Oda T, Heaney C, Hagopian J R, Okuda K, Griffin J D, Druker B J
Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland 97201.
J Biol Chem. 1994 Sep 16;269(37):22925-8.
The Philadelphia chromosome (Ph1), detected in virtually all cases of chronic myelogenous leukemia (CML), is formed by a reciprocal translocation between chromosome 9 and 22 that fuses Bcr-encoded sequences upstream of exon 2 of c-Abl. This oncogene produces a fusion protein, p210bcr-abl, in which the Abl tyrosine kinase activity is elevated. Using anti-phosphotyrosine immunoblotting, we have compared the pattern of phosphotyrosine-containing proteins from freshly prepared neutrophils of patients in the stable phase of CML to normal controls. The only consistent difference was the presence of a 39-kDa tyrosine-phosphorylated protein in 18 out of 18 neutrophil samples from CML patients that was not seen in normal controls. This same protein, as assessed by two-dimensional anti-phosphotyrosine immunoblotting, was also present in cell lines expressing p210bcr-abl, including K562 cells. Using K562 cells as a source of protein, the 39-kDa protein was purified and identified by microsequencing as Crkl, an SH2/SH3 adaptor protein related to the crk oncogene of the avian sarcoma virus, CT10. A direct interaction between Crkl and Abl has also been shown using a yeast two-hybrid screen.
费城染色体（Ph1）在几乎所有慢性粒细胞白血病（CML）病例中均可检测到，它由9号和22号染色体之间的相互易位形成，该易位使c-Abl外显子2上游的Bcr编码序列融合。这种致癌基因产生一种融合蛋白p210bcr-abl，其中Abl酪氨酸激酶活性升高。我们使用抗磷酸酪氨酸免疫印迹法，比较了处于CML稳定期患者新鲜制备的中性粒细胞与正常对照中含磷酸酪氨酸蛋白的模式。唯一一致的差异是，在18份CML患者的中性粒细胞样本中均存在一种39 kDa的酪氨酸磷酸化蛋白，而正常对照中未见。通过二维抗磷酸酪氨酸免疫印迹评估，表达p210bcr-abl的细胞系（包括K562细胞）中也存在同样的蛋白。以K562细胞作为蛋白质来源，纯化了这种39 kDa的蛋白，并通过微量测序鉴定为Crkl，它是一种与禽肉瘤病毒CT10的crk致癌基因相关的SH2/SH3衔接蛋白。使用酵母双杂交筛选也已证明Crkl与Abl之间存在直接相互作用。
