Activation of the c-abl oncogene by viral transduction or chromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties.

The v-abl protein of Abelson murine leukemia virus is a tyrosine-specific kinase. Its normal cellular homolog, murine c-abl, does not possess detectable tyrosine kinase activity in vitro. Previously, we have detected tyrosine kinase activity in vitro for an altered c-abl gene product (c-abl P210) in the K562 human chronic myelogenous leukemia cell line. The expression of this variant c-abl gene product correlates with chromosomal translocation and amplification of the c-abl gene in K562 cells. Like v-abl, c-abl P210 is a fusion protein containing non-abl sequences near the amino terminus of c-abl. We compared the in vitro tyrosine kinase activity of c-abl P210 with that of wild-type murine v-abl. The remarkable similarities of these two proteins with respect to cis-acting autophosphorylation, trans-acting phosphorylation of exogenous substrates, and kinase inhibition, using site-directed abl-specific antisera, suggested that c-abl P210 could function similarly to v-abl in vivo. In addition, c-abl P210 possessed an associated serine kinase activity in immunoprecipitates. The serine kinase activity was not inhibited by site-directed, abl-specific antisera that inhibit the tyrosine kinase activity, suggesting that the serine kinase activity is not an intrinsic property of c-abl P210. Thus, the activation of the c-abl gene in a human leukemia cell line may have functional consequences analogous to activation of the c-abl gene in Abelson murine leukemia virus.     

Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products.

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.     

Monoclonal antibodies specific for v-abl- and c-abl-encoded molecules.

Monoclonal antibodies specific for regions of the transforming protein of Abelson murine leukemia virus were prepared. Antibodies directed against the kinase domain inhibited the autophosphorylation of v-abl proteins, and all of the antibodies reacted with the products of the murine and human c-abl loci.     

The minimum transforming region of v-abl is the segment encoding protein-tyrosine kinase.

Only 1.2 kilobases (kb) at the 5' end of the 3.9-kb v-abl sequence in Abelson murine leukemia virus is required for fibroblast transformation. A precise delineation of this minimum transforming region was made by using small 5' or 3' deletions. Insertions of four amino acids, generated by putting synthetic DNA linkers into various restriction enzyme cleavage sites, abolished transforming activity, indicating that much of the internal sequence of the minimum transforming region plays a critical role in the transformation process. This 5' 1.2 kb of v-abl encodes protein-tyrosine kinase activity when expressed in Escherichia coli. Each of the mutations which caused a loss of transformation activity also resulted in a loss of protein-tyrosine kinase activity when expressed in E. coli. The minimum transforming region of v-abl contains amino acid homology to other protein-tyrosine kinase oncogenes, and a comparison with these oncogenes is presented.     

BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias.

The c-abl proto-oncogene encodes a cytoplasmic tyrosine kinase which is homologous to the src gene product in its kinase domain and in the upstream kinase regulatory domains SH2 (src homology region 2) and SH3 (src homology region 3). The murine v-abl oncogene product has lost the SH3 domain as a consequence of N-terminal fusion of gag sequences. Deletion of the SH3 domain is sufficient to render the murine c-abl proto-oncogene product transforming when myristylated N-terminal membrane localization sequences are also present. In contrast, the human BCR/ABL oncogene of the Philadelphia chromosome translocation has an intact SH3 domain and its product is not myristylated at the N terminus. To analyze the contribution of BCR-encoded sequences to BCR/ABL-mediated transformation, the effects of a series of deletions and substitutions were assessed in fibroblast and hematopoietic-cell transformation assays. BCR first-exon sequences specifically potentiate transformation and tyrosine kinase activation when they are fused to the second exon of otherwise intact c-ABL. This suggests that BCR-encoded sequences specifically interfere with negative regulation of the ABL-encoded tyrosine kinase, which would represent a novel mechanism for the activation of nonreceptor tyrosine kinase-encoding proto-oncogenes.     

Nucleic acid sequence and oncogenic properties of the HZ2 feline sarcoma virus v-abl insert.

