The NH ₂ -Terminal Coiled-Coil Domain and Tyrosine 177 Play Important Roles in Induction of a Myeloproliferative Disease in Mice by Bcr-Abl

Abstract

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.The bcr-abl oncogene, produced from the t(9;22)(q34;q11) chromosomal translocation known as the Philadelphia chromosome (Ph), is associated with 95% of the cases of chronic myelogenous leukemia (CML) and also with 20% of the adult and 5% of the pediatric cases of acute lymphoblastic leukemia (ALL) (23,33). Depending on the nature of the translocation and exactly how the bcr and abl sequences become spliced into a final bcr-abl mRNA, various Bcr-Abl fusion proteins, including p185, p210, and p230, can be generated that show a preferential association with different types of leukemia (33). Clinical and laboratory studies indicate that Bcr-Abl plays an essential role in initiation of the chronic phase of CML and also plays a critical role in the maintenance and progression of the disease (10,20,50).Bcr-Abl contains multiple functional domains and motifs. Abl-derived sequences in Bcr-Abl contain Src homology 3 (SH3), SH2, and tyrosine kinase domains in their N-terminal half, as well as a DNA binding domain, an actin binding domain, nuclear localization signals, and SH3 binding sites in their C-terminal region (41). The Bcr region (in the major p210 form) contains a coiled-coil (CC) oligomerization domain, a serine/threonine kinase domain, a Pleckstrin homology (PH) domain, a Dbl guanine-nucleotide exchange factor homology domain, and binding sites for the Abl SH2 domain and Grb2, Grb10, and 14-3-3 proteins (2,25,41,49). The multiple domains of Bcr-Abl work cooperatively to activate intracellular signaling pathways commonly used in hematopoietic growth fa...

Molecular and cellular bases of chronic myeloid leukemia

“…Mutations in the SH2 domain of ABL reduced the ability of BCR-ABL to induce a CML-like MPD in mice (Zhang et al, 2001). The Y1294F point mutation in SH2 domain of BCR-ABL also attenuated leukemogenesis by BCR-ABL (Smith et al, 2003).…”

“…A mutant form of BCR-ABL that lacks the BCR-CC domain (ΔCC-BCR-ABL) failed to induce MPD in mice, but, rather, induced a T-cell leukemia/ lymphoma only after a long latent period (Zhang et al, 2001;Smith et al, 2003). Reactivation of the kinase activity of ABL by mutating its SH3 domain (through deletion or a P1013L point mutation), rescued the ability of ΔCC-BCR-ABL to induce a CML-like MPD in mice (Smith et al, 2003).…”

“…Mutation of the tyrosine-177 residue of BCR-ABL to phenylalanine (Y177F) largely abolished its ability to bind GRB2 without affecting the kinase activity of ABL (Pendergast et al, 1993;Puil et al, 1994). In the BMT CML model, the Y177F mutant form of BCR-ABL has a greatly reduced ability to induce CML in mice, and these mice eventually developed T-ALL or abdominal T-cell lymphomas after a prolonged latent period (Million and Van Etten, 2000;Zhang et al, 2001). These results demonstrate that phosphorylation at Y177 is required for the induction of CML by BCR-ABL.…”

A Direct Binding Site for Grb2 Contributes to Transformation and Leukemogenesis by the Tel-Abl (ETV6-Abl) Tyrosine Kinase

“…Retroviral transduction of the BCR-ABL gene into murine bone marrow followed by transplantation into irradiated recipient mice results in the development of either CML-like myeloproliferative disease (30,44,62) or B-ALL (50) in all recipients, depending on the transduction conditions. The mouse retroviral bone marrow transduction/transplantation system provides accurate and quantitative models of human CML and Ph ϩ B-ALL (58) that have proven useful for analyzing the molecular pathophysiology of these diseases (15,29,30,39,50,63).Fusion of the ABL gene to a different partner, the TEL (ETV6) gene on chromosome 12p13, has been reported to occur in a small number of patients with leukemia, some who had acute leukemia of B-lymphoid (43), T-lymphoid (60), or myeloid (13, 40) origin and some who presented with atypical (3, 28) or typical (1, 60) CML. TEL encodes a ubiquitously expressed 452-amino-acid protein with homology to the Ets family of transcription factors (12).…”

“…Retroviral transduction of the BCR-ABL gene into murine bone marrow followed by transplantation into irradiated recipient mice results in the development of either CML-like myeloproliferative disease (30,44,62) or B-ALL (50) in all recipients, depending on the transduction conditions. The mouse retroviral bone marrow transduction/transplantation system provides accurate and quantitative models of human CML and Ph ϩ B-ALL (58) that have proven useful for analyzing the molecular pathophysiology of these diseases (15,29,30,39,50,63).…”