Uemura N, Salgia R, Li J L, Pisick E, Sattler M, Griffin J D
Division of Hematologic Malignancies, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA.
Leukemia. 1997 Mar;11(3):376-85. doi: 10.1038/sj.leu.2400590.
The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary leukemic neutrophils from patients with CML, the major tyrosine phosphorylated protein is CRKL, an SH2-SH3-SH3 adapter protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene. In cell lines transformed by BCR/ABL, CRKL was tyrosine phosphorylated, while CRK was not. We looked for changes in CRK- and CRKL-binding proteins in Ba/F3 hematopoietic cell lines which were transformed by BCR/ABL. Anti-CRK II or anti-CRKL immunoprecipitates were probed by far Western blotting with CRK II- or CRKL-GST fusion proteins to display CRK- and CRKL-coprecipitating proteins. There was a striking qualitative difference in the proteins coprecipitating with CRKL and CRK II. In untransformed cells, three major proteins coprecipitated with CRKL, identified as C3G, SOS and c-ABL. Each of these proteins was found to interact with the CRKL-SH3 domains, but not the SH2 domain. After BCR/ABL transformation, the CRKL SH3-domain binding proteins did not change, with the exception that BCR/ABL now coprecipitated with CRKL. Compared to CRKL, very few proteins coprecipitated with CRK II in untransformed, quiescent cells. After BCR/ABL transformation, both the CRKL- and CRK-SH2 domains bound to a new complex of proteins of approximate molecular weight 105-120 kDa. The major protein in this complex was identified as p120CBL. Thus, in these hematopoietic cell lines, CRKL is involved to a greater extent than CRK II in normal signaling pathways that involve c-ABL, C3G and SOS. In BCR/ABL-transformed cells, CRKL but not CRK II, appears to form complexes which potentially link BCR/ABL, c-ABL, C3G, and SOS to the protooncoprotein, p120CBL.
费城染色体易位产生一种嵌合致癌基因BCR/ABL，它会引发慢性粒细胞白血病（CML）。在CML患者的原发性白血病中性粒细胞中，主要的酪氨酸磷酸化蛋白是CRKL，它是一种SH2-SH3-SH3衔接蛋白，与v-crk致癌基因的人类同源物CRK总体同源性为60%。在由BCR/ABL转化的细胞系中，CRKL发生酪氨酸磷酸化，而CRK未发生。我们在由BCR/ABL转化的Ba/F3造血细胞系中寻找CRK和CRKL结合蛋白的变化。用CRK II或CRKL-GST融合蛋白进行远缘Western印迹法检测抗CRK II或抗CRKL免疫沉淀产物，以显示与CRK和CRKL共沉淀的蛋白。与CRKL和CRK II共沉淀的蛋白存在显著的定性差异。在未转化的细胞中，有三种主要蛋白与CRKL共沉淀，分别鉴定为C3G、SOS和c-ABL。发现这些蛋白中的每一种都与CRKL的SH3结构域相互作用，但不与SH2结构域相互作用。在BCR/ABL转化后，CRKL的SH3结构域结合蛋白没有变化，只是BCR/ABL现在与CRKL共沉淀。与CRKL相比，在未转化的静止细胞中，与CRK II共沉淀的蛋白很少。在BCR/ABL转化后，CRKL和CRK的SH2结构域都与一种分子量约为105 - 120 kDa的新蛋白复合物结合。该复合物中的主要蛋白被鉴定为p120CBL。因此，在这些造血细胞系中，在涉及c-ABL、C3G和SOS的正常信号通路中，CRKL比CRK II参与程度更高。在BCR/ABL转化的细胞中，似乎是CRKL而非CRK II形成复合物，这些复合物可能将BCR/ABL、c-ABL、C3G和SOS与原癌蛋白p120CBL联系起来。
