Drosophila abelson interacting protein (dAbi) is a positive regulator of abelson tyrosine kinase activity.

Human and mouse Abelson interacting proteins (Abi) are SH3-domain containing proteins that bind to the proline-rich motifs of the Abelson protein tyrosine kinase. We report a new member of this gene family, a Drosophila Abi (dAbi) that is a substrate for Abl kinase and that co-immunoprecipitates with Abl if the Abi SH3 domain is intact. We have identified a new function for both dAbi and human Abi-2 (hAbi-2). Both proteins activate the kinase activity of Abl as assayed by phosphorylation of the Drosophila Enabled (Ena) protein. Removal of the dAbi SH3 domain eliminates dAbi's activation of Abl kinase activity. dAbi is an unstable protein in cells and is present at low steady state levels but its protein level is increased coincident with phosphorylation by Abl kinase. Expression of the antisense strand of dAbi reduces dAbi protein levels and abolishes activation of Abl kinase activity. Modulation of Abi protein levels may be an important mechanism for regulating the level of Abl kinase activity in the cell.

Enhanced affinities and specificities of consolidated ligands for the Src homology (SH) 3 and SH2 domains of Abelson protein-tyrosine kinase.

The possible interrelationships between multiple domains of proteins involved in intracellular signal transduction are complex and not easily investigated. We have synthesized a series of bivalent consolidated ligands, which interact simultaneously with the SH2 and SH3 domain of Abelson kinase in a SH(32) dual domain construct, a portion of native Abelson kinase. Affinities were measured by quenching of intrinsic tryptophan fluorescence. Consolidated ligands have enhanced affinity and specificity compared to monovalent equivalents. Affinity is also dependent on the length of the linker joining the two parts, with an optimum distance similar to that expected from structural models of Abl (SH(32). These results suggest that consolidated ligands may be generally useful reagents for probing structural and functional activities of multidomain proteins.

Characterization of drug resistance mediated via the suppression of apoptosis by Abelson protein tyrosine kinase.

Constitutive activation of the Abelson (Abl) protein tyrosine kinase (PTK) is a causative event in chronic myeloid leukemia, where intense chemotherapy currently fails to eradicate the leukemic clone. Using a mouse mast cell line (IC.DP), we previously showed that v-Abl PTK induced resistance to the anti-cancer drugs melphalan and hydroxyurea by the suppression of apoptosis. Here, using this cell line, we demonstrate by alkaline elution that v-Abl PTK did not affect the levels of DNA damage induced by either drug. This confirms that v-Abl PTK acts downstream of the drug-target interaction to prevent the coupling of drug-induced damage to the apoptotic pathway. Although Abl PTK- and interleukin-3 (IL-3)-stimulated signaling events share common signaling pathways, a similar level of drug resistance was not provided by IL-3, implying that Abl PTK does not merely mimic an IL-3 survival signaling pathway. Previously we demonstrated translocation of protein kinase C-beta II stimulated by activation of Abl PTK. Drug sensitivity was restored in cells with active v-Abl PTK by simultaneous addition of calphostin C, an inhibitor of protein kinase C, suggesting a role for protein kinase C in the suppression of drug-induced apoptosis by v-Abl PTK. One novel strategy for the treatment of chronic myeloid leukemia could therefore include the use of a downstream modifier of the Abl PTK-mediated survival signaling pathway to render leukemic cells more sensitive to a second drug, such as a cytotoxic agent.

Inhibition of Abelson oncogene function by erbstatin analogues.

