STRUCTURAL AND SIGNALING REQUIREMENTS FOR BCR-ABL-MEDIATED TRANSFORMATION AND INHIBITION OF APOPTOSIS

BCR-AEL is a deregulated tyrosine kinase expressed in Philadelphia chr omosome-positive human leukemias, Prolongation of hematopoietic cell s urvival by inhibition of apoptosis has been proposed to be an integral component of BCR-ABL-induced chronic myelogenous leukemia. BCR-ABL el icits transformation of both fibroblast and hematopoietic cells and bl ocks apoptosis following cytokine deprivation in various factor-depend ent cells. To elucidate the mechanisms whereby BCR-ABL induces transfo rmation and blocks apoptosis in hematopoietic cells, we examined the b iological effects of expression of a series of BCR-ABL mutants, Single amino acid substitutions in the GRB2 binding site (Y177F), Src homolo gy 2 domain (R552L), or an autophosphorylation site in the is tyrosine kinase domain (Y793F) do not diminish the antiapoptotic and transform ing properties of BCR-ABL in hematopoietic cells, although these mutat ions were previously shown to drastically reduce the transforming acti vity of BCR-ABL in fibroblasts. A BCR-ABL molecule containing all thre e mutations (Y177F/R552L/Y793F) exhibits a severe decrease in transfor ming and antiapoptotic activities compared with the wild-type BCR-ABL protein in 32D myeloid progenitor cells, Pas is activated, the SHC ada pter protein is tyrosine phosphorylated and binds GRB2, and myc mRNA l evels are increased following expression of all kinase active BCR-ABL proteins with the exception of the Y177F/R552L/Y793F BCR-ABL mutant in 32D cells. We propose that BCR-ABL uses multiple pathways to activate Pas in hematopoietic cells and that this activation is necessary for the transforming and antiapoptotic activities of BCR-ABL. However, Ras activation is not sufficient for BCR ABL-mediated transformation. A B CR-ABL deletion mutant (Delta 176-427) that activates Pas and blocks a poptosis but has severely impaired transforming ability in 32D cells h as been identified. These data suggest that BCR-ABL requires additiona l signaling components to elicit tumorigenic growth which are distinct from those required to block apoptosis.