The abl oncogene family and apoptosis

The role of the abl oncogene family in cellular transformation has been well established, but knowledge of its role in apoptosis is limited. Recent studies demonstrate that it may act as a suppressor of apoptosis in certain circumstances. The growth factor independence conferred on IL-3 dependent myeloid progenitor cell lines following v-Abl transformation is due to the suppressive effects of this oncogene on apoptosis. Similarly, inhibition of the deregulated activity of the p210(bcr-abl) protein in both myeloid progenitor lines and CML granulocytes has proven effective in reversing resistance to apoptosis in such cells. The Bcr-Abl fusion protein might therefore promote myeloid expansion by suppression of apoptotic cell death rather than through promoting proliferation. While oncogenic forms of Abl appear to be anti-apoptotic, the function of c-Abl remains elusive. Through the elucidation of the roles in cell growth and survival of the Abl family members we may gain valuable insights into the regulation of apoptosis and the mechanisms of oncogenesis.     

Structure and Dynamic Regulation of Abl Kinases

The c-abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes, and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as in the regulation of the actin cytoskeleton. Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia. Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in chronic myelogenous leukemia and revolutionized the field of targeted cancer therapy. In this minireview, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity.     

Bcr-Abl with an SH3 deletion retains the ability To induce a myeloproliferative disease in mice, yet c-Abl activated by an SH3 deletion induces only lymphoid malignancy

The bcr-abl oncogene plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). The fusion of Bcr sequences to Abl constitutively activates the Abl protein tyrosine kinase. We have recently shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces in mice a myeloproliferative disease resembling human CML and that Abl kinase activity is essential for Bcr-Abl to induce a CML-like myeloproliferative disease. However, it is not known if activation of the Abl kinase alone is sufficient to induce a myeloproliferative disease. In this study, we examined the role of the Abl SH3 domain of Bcr-Abl in induction of myeloproliferative disease and tested whether c-Abl activated by SH3 deletion can induce a CML-like disease. We found that Bcr-Abl with an Abl SH3 deletion still induced a CML-like disease in mice. In contrast, c-Abl activated by SH3 deletion induced only lymphoid malignancies in mice and did not stimulate the growth of myeloid colonies from 5-fluorouracil-treated bone marrow cells in vitro. These results indicate that Bcr sequences in Bcr-Abl play additional roles in inducing myeloproliferative disease beyond simply activating the Abl kinase domain and that functions of the Abl SH3 domain are either not required or redundant in Bcr-Abl-induced myeloproliferative disease. The results also suggest that the type of hematological neoplasm induced by an abl oncogene is influenced not only by what type of hematopoietic cells the oncogene is targeted into but also by the intrinsic oncogenic properties of the particular abl oncogene. In addition, we found that DeltaSH3 c-Abl induced less activation of Akt and STAT5 than did Bcr-Abl, suggesting that activation of these pathways plays a critical role in inducing a CML-like disease.     

The NH(2)-terminal coiled-coil domain and tyrosine 177 play important roles in induction of a myeloproliferative disease in mice by Bcr-Abl

Bcr-Abl, a fusion protein generated by t(9;22)(q34;q11) translocation, plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). It has been shown that Bcr-Abl contains multiple functional domains and motifs and can disrupt regulation of many signaling pathways and cellular functions. However, the role of specific domains and motifs of Bcr-Abl or of specific signaling pathways in the complex in vivo pathogenesis of CML is not completely known. We have previously shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder (MPD) in mice resembling human CML. We have also shown that the Abl kinase activity within Bcr-Abl is essential for Bcr-Abl leukemogenesis, yet activation of the Abl kinase without Bcr sequences is not sufficient to induce MPD in mice. In this study we investigated the role of Bcr sequences within Bcr-Abl in inducing MPD using this murine model for CML. We found that the NH(2)-terminal coiled-coil (CC) domain was both essential and sufficient, even though not efficient, to activate Abl to induce an MPD in mice. Interestingly, deletion of the Src homology 3 domain complemented the deficiencies of the CC-deleted Bcr-Abl in inducing MPD in mice. We further demonstrated that the Grb2 binding site at Y177 played an important role in efficient induction of MPD. These studies directly demonstrated the important roles of Bcr sequences in induction of MPD by Bcr-Abl.     

An actin-binding function contributes to transformation by the Bcr-Abl oncoprotein of Philadelphia chromosome-positive human leukemias.