The authors examined the effect of a tyrosine kinase inhibitor, erbstatin, and its analogues on abl oncogene functions. Erbstatin and its stable analogue methyl 2,5-dihydroxycinnamate (2,5-MeC) inhibited the growth of v-ablts-NIH3T3 cells at the permissive temperature (33 degrees C) at lower concentrations than at the non-permissive temperature (39 degrees C). 2,5-MeC inhibited the morphological transformation and the activation of v-abl tyrosine kinase by the temperature shift (39 degrees C to 33 degrees C) more effectively than erbstatin. Previously the authors reported that erbstatin induced erythroid differentiation of K562 human chronic myelogenous leukaemia cells, so they examined the effect of erbstatin analogues on the erythroid differentiation. Among eight erbstatin analogues studied, ethyl 2,5-dihydroxycinnamate induced erythroid differentiation of K562 cells most effectively. Ethyl 2,5-dihydroxycinnamate also inhibited bcr-abl tyrosine kinase. These results indicate that the stable analogues of erbstatin suppress oncogene functions of Abl by inhibiting its tyrosine kinase.

Temperature-sensitive mutants of Abelson murine leukemia virus deficient in protein tyrosine kinase activity.

The effect of two missense mutations in abl on transformation by Abelson murine leukemia virus was evaluated. These mutations led to the substitution of a histidine for Tyr-590 and a glycine for Lys-536. Both changes gave rise to strains that were temperature dependent for transformation of both NIH 3T3 cells and lymphoid cells when expressed in the context of a truncated Abelson protein. In the context of the prototype P120 v-abl protein, the Gly-536 substitution generated a host range mutant that induced conditional transformation in lymphoid cells but had only a subtle effect on NIH 3T3 cells. The combination of both substitutions gave rise to a P120 strain that was temperature sensitive for both NIH 3T3 and lymphoid cell transformation. The Abelson proteins encoded by the temperature-sensitive strain displayed in vitro kinase activities that were reduced when compared with those of wild-type proteins. In vivo, levels of phosphotyrosine were reduced only at the restrictive temperature. Analysis of cells expressing either the wild-type P160 v-abl protein or the P210 bcr/abl protein and an Abelson protein encoded by a temperature-sensitive strain failed to correct this defect, suggesting either that tyrosine phosphorylation in vivo is an intramolecular reaction or that the protein encoded by the temperature-sensitive strain is a poor substrate for tyrosine phosphorylation in vivo. These results raise the possibility that tyrosine phosphorylation of Abelson protein plays a role in transformation.

An E. coli expression system for the rapid purification and characterization of a v-abl tyrosine protein kinase.

A bacterial expression vector containing a segment of the v-abl gene from Abelson murine leukemia virus (A-MuLV) was constructed such that the gag region of v-abl was replaced by a sequence encoding the IgG-binding domain of the S. aureus protein A. pabl HP, a fusion protein encoded by this vector was rapidly purified to near homogeneity by affinity chromatography on IgG-Affigel and Mono Q FPLC. The Km of the pabl HP kinase for ATP varied with [Val5]-angiotensin II concentration and was 21.2 microM at saturating concentrations of [Val5]-angiotensin II. The Km for [Val5]-angiotensin II at saturating concentrations of ATP was 3.8 mM. The turnover number, at 20 degrees C, was 62 mumol min-1 mumol-1. Initial rate studies support a ternary complex kinetic mechanism for phosphoryl transferase. The substrate specificity of the pabl HP kinase was further characterized using synthetic peptides. This expression system, which enables the rapid purification of recombinant v-abl kinase is suitable for the comparative enzymological study of mutant v-abl enzymes generated by site-directed mutagenesis.

Isolation and characterization of temperature-sensitive mutants of Abelson murine leukaemia virus that exhibit dissociation among morphological transformation, soft agar colony-forming ability and tyrosine kinase activity.

Seven temperature-sensitive (ts) mutants of Abelson murine leukaemia virus (A-MuLV) were isolated on the basis of the temperature dependence of their soft agar colony-forming ability. These seven ts mutants exhibited similar characteristics and were not ts for morphological transformation and autophosphorylation of P120gag-abl protein. The dissociation of the properties of morphology, soft agar colony formation and tyrosine kinase activity might suggest that the v-abl product has more than one primary intracellular target.

