (Btk, Middle), and BAP-135 was detected by amido black staining (p135m [stain], Bottom).