In Philadelphia chromosome-positive human leukemias, which include chronic myelogenous leukemia and some acute lymphocytic leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene on chromosome 22, and Bcr-Abl fusion proteins are produced. The Bcr sequences activate the Abl tyrosine kinase which is required for the transforming function of Bcr-Abl. The Bcr sequences also enhance an F-actin-binding activity associated with c-Abl. Here, we show that binding of c-Abl and Bcr-Abl proteins to actin filaments in vivo and in vitro is mediated by an evolutionarily conserved domain at the C-terminal end of c-Abl. The c-Abl F-actin-binding domain contains a consensus motif found in several other actin-crosslinking proteins. Mutations in the consensus motif are shown to abolish binding to F-actin. Bcr-Abl proteins unable to associate with F-actin have a reduced ability to transform Rat-1 fibroblasts and to abrogate the requirement for interleukin-3 in the lymphoblastoid cell line Ba/F3. In transformed cells, Bcr-Abl induces a redistribution of F-actin into punctate, juxtanuclear aggregates. The binding to actin filaments has important implications for the pathogenic and physiological functions of the Bcr-Abl and c-Abl proteins.     

Role of the IkappaB kinase complex in oncogenic Ras- and Raf-mediated transformation of rat liver epithelial cells

NF-kappaB/Rel factors have been implicated in the regulation of liver cell death during development, after partial hepatectomy, and in hepatocytes in culture. Rat liver epithelial cells (RLEs) display many biochemical and ultrastructural characteristics of oval cells, which are multipotent cells that can differentiate into mature hepatocytes. While untransformed RLEs undergo growth arrest and apoptosis in response to transforming growth factor beta1 (TGF-beta1) treatment, oncogenic Ras- or Raf-transformed RLEs are insensitive to TGF-beta1-mediated growth arrest. Here we have tested the hypothesis that Ras- or Raf-transformed RLEs have altered NF-kappaB regulation, leading to this resistance to TGF-beta1. We show that classical NF-kappaB is aberrantly activated in Ras- or Raf-transformed RLEs, due to increased phosphorylation and degradation of IkappaB-alpha protein. Inhibition of NF-kappaB activity with a dominant negative form of IkappaB-alpha restored TGF-beta1-mediated cell killing of transformed RLEs. IKK activity mediates this hyperphosphorylation of IkappaB-alpha protein. As judged by kinase assays and transfection of dominant negative IKK-1 and IKK-2 expression vectors, NF-kappaB activation by Ras appeared to be mediated by both IKK-1 and IKK-2, while Raf-induced NF-kappaB activation was mediated by IKK-2. NF-kappaB activation in the Ras-transformed cells was mediated by both the Raf and phosphatidylinositol 3-kinase pathways, while in the Raf-transformed cells, NF-kappaB induction was mediated by the mitogen-activated protein kinase cascade. Last, inhibition of either IKK-1 or IKK-2 reduced focus-forming activity in Ras-transformed RLEs. Overall, these studies elucidate a mechanism that contributes to the process of transformation of liver cells by oncogene Ras and Raf through the IkappaB kinase complex leading to constitutive activation of NF-kappaB.     

Urea derivatives of STI571 as inhibitors of Bcr-Abl and PDGFR kinases

The constitutively active Abl kinase activity of the Bcr-Abl oncoprotein is causative for chronic myelogenous leukemia. Urea derivatives, structurally related to the therapeutic agent STI571, have been identified, which potently inhibit the tyrosine kinase activity of recombinant Abl. In particular a dimethylamino-aniline derivative (18) inhibited c-Abl transphosphorylation with an IC(50) value of 56 nM. Although this activity was not translated into cellular activity against the constitutively activated oncogenic Bcr-Abl, a number of compounds from this series potently inhibited cellular PDGFR autophosphorylation. It was also possible to differentiate between c-Abl and PDGFR kinase inhibition, with compound 22 being selective towards Abl and 23 selective for PDGFR.     

Targeting the SH2-kinase interface in Bcr-Abl inhibits leukemogenesis

Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.     

