The Philadelphia chromosome translocation generates a chimeric oncogene in which the BCR gene and the c-ABL genes are fused. The product of this oncogene, p210BCR/ABL, has elevated ABL tyrosine kinase activity, relocates to the cytoskeleton, and phosphorylates several cellular proteins. BCR/ABL transforms hematopoietic cells in vitro and in vivo and exerts a wide variety of biological effects, including induction of factor-independence, reduction of apoptosis, and altering adhesion of CML cells to marrow stroma. However, at a biochemical level, the mechanisms by which BCR/ABL transforms myeloid cells are poorly understood. When introduced into factor-dependent myeloid cell lines, BCR/ABL induces the tyrosine phosphorylation of many cellular proteins, some of which have been identified by our group and others. These include c-BCR, p120rasGAP, c-CBL, p52SHC, p93FES, p95VAV, p125FAK, p68paxillin, and p72SHPTP2. Also, BCR/ABL has been shown to bind directly to GRB2 at Y177 of BCR, and therefore potentially activating p21ras. However, it has been difficult to determine the significance of any of these potential BCR/ABL substrates, in part due to the complexity of studying a large protein with many potential signaling motifs, and in part due to the fact that there are so many BCR/ABL substrates in these cell lines. One approach to simplifying BCR/ABL biology has been to examine primary human CML cells, rather than cell lines made to overexpress BCR/ABL. Interestingly, in primary leukemic cells, there are only a few proteins which either interact with BCR/ABL or are phosphorylated by BCR/ABL. This suggests that studies in primary CML cells, rather than tissue culture cell lines, may be more reliable in terms of identifying important signaling pathways. In preliminary studies it was found that there is only a single tyrosine phosphoprotein complexed with BCR/ABL in CML neutrophils, and this protein has been identified as CRKL, an SH2/SH3 """"""""adapter"""""""" protein. CRKL binds to BCR/ABL at least partly through its SH3 domain. In additional studies, we have identified three cellular proteins which bind to CRKL through its SH2 domain in CML cells. The first protein is a component of focal adhesions, p68 paxillin, the second is a 130 Kda protein termed CAS for """"""""CRK associated substrate,"""""""" and the third is a 120kDa signalling protein termed Cbl. In preliminary studies, we cloned the human and chicken paxillin genes, and identified multiple protein/protein interaction domains, including LIM domains and sites for binding CRKL SH2 and other proteins. The central hypothesis to be tested here is that the interaction of BCR/ABL with the CRKL adapter protein is important in the pathogenesis of stable phase CML. The preliminary data suggest that CRKL, through binding to paxillin, Cbl, or CAS, links BCR/ABL to these proteins and that this event may be activating pathways which regulate integrin function, viability, or proliferation, and these hypotheses will be tested. It is anticipated that these results will improve our understanding of the pathogenesis of CML.
