Lionberger J M, Wilson M B, Smithgall T E
Department of Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
J Biol Chem. 2000 Jun 16;275(24):18581-5. doi: 10.1074/jbc.C000126200.
Bcr-Abl is the constitutively active protein-tyrosine kinase expressed as a result of the Philadelphia translocation in chronic myelogenous leukemia. Bcr-Abl is coupled to many of the same signaling pathways normally regulated by hematopoietic cytokines. Recent work shows that Hck, a member of the Src tyrosine kinase family with myeloid-restricted expression, associates with and is activated by Bcr-Abl. Here we investigated the mechanism of Hck interaction with Bcr-Abl and the requirement for Hck activation in Bcr-Abl transformation signaling. Binding studies demonstrated that the Hck SH3 and SH2 domains are sufficient for interaction with Bcr-Abl in vitro. Hck binding localizes to the Abl SH2, SH3, and kinase domains as well as the distal portion of the C-terminal tail. To address the requirement for endogenous Src family kinase activation in Bcr-Abl signaling, a kinase-defective mutant of Hck was stably expressed in the cytokine-dependent myeloid leukemia cell line DAGM. Kinase-defective Hck dramatically suppressed Bcr-Abl-induced outgrowth of these cells in the absence of cytokine compared with a control cell line expressing beta-galactosidase. In contrast, kinase-defective Hck did not affect cell proliferation in response to interleukin-3, suggesting that the effect is specific for Bcr-Abl. These data show that Hck interacts with Bcr-Abl through a complex mechanism involving kinase-dependent and -independent components and that interaction with Hck or other Src family members is essential for transformation signaling by Bcr-Abl.
Bcr-Abl是一种组成型活性蛋白酪氨酸激酶，由慢性粒细胞白血病中的费城染色体易位产生。Bcr-Abl与许多通常由造血细胞因子调节的相同信号通路相关联。最近的研究表明，Hck是Src酪氨酸激酶家族的成员，具有髓系限制性表达，与Bcr-Abl相关联并被其激活。在这里，我们研究了Hck与Bcr-Abl相互作用的机制以及Bcr-Abl转化信号中Hck激活的必要性。结合研究表明，Hck的SH3和SH2结构域足以在体外与Bcr-Abl相互作用。Hck的结合定位于Abl的SH2、SH3和激酶结构域以及C末端尾巴的远端部分。为了研究内源性Src家族激酶激活在Bcr-Abl信号传导中的必要性，在细胞因子依赖性髓系白血病细胞系DAGM中稳定表达了Hck的激酶缺陷突变体。与表达β-半乳糖苷酶的对照细胞系相比，激酶缺陷的Hck在没有细胞因子的情况下显著抑制了Bcr-Abl诱导的这些细胞的生长。相反，激酶缺陷的Hck不影响细胞对白介素-3的增殖反应，表明这种作用对Bcr-Abl具有特异性。这些数据表明，Hck通过一种涉及激酶依赖性和非依赖性成分的复杂机制与Bcr-Abl相互作用，并且与Hck或其他Src家族成员的相互作用对于Bcr-Abl的转化信号传导至关重要。
