PMC

Reduced efficiency and extended latency of the Jak1 binding mutant of v-Abl in tumorigenesis. Nude mice were injected with either parental IL-3-dependent BAF/3 cells (■) or BAF/3 transfectants expressing wild-type (•) or Δ858–1080 mutant (▴) v-Abl and monitored for visible signs of growth during the 19-day period postinjection. Data from three experiments are pooled. The total numbers of mice injected per cell line were as follows: 5 for parental IL-3-dependent BAF/3 cells, 16 for BAF/3 transfectants expressing p160 v-Abl, and 22 for BAF/3 transfectants expressing Δ858–1080.

Functional requirement of Jak1 in v-Abl-induced mitogenesis and STAT activation independent of cytokines. (A) Induction of a kinase-inactive Jak1 leads to decreased v-Abl-induced, cytokine-independent proliferation. BAF/3 cells stably expressing p160 wild-type v-Abl (160.8) or two independently derived clones expressing p160 v-Abl and a construct encoding a kinase-inactive/dominant negative Jak1 (160.8/DNJ1.7 and 160.8/DNJ1.14) under the control of the metallothionine promoter were incubated overnight in 10⁻⁴ M (final concentration) ZnSO₄. [³H]thymidine incorporation assays were performed after 24 h, as in Fig. A. Extracts were made from the same sample of cells to examine the induction of DNJ1 protein by Western blotting (insets). (B) Induction of dominant negative Jak1 leads to decreased STAT activation. Lysates from 160.8/DNJ1.14 cells were precleared, and Stat1, Stat3, and Stat5 were immunoprecipitated (IP); their phosphorylation was analyzed as described in the legend to Fig. C. WB, Western blotting.

Activation of other signaling pathways by the Δ858–1080 Jak1 binding mutant. (A and C) Lysates prepared from BAF/3 transfectants expressing p160 v-Abl or the Δ858–1080 mutant form of the protein were immunoprecipitated with anti-Shc (A) or anti-p62 Dok (C) antibodies. Immune complexes were fractionated by SDS-PAGE. The activation state of the proteins was examined by blotting the membranes with an antiphosphotyrosine antibody. The membranes were then stripped and reprobed with anti-Shc or anti-Dok antibodies. (B) A 30-μg portion of total protein cell lysate was fractionated on SDS-PAGE and immunoblotted with an antibody to c-Myc. Membranes were stripped and reprobed with an antibody to β-actin to control for protein loading. (D) Extracts from cytokine-starved cells (lanes 1 to 4) or pre-B cells transformed with the constitutively active form of Ras (v-H Ras [lane 5]) were incubated with glutathione beads coupled to GST Raf RBD fusion protein. Bound materials were washed, fractionated on SDS-PAGE (12.5% gel), and immunoblotted with an anti-Ras antibody. As a positive control for Ras protein, Ras was immunoprecipitated from v-H Ras-transformed pre-B cells (lane 6). The GST moiety alone does not bind Ras (data not shown).

Requirement for the Jak1 binding domain of v-Abl in cytokine-independent proliferation, and the activation of Jak1 and STAT proteins in BAF/3 pro-B cells. (A) The Jak1 binding mutant (Δ858–1080) of v-Abl cannot support the proliferation of BAF/3 pro-B cells in the absence of IL-3. IL-3-dependent parental BAF/3 cells or BAF/3 transfectants expressing p160 v-Abl (160.8) or the Jak1 binding mutant (Δ858–1080) were washed and used in [³H]thymidine incorporation assays in the absence of IL-3 for 24 or 48 h. (B) The Δ858–1080 mutant of v-Abl cannot activate Jak1 in the absence of cytokines. Jak1 was immunoprecipitated (IP) from precleared lysates prepared from BAF/3 transfectants stably expressing wild-type (160.8 and 160.6) or Jak1 binding mutant (Δ858–1080) forms of v-Abl. Immune complexes were subjected to an in vitro kinase assay. Half of each sample was analyzed by SDS-PAGE (7% gel) and autoradiography, and the other half was blotted (WB) with an antibody to Jak1 to control for equal amounts of protein immunoprecipitated. (C) The Δ858–1080 mutant of v-Abl cannot activate STATs in the absence of cytokines. Stat1, Stat3, and Stat5 were immunoprecipitated (IP) from precleared lysates prepared from BAF/3 cells stably expressing the wild-type or Δ858–1080 mutant forms of v-Abl. Immune complexes were fractionated by SDS-PAGE and immunoblotted (WB) with an antiphosphotyrosine antibody. The blots were then stripped and reprobed with antibodies to specific STATs as indicated. As a control for tyrosine phosphorylation of specific STATs, parental BAF/3 cells were washed extensively and starved of IL-3 for 2 h. They were then either left untreated or treated for 15 min with WEHI (IL-3) conditioning medium, TPO (for BAF/3 cells stably expressing the TPO receptor), or gamma interferon (IFNγ).

Mapping the Jak1 interaction domain of v-Abl in vitro and in vivo. (A) GST fusion proteins spanning various domains of v-Abl, including the src homology 2 and the protein tyrosine kinase domains (SH2-PTK, aa 237 to 645), the proline-rich and the DNA binding domains (aa 706 to 1080), and the F-actin binding domain (aa 1081 to 1244), or smaller GST fusion proteins spanning aa 706 to 857, 858 to 981, and 982 to 1080 were expressed in bacteria and bound to glutathione beads. (B and C) Equal amounts of fusion proteins were incubated with 1 mg of whole-cell extracts prepared from a murine pre-B-cell line (clone K) (B) or 1 to 3 μg of purified Jak1 protein (C). Bound materials were washed extensively, eluted off the beads, fractionated by SDS-PAGE (7% polyacrylamide), and blotted with an antibody to Jak1. The GST moiety alone and the SH2 domain of Crk were used as controls for the specificity of the GST fusion protein-Jak1 interaction. Jak1 immunoprecipitates from the same whole-cell extracts (panel B, lane 6) were included as control for the Jak1 protein. (D) IL-3-dependent BAF/3 cells were stably transfected with v-Abl expression constructs encoding the wild-type p160 v-Abl or the Jak1 binding mutant Δ858–1080. A 1-mg portion of total protein extracts from the transfectants was immunoprecipitated (IP) with an antibody to Jak1. Immune complexes were fractionated by SDS-PAGE (7% polyacrylamide) and subjected to Western blotting (WB) with an Abl antibody. The blot was stripped and probed with a Jak1-specific antibody to control for the amount of Jak1 immunoprecipitated.