Inhibition of tyrosine protein kinases by the antineoplastic agent adriamycin.

Adriamycin, a lipid-interacting anti-cancer agent, was found to inhibit the phosphorylation of polyGlu/Tyr (4:1) by tyrosine protein kinases either from spleen or expressed by the oncogene of Abelson murine leukemia virus. The dose dependent inhibition by adriamycin is accounted for by competition for the ATP binding site, but it is also deeply influenced by the nature and concentration of the phosphorylatable substrate, suggesting multiple interactions with the enzyme. The phosphorylation at tyrosine residues of cytosolic proteins from cells transformed by Abelson leukemia virus and the autophosphorylation of tyrosine protein kinases are also inhibited by adriamycin. Unlike tyrosine protein kinases most serine/threonine specific protein kinases, with the notable exception of protein kinase-C, appear to be relatively insensitive to adriamycin.

A truncated v-abl-derived tyrosine-specific tyrosine kinase expressed in Escherichia coli.

Several biochemical properties of a 43 kDa v-abl-encoded tyrosine-specific protein kinase (p43v-abl) expressed in Escherichia coli were examined. p43v-abl is a fragment of a 60 kDa v-abl-encoded precursor, p60v-abl, and could be generated by limited proteolysis of a purified p60v-abl with trypsin. Tryptic cleavage of p60v-abl was prevented in the presence of ATP. These results suggest that the catalytic kinase domain of v-abl-derived protein can be separated from other (regulatory) domains by limited proteolysis. p43v-abl readily phosphorylated tyrosine residues on several different protein and peptide substrates, including peptides containing only two amino acid residues. However, the local sequence of the tyrosine-containing peptide substrate significantly affected its rate of phosphorylation. Thus the primary structure and local conformation at the tyrosine acceptor site can play an important role in determining the substrate specificity of v-abl-derived kinase. Phosphorylation by p43v-abl requires Mn2+, Co2+ or Mg2+ and exhibits a strong preference for ATP as phosphate donor. Analogues of ATP and the thiol-reactive reagent N-ethylmaleimide inhibited p43v-abl kinase activity. Purified p43v-abl is intrinsically thermolabile (t1/2 = 5 min at 40 degrees C) and phosphorylates glycerol inefficiently (Km = 1.4 M).

Synthesis and evaluation of multisubstrate inhibitors of an oncogene-encoded tyrosine-specific protein kinase. 1.

The synthesis and testing of potential multisubstrate inhibitors of tyrosine-specific protein kinases are described. One of the substrates, ATP, was mimicked by the known kinase inhibitor 5'-[4-(fluorosulfonyl)benzoyl]adenosine, which was covalently linked via the sulfonyl moiety to tyrosine mimics. The resulting multisubstrate inhibitors were tested for their ability to inhibit the transfer of phosphate from ATP to a protein acceptor by p60v-abl, the tyrosine kinase encoded by the transforming gene (v-abl) of the Abelson murine leukemia virus (A-MuLV). Although the series of inhibitors displayed moderately potent activity (IC50 values as low as 19 microM), the absence of large effects produced by modification of the tyrosine mimic suggests that they do not behave as multisubstrate inhibitors but bind primarily through the adenosine moiety common to all the inhibitors. This interpretation is strengthened by the finding that the inhibitors lack specificity, inhibiting a serine kinase at comparable concentrations.

Synthesis and evaluation of multisubstrate inhibitors of an oncogene-encoded tyrosine-specific protein kinase. 2.

Tyrosine-specific protein kinases that transfer the terminal phosphate from ATP to protein acceptors are associated with certain transforming viruses and cell surface growth factor receptors. Here we describe the synthesis and testing of potential multisubstrate inhibitors of this class of enzymes. The inhibitors were prepared by covalent attachment of the terminal phosphate of ATP or its tetraphosphate analogue to tyrosine mimics. Testing against p60v-abl, the tyrosine kinase from the Abelson murine leukemia virus, showed that the series of inhibitors was moderately potent (IC50 values as low as 13 microM). However, structural modification of the tyrosine mimic, including replacement with a serine-like moiety, had little effect on potency. It is therefore concluded that the ATP moiety is largely responsible for binding and that the enzyme requires additional structural features for recognition of the tyrosine-containing substrate.