STI-571: an anticancer protein-tyrosine kinase inhibitor

STI-571 (imatinib, Gleevec, Glivec, CGP 57148) is an inhibitor of the Abl group of protein-tyrosine kinases. One of these enzymes, the Bcr-Abl oncoprotein, results from the fusion of the BCR and ABL genes that result from the reciprocal chromosomal translocation that forms the Philadelphia chromosome. The Philadelphia chromosome occurs in 95% of people with chronic myeloid leukemia. ABL is the cellular homologue of the oncogene found in murine Abelson leukemia virus, and BCR refers to breakpoint cluster region. The Bcr-Abl oncoprotein exhibits elevated protein-tyrosine kinase activity, which is strongly implicated in the mechanism of development of chronic myeloid leukemia. STI-571 is effective in the treatment of the stable phase of chronic myeloid leukemia. The c-Abl protein kinase domain exists in an active and inactive conformation. STI-571 binds only to the inactive state of the enzyme as shown by X-ray crystallography. The drug binds to a portion of the ATP-binding site and extends from there into adjacent hydrophobic regions. STI-571 is a competitive inhibitor of Abl kinase with respect to ATP. Resistance to STI-571 is often the result of mutations in residues of the Bcr-Abl kinase that ordinarily bind to the drug. Inhibition of target protein kinases represents an emerging therapeutic strategy for the treatment of cancer.     

The tyrosine phosphatase TC48 interacts with and inactivates the oncogenic fusion protein BCR-Abl but not cellular Abl

The chimeric oncoprotein BCR-Abl exhibits deregulated protein tyrosine kinase activity and is responsible for the pathogenesis of certain human leukemias, such as chronic myelogenous leukemia. The activities of cellular Abl (c-Abl) and BCR-Abl are stringently regulated and the cellular mechanisms involved in their inactivation are poorly understood. Protein tyrosine phosphatases can negatively regulate Abl mediated signaling by dephosphorylating the kinase and/or its substrates. This study investigated the ability of the intracellular T cell protein tyrosine phosphatase (TCPTP/PTPN2) to dephosphorylate and regulate the functions of BCR-Abl and c-Abl. TCPTP is expressed as two alternately spliced isoforms — TC48 and TC45, which differ in their C-termini and localize to the cytoplasm and nucleus respectively. We show that TC48 dephosphorylates BCR-Abl but not c-Abl and inhibits its activity towards its substrate, CrkII. Y1127 and Y1294 residues whose phosphorylation corresponds with BCR-Abl activation status were the primary sites targeted by TC48. Co-localization and immunoprecipitation experiments showed that TC48 interacted with BCR-Abl but not with c-Abl, and BCR domain was sufficient for interaction. TC48 expression resulted in the stabilization of Bcr-Abl protein dependent on its phosphatase activity. Inactivation of cellular TC48 in K562 cells by stable expression of a dominant negative catalytically inactive mutant TC48, enhanced proliferation. TC48 expressing K562 clones showed reduced proliferation and enhanced sensitivity to STI571 compared to control clones suggesting that TC48 can repress the growth of CML cells. This study identifies a novel cellular regulator that specifically inhibits the activity of oncogenic BCR-Abl but not that of the cellular Abl kinase.     

Reduction of Raf Kinase Inhibitor Protein Expression by Bcr-Abl Contributes to Chronic Myelogenous Leukemia Proliferation

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl-transformed cells, and Ras activity enhances the oncogenic ability of Bcr-Abl. However, the mechanism by which Bcr-Abl activates the Ras pathway is not completely understood. Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction, resulting in blocking the MAP kinase pathway. In the present study, we found that RKIP was depleted in CML cells. We investigated the interaction between RKIP and Bcr-Abl in CML cell lines and Bcr-Abl⁺ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of RKIP and reduced the pERK1/2 status, resulting in inhibited proliferation of CML cells. Moreover, RKIP up-regulated cell cycle regulator FoxM1 expression, resulting in G₁ arrest via p27^Kip1 and p21^Cip1 accumulation. In colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte, colony-forming unit-granulocyte macrophage, and burst-forming unit erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced RKIP and reduced FoxM1 expressions, and inhibited colony formation of Bcr-Abl⁺ progenitor cells, whereas depletion of RKIP weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl⁺ progenitor cells. Thus, Bcr-Abl represses the expression of RKIP, continuously activates pERK1/2, and suppresses FoxM1 expression, resulting in proliferation of CML cells.     

A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates

Regulated phosphorylation by protein tyrosine kinases (PTKs), such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the chronic myeloid leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogel-immobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semiquantitative detection of peptide phosphorylation by recombinant c-Abl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay are demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening.     