Hardy-Zuckerman 2 feline sarcoma virus (HZ2-FeSV), isolated from a multicentric feline fibrosarcoma is a replication-defective acute transforming feline retrovirus which originated by transduction of feline c-abl sequences with feline leukemia virus (FeLV) and is known to encode a 110-kilodalton gag-abl fusion protein with tyrosine-specific protein kinase activity (P. Besmer, W. D. Hardy, E. E. Zuckerman, P. J. Bergold, L. Lederman, and H. W. Snyder, Nature (London) 303:825-828, 1983). The nucleotide sequence of the abl segment in the HZ2-FeSV genome was determined and compared with the murine and human v-abl and c-abl sequences. The predicted transforming protein consists of 344 amino acids (aa) of FeLV gag origin, 439 aa of abl origin, and at least 200 aa of FeLV pol origin (p110gag-abl-pol). The 1,317-base-pair HZ2-FeSV v-abl segment (fv-abl) corresponds to 5' abl sequences which include the region known to specify the protein kinase domain. The 5' 189 base pairs of fv-abl correspond to 5' c-abl sequences not contained in Abelson murine leukemia virus (MuLV) v-abl. The mouse c-abl exon which contains these segments was identified, and its nucleotide sequence was determined. Comparison of the predicted amino acid sequence of fv-abl with those of Abelson MuLV v-abl and c-abl revealed five aa differences. The 5' junction between FeLV and abl was found to involve a preferred region in FeLV gag p30 (P. Besmer, J. E. Murphy, P. C. George, F. H. Qiu, P. J. Bergold, L. Lederman, H. W. Snyder, D. Brodeur, E. E. Zuckerman, and W. D. Hardy, Nature (London) 320:415-421, 1986). A six-base homology exists at the recombination site between the parental FeLV and the c-abl sequences. The 3' junction between fv-abl and FeLV pol predicts an in-frame fusion of fv-abl and FeLV pol. A transformed cell line containing a truncated gag-abl-pol protein, p85, that lacks most of the FeLV pol sequences was obtained by transfection of NIH 3T3 mouse cells. This result implies that the pol sequences of the p110gag-abl-pol protein are dispensable for fibroblast transformation. To assess whether the fv-abl segment specifies the unique biological properties of HZ2-FeSV, we constructed a Moloney MuLV-based version of HZ2-FeSV, Mo-MuLV(fv-abl), in which the fv-abl sequences were contained in a genetic context similar to that in HZ2-FeSV.(ABSTRACT TRUNCATED AT 400 WORDS)     

A membrane-associated, carbohydrate-modified form of the v-abl protein that cannot be phosphorylated in vivo or in vitro.

Abelson murine leukemia virus encodes a transforming protein which contains tyrosine kinase activity and is phosphorylated in vivo and in vitro. We found that P160 and P160-derived virus strains expressed an additional, altered v-abl protein which could not be phosphorylated. The altered v-abl protein (L-v-abl) differed from the phosphorylated form (K-v-abl) in that it was glycosylated and localized exclusively to the membrane fraction. Tunicamycin inhibition of N-linked carbohydrate addition did not restore phosphorylation. It did, however, reveal that L-v-abl had additional sequences relative to K-v-abl. The coding sequences required for this region and for the expression of L-v-abl were identified by replacing sequences in the P120 virus genome, which did not express L-v-abl, with sequences from the P160 virus genome. The necessary sequences were localized to the Moloney murine leukemia virus-derived gag gene. Comparison between the in vitro altered P120 and wild-type P120 virus strains indicated that expression of L-v-abl did not increase the efficiency of lymphoid transformation. Although the biological role of L-v-abl is not clear, our analyses have revealed that a specific amino terminal gag sequence can prevent v-abl from acting as a kinase substrate and can alter the cellular localization and modification of v-abl. These properties distinguish L-v-abl from previously reported v-abl proteins.     

An alteration of the human c-abl protein in K562 leukemia cells unmasks associated tyrosine kinase activity

The v-abl protein is known to be a tyrosine-specific protein kinase. However, its normal cellular homolog, c-abl P150, is not detectably phosphorylated on tyrosine in vivo or in vitro. The lack of associated tyrosine kinase activity for the c-abl protein seems paradoxical since it is the c-abl-derived sequences of the v-abl protein that encode the kinase activity. We have detected an altered human c-abl protein (P210) with associated tyrosine kinase activity in the K562 leukemia cell line. K562 cells are known to have a 9:22 chromosomal translocation involving the c-abl locus and have amplified the c-able gene 4 to 8 fold. The altered P210 human c-abl is serologically and structurally related to the normal c-abl protein. A structural alteration of the human c-abl protein. K562 cells may have unmasked its associated tyrosine kinase activity. This altered c-abl protein may have important implications for a mechanism of activation of this oncogene.     

Conservation of function of Drosophila melanogaster abl and murine v-abl proteins in transformation of mammalian cells.