Insertional mutagenesis of the Abelson murine leukemia virus genome: identification of mutants with altered kinase activity and defective transformation ability.

A library of Abelson murine leukemia virus (A-MuLV) proviral DNAs with 12- or 6-base-pair (bp) insertional mutations was constructed. The 29 mutations characterized spanned the entire protein-coding region of the provirus. We tested the effects of these mutations both on the kinase activity of the gag-abl fusion protein encoded by the provirus and on the ability of the provirus to transform NIH 3T3 fibroblasts. To simplify assessment of the mutant kinases, we expressed the A-MuLV-encoded kinase in the bacterial expression vector pATH2, resulting in production of a trpE-gag-abl fusion protein in Escherichia coli. We used an immunoprecipitation kinase assay to measure both autophosphorylation and artificial substrate phosphorylation by the mutant kinases. To assay transformation ability of the mutant proviruses, we transfected NIH 3T3 fibroblasts with the mutants and with helper virus (Moloney MuLV) by the DEAE-dextran method. Our analysis of these A-MuLV insertional mutants allows the division of the protein-coding region of the provirus into four domains: domain A (proviral bp 1068 to 1685), in which insertions have no effect on the bacterially expressed kinase, but diminish both kinase activity and transformation efficiency in fibroblasts; domain B (bp 1750 to 2078), in which insertions have no effect on the provirus; domain C (bp 2181 to 2878), the critical kinase domain, in which 12-bp or even 6-bp insertions completely inactivate the A-MuLV kinase and result in transformation-defective proviruses; and domain D (bp 2956 to 4610), the large C-terminal domain in which mutations are silent.

Abelson-transformed fibroblasts contain nuclear phosphotyrosyl-proteins which preferentially bind to murine DNA.

Protein-tyrosine kinases, either in the form of growth-factor receptors or as the polypeptide products of oncogenes, appear to be important in the regulation of cell growth and transformation. A major question, however, is how their substrates mediate changes in gene expression (reviewed in refs 6-8). Because binding of proteins to specific DNA sequences represents the most direct mechanism for regulating transcription, we have investigated the possibility that some DNA-binding proteins may be substrates of protein-tyrosine kinases. Here, we present evidence for nuclear phosphotyrosyl-proteins in murine fibroblasts transformed by the v-abl protein-tyrosine kinase. Furthermore, we have found that these proteins are not significantly phosphorylated in normal NIH 3T3 cells. Finally, using affinity competition chromatography with bacterial and mouse DNA, we have demonstrated that some of these proteins preferentially bind to mouse DNA. The identification of phosphotyrosyl-proteins with selective DNA-binding properties suggests a possible mechanism through which protein-tyrosine kinases may effect changes in gene transcription.

Possible target of Abelson virus phosphokinase in cell transformation.

By fusing interphase cells to cells undergoing mitosis, the interphase chromosomes can be visualized. When analyzed in this way, chromosomes of normal mouse cells show characteristic undercondensed centromeric regions. We have found that the centromeric regions of chromosomes from Abelson virus-transformed cells are fully condensed. Abelson virus transforms mouse cells by introducing into them a virally encoded phosphokinase that is expressed constitutively. Thus, we propose that the condensation of centromeric chromatin is a result of overphosphorylation by the Abelson virus phosphokinase, and that the centromeric region is the relevant target of overphosphorylation in transformed cell growth.

Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus.

