The long -term objective of this research is to reconstitute the signaling pathways utilized by altered forms of the ABL tyrosine kinases in human leukemias. Oncogenic activation of the cellular ABL (cABL) gene in human leukemias occurs as a result of chromosomal translocation event that fuses exons from the BCR gene on chromosome 22 with exons from the cABL gene on chromosome 9. The translocation results in the formation of a chimeric BCR/ABL gene. Expression of BCR/ABL in hematopoietic cells is implicated as a key event in the development of a number of leukemias. To understand the mechanism(s) whereby BCR/ABL induces leukemia it is imperative to identify the cellular proteins which transduce the BCR/ABL transforming signal. Recently, we have succeeded in defining the domains required for activation of the transforming properties of BCR/ABL. These domains are sites of potential interactions with downstream effectors. The main specific aim of this proposal is the identification of these effectors. A combination of expression cloning and affinity purification techniques will be employed to achieve this end. In parallel, cloned cellular proteins which are implicated in the regulation of cell growth will be tested as effectors of BCR/ABL. The second specific aim is to define the role of BCR in the pathogenesis of human leukemias and other cellular processes. BCR is a multifunctional molecule. Amino terminal sequences in BCR which are essential for oncogenic activation of the BCR/ABL chimera bind with high affinity to the ABL src homology 2 (SH2) domain as well as to SH2 domains from other proteins. Carboxy-terminal sequences of BCR function as a GTPase activating protein (GAP) for the ras-related p2l rac protein, while sequences in the central portion of BCR show striking homology with a family of GDP-GTP exchange factors. These functional/structural motifs suggest that BCR may play multiple roles in normal and leukemic cells. Several approaches will be undertaken to study the role of BCR in cellular processes. Proteins that interact with the SH2-binding domain of BCR will be identified by expression cloning and affinity purification techniques and their role in cellular transformation by BCR/ABL will be examined using in vitro cell culture assays. Also, the potential tyrosine phosphorylation of BCR by BCR/ABL in leukemic cells will be analyzed. Tyrosine phosphorylated BCR may exhibit deregulated GAP functions. Results from the proposed experiments will permit the reconstruction of signaling pathways involving the BCR/ABL and BCR proteins. With the definition of these pathways it will be possible to achieve a molecular understanding of the mechanisms underlying the development of certain human leukemias while gaining insight into the range of growth regulatory mechanisms employed by hematopoietic cells.
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