The Drosophila melanogaster abl and the murine v-abl genes encode tyrosine protein kinases (TPKs) whose amino acid sequences are highly conserved. To assess functional conservation between the two gene products, we constructed Drosophila abl/v-abl-chimeric Abelson murine leukemia viruses. In these chimeric Abelson murine leukemia viruses, the TPK and carboxy-terminal regions of v-abl were replaced with the corresponding regions of D. melanogaster abl. The chimeric Abelson murine leukemia viruses were able to mediate morphological and oncogenic transformation of NIH 3T3 cells and were able to abrogate the interleukin-3 dependence of a lymphoid cell line. We also found that a virus that contained both TPK and carboxy-terminal Drosophila abl regions had no in vitro transforming activity for primary bone marrow cells and lacked the ability to induce tumors in susceptible mice. A virus that replaced only a portion of the v-abl TPK region with that of Drosophila abl had low activity in in vitro bone marrow transformation and tumorigenesis assays. These results indicate that the transforming functions of abl TPKs are only partially conserved through evolution. These results also imply that the TPK region of v-abl is a major determinant of its efficient lymphoid cell-transforming activity.     

Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.     

Characterization of the feline c-abl proto-oncogene

Analysis of total feline DNA by genomic blot hybridization, using the viral oncogene of Abelson murine leukemia virus as a specific probe, has led to the identification of multiple v-abl homologous genetic sequences in the cat genome. Upon restriction endonuclease BamHI digestion, the combined size of the v-abl homologous DNA fragments was about 31 kbp. To characterize these sequences further, four independent v-abl homologous cosmid clones with overlapping cellular inserts have been isolated from a gene library of cat lung genomic DNA. These inserts represent a contiguous region of cellular DNA sequences of 56 kbp in length. Within this region of the feline genome, the v-abl homologous sequences are discontinuously dispersed over a region of about 34 kbp. They represent the complete feline v-abl cellular homolog and are colinear with the viral v-abl oncogene. Nine regions of highly repetitive DNA sequences have been mapped in close proximity to v-abl homologous sequences. These results establish the presence of only a single c-abl proto-oncogene in the cat genome and present its genetic organization.     

Isolation and analysis of an Abelson murine leukemia virus-encoded tyrosine-specific kinase produced in Escherichia coli

A segment of the coding sequence of the Abelson murine leukemia virus transforming gene (v-abl) has been inserted into a plasmid vector that allows its efficient and regulated expression in Escherichia coli. The product of the v-abl-derived coding sequence, designated p60v-abl, accumulated to a level of approximately 10% of total E. coli protein. A procedure is described for the isolation of p60v-abl from E. coli that yields about 50 micrograms of p60v-abl/g wet weight of E. coli. p60v-abl was capable of autophosphorylation and phosphorylating certain E. coli proteins specifically at tyrosine residues. The E. coli-expressed p60v-abl specifically phosphorylated tyrosine residues on casein and angiotensin II. The Km and Vmax values for ATP, casein, and angiotensin II in the p60v-abl kinase reaction have been determined and compared to values reported for other tyrosine-specific kinases. The expression system and isolation procedure described here permit the preparation of functional p60v-abl in quantities sufficient for detailed physical and biochemical characterization and examination of its biological action(s).     

N-terminal mutations activate the leukemogenic potential of the myristoylated form of c-abl.

The two major forms of the c-abl gene differ from their activated counterpart, the v-abl oncogene of the Abelson murine leukemia virus by the replacement of their N-terminal sequences with viral gag sequences. Overexpression of p150c-abl type IV in a retroviral vector similar to Abelson virus does not transform NIH 3T3 fibroblasts, even though it is expressed and myristoylated at levels comparable to pp160v-abl. Members of a nested set of deletion mutations of the N-terminus of c-abl type IV in this expression system will activate abl to transform murine fibroblasts. The smallest of these deletions, delta XB, efficiently transforms lymphoid cells in vitro and causes leukemia in vivo demonstrating that gag sequences are not necessary for abl-induced leukemogenesis. The delta XB mutation defines an N-terminal regulatory domain, which shares a surprising homology with chicken oncogene v-crk and phospholipase C-II. Although overexpression of the myristoylated form of c-abl does not transform cells, it nonetheless has a profound effect on cell growth.     

Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells.

The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1). Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA. The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus. Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome. This substituted region contains v-fos, the transforming gene of FBJ-MSV. Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA. Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region. However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome. The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human).     

Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations

The c-abl gene, originally identified as the cellular homolog of the transforming gene of the Abelson murine leukemia virus, encodes a protein-tyrosine kinase of unknown function that is expressed in all mammalian tissues. We have previously described the introduction of a mutation in the c-abl gene into the mouse germline via targeted gene disruption of embryonic stem cells. We now show that mice homozygous for this mutation are severely affected, displaying increased perinatal mortality, runtedness, and abnormal spleen, head, and eye development. We have examined components of the immune system and have found major reductions in B cell progenitors in the adult bone marrow, with less dramatic reductions in developing T cell compartments.     

Abelson virus transformation of an interleukin 2-dependent antigen-specific T-cell line.

Abelson murine leukemia virus (A-MuLV) carries the gene v-abl, one of a group of oncogenes with structural and functional (tyrosine kinase) homology to three growth factor receptors. Work in this and other laboratories has shown that A-MuLV infection can render myeloid and lymphoid cells independent of the growth factors interleukin 3 and granulocyte-macrophage colony-stimulating factor. We have now shown that v-abl can also relieve interleukin 2 (IL-2) dependence in T cells. We infected a cloned IL-2-dependent antigen-specific cell line. Transformed cells were generated which were factor independent and tumorigenic. The transformants each bore unique v-abl DNA inserts and expressed v-abl mRNA. No elevation of expression of either IL-2 or its receptor could be detected in these cells. Thus, A-MuLV can short-circuit the dependence of hematopoietic cells on IL-2, IL-3, and possibly granulocyte-macrophage colony-stimulating factor, none of whose receptors are known to be of the tyrosine kinase type.     

Drosophila melanogaster DNA clones homologous to vertebrate oncogenes: evidence for a common ancestor to the src and abl cellular genes

We have isolated phage clones containing the D. melanogaster sequence homologous to the v-abl oncogene, and two types of phage clones containing sequences homologous to the v-src probe. The D. melanogaster abl clone (lambda Dabl1) and one of the src clones (lambda Dsrc1) hybridize with both v-abl and v-src probes, and both map in situ to the same chromosomal position, 73B, on chromosome arm 3L. The second D. melanogaster src clone (lambda Dsrc2) does not react with the v-abl probe and hybridizes in situ to chromosomal position 64B. The hybridization pattern suggests that the src and abl cellular oncogenes have evolved from a common prototype sequence. The homologous sequences in D. melanogaster exhibit hybridization to regions in the vertebrate v-abl and v-src that are important for kinase activity and transforming potential of the viral gene products.     

Molecular cloning of the feline c-fes proto-oncogene and construction of a chimeric transforming gene

The feline c-fes proto-oncogene, different parts of which were captured in feline leukemia virus (FeLV) to generate the transforming genes (v-fes) of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) and the Snyder-Theilen strain (ST) of FeSV, was cloned and its genetic organization determined. Southern blot analysis revealed that the c-fes genetic sequences were distributed discontinuously and colinearly with the v-fes transforming gene over a DNA region of around 12.0 kb. Using cloned c-fes sequences, complementation of GA-FeSV transforming activity was studied. Upon replacement of the 3' half of v-fesGA with homologous feline c-fes sequences and transfection of the chimeric gene, morphological transformation was observed. Immunoprecipitation analysis of these transformed cells revealed expression of high Mr fusion proteins. Phosphorylation of these proteins was observed in an in vitro protein kinase assay, and tyrosine residues appeared to be involved as acceptor amino acid.     

Molecular analysis of both translocation products of a Philadelphia-positive CML patient.

The breakpoint regions of both translocation products of the (9;22) Philadelphia translocation of CML patient 83-H84 and their normal chromosome 9 and 22 counterparts have been cloned and analysed. Southern blotting with bcr probes and DNA sequencing revealed that the breaks on chromosome 22 occurred 3' of bcr exon b3 and that the 88 nucleotides between the breakpoints in the chromosome 22 bcr region were deleted. Besides this small deletion of chromosome 22 sequences a large deletion of chromosome 9 sequences (greater than 70 kb) was observed. The chromosome 9 sequences remaining on the 9q+ chromosome (9q+ breakpoint) are located at least 100 kb upstream of the v-abl homologous c-abl exons whereas the translocated chromosome 9 sequences (22q-breakpoint) could be mapped 30 kb upstream of these c-abl sequences. The breakpoints were situated in Alu-repetitive sequences either on chromosome 22 or on chromosome 9, strengthening the hypothesis that Alu-repetitive sequences can be hot spots for recombination.