Sequences termed v-abl, which encode the protein-tyrosine kinase activity of Abelson murine leukemia virus, have been expressed in Escherichia coli as a fusion product (ptabl50 kinase). This fusion protein contains 80 amino acids of SV40 small t and the 403 amino acid protein kinase domain of v-abl. We report here the purification and characterization of this kinase. The purified material contains two proteins (Mr = 59,800 and 57,200), both of which possess sequences derived from v-abl. Overall purification was 3,750-fold, with a 31% yield, such that 117 micrograms of kinase could be obtained from 40 g of E. coli within 6-7 days. The specific kinase activity is over 170 mumol of phosphate min-1 mumol-1, comparable to the most active protein-serine kinases. Kinase activity is insensitive to K+, Na+, Ca2+, Ca2+-calmodulin, cAMP, or cAMP-dependent protein kinase inhibitor. The Km for ATP is dependent on the concentration of the second substrate. GTP can also be used as a phosphate donor. The enzyme can phosphorylate peptides consisting of as few as two amino acids and, at a very low rate, free tyrosine. Incubation of the kinase with [gamma-32P]ATP results in incorporation of 1.0 mol of phosphate/mol of protein. This reaction, however, cannot be blocked by prior incubation with unlabeled ATP. Incubation of 32P-labeled kinase with either ADP or ATP results in the synthesis of [32P]ATP. This suggests the phosphotyrosine residue on the Abelson kinase contains a high energy phosphate bond.

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).

Phosphorylation of ribosomal protein S6 on serine after microinjection of the Abelson murine leukemia virus tyrosine-specific protein kinase into Xenopus oocytes.

Phosphorylation of ribosomal protein S6 in NIH 3T3 fibroblasts is dependent on the presence of serum, but after transformation of these cells by Abelson murine leukemia virus (Ab-MuLV), S6 remained highly phosphorylated on serine residues either in the absence or the presence of serum. To investigate whether S6 phosphorylation in this system was a consequence of the action of the Ab-MuLV tyrosine-specific protein kinase, purified Ab-MuLV kinase made in Escherichia coli was microinjected into Xenopus oocytes and was observed to cause a 7- to 15-fold increase in the phosphorylation of S6 on serine residues. Two-dimensional phosphopeptide maps of S6 phosphorylated in Ab-MuLV-transformed NIH cells in the absence of serum were identical to those of S6 isolated from normal cells grown in the presence of serum. In addition, S6 from oocytes injected with Ab-MuLV kinase yielded an S6 phosphopeptide map indistinguishable from that of serum-stimulated NIH 3T3 cells, whereas S6 from control oocytes lacked several phosphopeptides. Ab-MuLV kinase did not phosphorylate S6 directly in vitro, and microinjection of a mutant Ab-MuLV protein lacking kinase activity had no effect. These results indicate that the Ab-MuLV kinase interacts with a cellular pathway to enhance S6 phosphorylation by directly or indirectly activating an S6 protein kinase and/or inactivating an S6 protein phosphatase.

Nucleotide sequence of Abelson murine leukemia virus genome: structural similarity of its transforming gene product to other onc gene products with tyrosine-specific kinase activity.

The nucleotide sequence of the proviral genome of Abelson murine leukemia virus (A-MuLV), an acute transforming virus of murine origin, has been determined. Like other transforming viruses, A-MuLV contains sequences derived from its helper virus, Moloney murine leukemia virus (M-MuLV), and a cell-derived protooncogene (abl) insertion sequence. By comparison of the A-MuLV sequence with that of M-MuLV, it was possible to precisely localize and define sequences contributed by the host cellular DNA. From the nucleotide sequence, we have predicted the amino acid sequence of p120gag-abl, the product of the A-MuLV gag-abl hybrid gene. The amino acid sequence of the putative abl gene, when compared with the sequences of other tyrosine-specific protein kinases (src, fes, fps, and yes), revealed significant homologies, indicating that all these functionally related transforming genes are derived from divergent members of the same protooncogene family. In addition to the gag-abl sequence, the proviral genome was found to contain an additional open reading frame that could code for an 18,000-dalton protein, whose role is at present undetermined.