A direct binding site for Grb2 contributes to transformation and leukemogenesis by the Tel-Abl (ETV6-Abl) tyrosine kinase

A direct binding site for the Grb2 adapter protein is required for the induction of fatal chronic myeloid leukemia (CML)-like disease in mice by Bcr-Abl. Here, we demonstrate direct binding of Grb2 to the Tel-Abl (ETV6-Abl) fusion protein, the product of complex (9;12) chromosomal translocations in human leukemia, via tyrosine 314 encoded by TEL exon 5. A Tel-Abl point mutant (Y314F) and a splice variant without TEL exon 5 sequences (Deltae5) lacked Grb2 interaction and exhibited decreased binding and phosphorylation of the scaffolding protein Gab2 and impaired activation of phosphatidylinositol 3-kinase, Akt, and extracellular signal-regulated kinase/mitogen-activated protein kinase in hematopoietic cells. Tel-Abl Y314F and Deltae5 were unable to transform fibroblasts to anchorage-independent growth and were defective for B-lymphoid transformation in vitro and lymphoid leukemogenesis in vivo. Previously, we demonstrated that full-length Tel-Abl induced two distinct myeloproliferative diseases in mice: CML-like leukemia similar to that induced by Bcr-Abl and a novel syndrome of small-bowel myeloid infiltration endotoxemia and hepatic and renal failure. Lack of the Grb2 binding site had no effect on development of small bowel syndrome but significantly attenuated the induction of CML-like disease by Tel-Abl. These results suggest that direct binding of Grb2 is a common mechanism contributing to leukemogenesis by oncogenic Abl fusion proteins.     

Transformation of myeloid leukemia cells to cytokine independence by Bcr-Abl is suppressed by kinase-defective Hck

Bcr-Abl is the constitutively active protein-tyrosine kinase expressed as a result of the Philadelphia translocation in chronic myelogenous leukemia. Bcr-Abl is coupled to many of the same signaling pathways normally regulated by hematopoietic cytokines. Recent work shows that Hck, a member of the Src tyrosine kinase family with myeloid-restricted expression, associates with and is activated by Bcr-Abl. Here we investigated the mechanism of Hck interaction with Bcr-Abl and the requirement for Hck activation in Bcr-Abl transformation signaling. Binding studies demonstrated that the Hck SH3 and SH2 domains are sufficient for interaction with Bcr-Abl in vitro. Hck binding localizes to the Abl SH2, SH3, and kinase domains as well as the distal portion of the C-terminal tail. To address the requirement for endogenous Src family kinase activation in Bcr-Abl signaling, a kinase-defective mutant of Hck was stably expressed in the cytokine-dependent myeloid leukemia cell line DAGM. Kinase-defective Hck dramatically suppressed Bcr-Abl-induced outgrowth of these cells in the absence of cytokine compared with a control cell line expressing beta-galactosidase. In contrast, kinase-defective Hck did not affect cell proliferation in response to interleukin-3, suggesting that the effect is specific for Bcr-Abl. These data show that Hck interacts with Bcr-Abl through a complex mechanism involving kinase-dependent and -independent components and that interaction with Hck or other Src family members is essential for transformation signaling by Bcr-Abl.     

Enterovirus 71 triggering of neuronal apoptosis through activation of Abl-Cdk5 signalling

The molecular mechanism behind what causes an infection of Enterovirus 71 (EV71) in young children to result in severe neurological diseases is unclear. Herein, we show that Cdk5, a critical signalling effector of various neurotoxic insults in the brain, is activated by EV71 infection of neuronal cells. EV71-induced neuronal apoptosis could be effectively repressed by blocking either Cdk5 kinase activity or its protein expression. Moreover, EV71-induced Cdk5 activation was modulated by c-Abl. The suppression of c-Abl kinase activity by STI571 notably repressed both the Cdk5 activation and neuronal apoptosis in cells infected with EV71. Although EV71 also induces apoptosis in non-neuronal cells, it did not affect Abl and Cdk5 activities in several non-neuronal cell lines. Intriguingly, coxsackievirus A16 (CA16), a genetically closely related serotype to EV71 that usually does not induce severe neurological disorders, could only weakly stimulate Abl, but not Cdk5 kinase activity. Taken together, our data suggest a serotype- and cell type-specific mechanism, by which EV71 induces Abl kinase activity, which in turn triggers Cdk5-signalling for neuronal apoptosis.     

