Fig. 1. STAT5 is phosphorylated by BCR/ABL immunoprecipitates. (A) STAT5 phosphorylation was examined in anti-ABL immunoprecipitates obtained from IL-3- and serum-starved 32Dcl3 cells expressing BCR/ABL wild-type (WT) or the kinase-defective mutant (K1172R), using full-length STAT5 or the C5 (amino acids 547–787) and N5 (amino acids 1–546) fragments conjugated to GST as substrates and [γ-³²P]ATP (upper box). GST alone was not phosphorylated (data not shown). GST–STAT5 proteins loaded onto the gel were detected by western blot using anti-STAT5B antibodies (C-17 recognizes the C- terminus and N-20 recognizes the N-terminus) (middle box). Arrows 1, 2 and 3 indicate the position of full-length GST–STAT5, GST–N5 and GST–C5, respectively. GST was detected by Ponceau red staining (not shown). Immunoprecipitated BCR/ABL proteins (arrow 4) were visualized by western analysis using anti-ABL antibody (bottom box). (B) Phosphorylation of GST–C5 (C5) and enolase (Eno) was examined in anti-ABL immunoprecipitates obtained from IL-3- and serum-starved 32Dcl3 cells (Parental) and cells expressing BCR/ABL kinase in the absence (–) or presence (+) of 1 µM STI571. C5 and enolase substrates were detected by Coomassie Blue staining of the gel. Results represent three independent experiments.

Fig. 2. Pattern of phosphotyrosine proteins co-immunoprecipitating with STAT5. STAT5 was immunoprecipitated from the lysates of 32Dcl3 cells (Parental) and cells expressing BCR/ABL or the BCR/ABLΔΔ mutant, after being starved of IL-3 for 12 h. The immunoprecipitates were analyzed by western analysis with anti-P.Tyr antibodies (upper panel). Subsequent blotting with anti-STAT5, anti-ABL and anti-Hck confirmed the localization of the indicated proteins (lower panels). An equal amount of STAT5 was immunoprecipitated in each sample. Results represent two independent experiments.

Fig. 3. The BCR/ABL SH3 + SH2 region interacts with the Hck–STAT5 complex. 32Dcl3 cells (Parental) and cells expressing BCR/ABL, or the indicated BCR/ABL mutants, were starved of IL-3 for 5 h. STAT5 immunoprecipitates (A) and Hck immunoprecipitates (B) were examined by SDS–PAGE followed by western analysis with anti-STAT5, anti-Hck, anti-P.Tyr and anti-ABL antibodies. Hck immuno precipitation and kinase assay (C) were performed to detect its association with BCR/ABL and to measure its catalytic activity. Hck immunoprecipitates were examined by SDS–PAGE followed by western analysis with anti-ABL (upper panel) and anti-anti-Hck (bottom panel) antibody. An in vitro kinase reaction was performed in these immunoprecipitates using [γ-³²P]ATP and 5 µg of the standard substrate for the Src-related family of tyrosine kinases, Sam68 (middle panel). Results represent two or three independent experiments.

Fig. 4. The BCR/ABL SH3 + SH2 region is required for activation of Hck and phosphorylation of STAT5B on Y699. (A) Hck was immunoprecipitated from cell lysates obtained from 32Dcl3 parental cells (Parental) and from cells expressing BCR/ABL or the BCR/ABLΔΔ mutant after being starved of IL-3 for 5 h. An in vitro kinase reaction was performed using [γ-³²P]ATP and 5 µg of the substrates: Sam68 (lower panel) or GST–C5 (upper panel). (B) Hck was immunoprecipitated from BCR/ABL cells. GST–C5 (C5) or GST–C5[Y699F] mutant (C5-YF) proteins were added as substrates along with [γ-³²P]ATP. (C) Wild-type Hck and Hck-KE were immunopurified from infected Sf9 cell lysates and incubated in vitro with [γ-³²P]ATP alone (Control) or together with the C5 or C5-YF proteins. The kinase reactions were resolved by SDS–PAGE. Phosphorylation of GST fusion proteins was assessed by autoradiography (³²P.C5 and ³²P.Sam68 boxes). The presence of Hck in each reaction was verified by immunoblotting (Hck panel). Results represent three independent experiments.

Fig. 5. Analysis of STAT5 activation by Hck in Sf9 insect cells. (A) STAT5, wild-type Hck (Hck-WT) and kinase-defective Hck (Hck-KE) were expressed in Sf9 insect cells. Uninfected Sf9 cells were included as a negative control (Control). STAT5 was immunoprecipitated from infected cell lysates and analyzed for phosphotyrosine content by immunoblotting with anti-phosphotyrosine antibodies (P-Tyr. panel). Equivalent recovery of STAT5 was verified by immunoblotting an aliquot of each immunoprecipitate with anti-STAT5 antibodies (STAT5 panel). Hck protein expression was verified by immunoblotting of the cell lysates (bottom). (B) Sf-9 cells were infected with recombinant baculoviruses carrying the indicated cDNAs. Aliquots of the nuclear extracts were tested for STAT5–DNA binding activity by EMSA using a [γ-³²P]ATP-labeled MGFE probe. Results are representative of three independent experiments.

Fig. 6. Hck is a required intermediate in STAT5 activation by BCR/ABL. (A) Parental 32Dcl3 cells and BCR/ABL-positive cells were transfected with IRES–GFP (E), [Hck-YF]-IRES–GFP or [Hck-KE]-IRES–GFP retroviral particles or incubated with STI571, PP2 and PP3. GFP-positive cells and inhibitor-treated cells were examined for STAT5 binding ability by EMSA using a [γ-³²P]ATP-labeled fragment of the FcγRI probe (upper large boxes). STAT5 tyrosine phosphorylation (P-Tyr.STAT5 boxes) was analyzed by immunoprecipitation followed by western analysis using anti-P.Tyr antibodies. Hck and BCR/ABL kinase activities were assayed as described in Figures 4A and 1B, respectively (*P.Sam68 and ³²P.Eno boxes, respectively). STAT5, Hck and BCR/ABL proteins were detected in the immunoprecipitates by western analysis (STAT5, Hck and BCR/ABL boxes, respectively). (B) A transactivation assay was performed in Tk^–ts13 cells transfected with the β-casein–luciferase reporter construct and the indicated constructs, and treated with the inhibitors. Activation of the luciferase gene is shown in arbitrary units in comparison with the control group transfected with the reporter plasmid. (C) Western analysis of the levels of A1 and pim-1 proteins in 32Dcl3 parental cells and cells expressing the indicated BCR/ABL and/or Hck proteins. The cells eventually were treated with the inhibitors as indicated. Actin was detected as a loading control. Results represent the mean ± SD from two or three independent experiments.