Bcr-Abl-mediated molecular mechanism for apoptotic suppression in multipotent haemopoietic cells: a role for PKCbetaII

Bcr-Abl protein tyrosine kinase (PTK) activity is a feature of chronic myeloid leukaemia and confers a survival advantage on haemopoietic progenitor cells. We have expressed conditional mutant of the Bcr-Abl PTK in the FDCP-Mix A4 multipotent haematopoietic cell line in order to examine the molecular mechanisms whereby Bcr-Abl PTK leads to enhanced cell survival under conditions in which normal cells die. Activation of Bcr-Abl PTK does not phosphorylate or activate either ERK-1/2 or JAK-2/STAT-5b, suggesting that these signal transduction pathways are not involved in Abl PTK-mediated suppression of apoptosis in FDCP-Mix cells. However, protein kinase C (PKC) does have a role to play. Inhibition of PKC results in a reversal of Bcr-Abl PTK-mediated survival in the absence of growth factor and Bcr-Abl stimulates translocation of the PKCbetaII isoform to the nucleus. Furthermore, expression of a constitutively activated PKCbetaII in haemopoietic progenitor FDCP-Mix cells stimulates enhanced cell survival when IL-3 is withdrawn. However, expression of this constitutively activated PKC isoform does not suppress cytotoxic drug-induced apoptosis. Thus Bcr-Abl PTK has pleiotropic effects which can suppress cell death induced by a number of stimuli.     

Role of Abl Kinase and the Wave2 Signaling Complex in HIV-1 Entry at a Post-Hemifusion Step

Entry of human immunodeficiency virus type 1 (HIV-1) commences with binding of the envelope glycoprotein (Env) to the receptor CD4, and one of two coreceptors, CXCR4 or CCR5. Env-mediated signaling through coreceptor results in Gαq-mediated Rac activation and actin cytoskeleton rearrangements necessary for fusion. Guanine nucleotide exchange factors (GEFs) activate Rac and regulate its downstream protein effectors. In this study we show that Env-induced Rac activation is mediated by the Rac GEF Tiam-1, which associates with the adaptor protein IRSp53 to link Rac to the Wave2 complex. Rac and the tyrosine kinase Abl then activate the Wave2 complex and promote Arp2/3-dependent actin polymerization. Env-mediated cell-cell fusion, virus-cell fusion and HIV-1 infection are dependent on Tiam-1, Abl, IRSp53, Wave2, and Arp3 as shown by attenuation of fusion and infection in cells expressing siRNA targeted to these signaling components. HIV-1 Env-dependent cell-cell fusion, virus-cell fusion and infection were also inhibited by Abl kinase inhibitors, imatinib, nilotinib, and dasatinib. Treatment of cells with Abl kinase inhibitors did not affect cell viability or surface expression of CD4 and CCR5. Similar results with inhibitors and siRNAs were obtained when Env-dependent cell-cell fusion, virus-cell fusion or infection was measured, and when cell lines or primary cells were the target. Using membrane curving agents and fluorescence microscopy, we showed that inhibition of Abl kinase activity arrests fusion at the hemifusion (lipid mixing) step, suggesting a role for Abl-mediated actin remodeling in pore formation and expansion. These results suggest a potential utility of Abl kinase inhibitors to treat HIV-1 infected patients.     

Src family kinases phosphorylate the Bcr-Abl SH3-SH2 region and modulate Bcr-Abl transforming activity

Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for chronic myelogenous leukemia. Recently, we observed that inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, and Fyn) induces growth arrest and apoptosis in Bcr-Abl-transformed cells, suggesting that cell transformation by Bcr-Abl involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075-8088). Here, we report the unexpected observation that Hck, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of Bcr-Abl. Seven phosphorylation sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Tyr89 and Tyr134 in the Abl-derived SH3 domain; Tyr147 in the SH3-SH2 connector; and Tyr158, Tyr191, Tyr204, and Tyr234 in the SH2 domain. SH3 domain Tyr89, the most prominent phosphorylation site in vitro, was strongly phosphorylated in chronic myelogenous leukemia cells in a Src family kinase-dependent manner. Substitution of the SH3-SH2 tyrosine phosphorylation sites with phenylalanine substantially reduced Bcr-Abl-mediated transformation of TF-1 myeloid cells to cytokine independence. The positions of these tyrosines in the crystal structure of the c-Abl core and the transformation defect of the corresponding Bcr-Abl mutants together suggest that phosphorylation of the SH3-SH2 region by Src family kinases impacts Bcr-Abl protein conformation and signaling.