Ras complements the carboxyl terminus of v-Abl protein in lymphoid transformation.

Abelson murine leukemia virus (Ab-MLV) mutants expressing v-Abl proteins lacking the carboxyl terminus are compromised in the ability to transform lymphoid but not NIH 3T3 cells. This feature correlates with the presence of low levels of phosphotyrosine in lymphoid cells infected with carboxyl-terminal truncation mutants. In contrast, high levels of phosphotyrosine are observed in NIH 3T3 cells infected with wild-type and mutant Ab-MLV. Two downstream targets affected in lymphoid transformants are the GTPase-activating protein and GTPase-activating protein-associated protein p62, molecules which are heavily tyrosine phosphorylated in lymphoid cells transformed by wild-type Ab-MLV but not carboxyl-terminal truncation mutants of Ab-MLV. This difference suggested that signaling mediated via the Ras pathway may be compromised in lymphoid cells expressing the carboxyl-terminal truncation mutants. Consistent with this idea, expression of v-Ha-ras complemented these mutants in primary bone marrow transformation assays and increased transformation frequencies obtained with the Ab-MLV mutants 8- to 20-fold. These data suggest that a biologically important link exists between the carboxyl terminus of v-Abl protein and the Ras pathway. Signals transmitted via this connection may enhance those mediated via other regions of the v-Abl protein and facilitate transformation of primary, nonimmortalized cells such as pre-B lymphocytes.     

The carboxyl terminus of v-Abl protein can augment SH2 domain function

Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway.     

p53 deficiency increases transformation by v-Abl and rescues the ability of a C-terminally truncated v-Abl mutant to induce pre-B lymphoma in vivo

Abelson murine leukemia virus (A-MuLV) is an acute transforming retrovirus that preferentially transforms early B-lineage cells both in vivo and in vitro. Its transforming protein, v-Abl, is a tyrosine kinase related to v-Src but containing an extended C-terminal domain. Many mutations affecting the C-terminal portion of the molecule block the pre-B-transforming activity of v-Abl without affecting the fibroblast-transforming ability. In this study we have determined the abilities of both wild-type and C-terminally truncated (p90) forms of v-Abl to transform cells from p53(-/-) mice. Lack of p53 increases the susceptibility of bone marrow cells to transformation by v-Abl by a factor of more than 7 but does not alter v-Abl's preference for B220(+) IgM(-) pre-B cells. p53-deficient mice have earlier tumor onset, more rapid tumor progression, and decreased survival time following A-MuLV infection, but all of the tumors are pre-B lymphomas. Thus, p53-dependent pathways inhibit v-Abl transformation but play no role in conferring preferential transformation of pre-B cells. Surprisingly, the C-terminally truncated form of v-Abl (p90) transforms pre-B cells very efficiently in mice lacking p53, thus demonstrating that the C terminus of v-Abl does not determine preB tropism but is necessary to overcome p53-dependent inhibition of transformation.     

Gag influences transformation by Abelson murine leukemia virus and suppresses nuclear localization of the v-Abl protein

Like the v-Onc proteins encoded by many transforming retroviruses, the v-Abl protein is expressed as a Gag-Onc fusion. Although the Gag-derived myristoylation signal targets the v-Abl protein to the plasma membrane, the protein contains the entire MA and p12 sequences and a small number of CA-derived residues. To understand the role of Gag sequences in transformation, mutants lacking portions of these sequences were examined for the effects of these deletions on v-Abl function and localization. Deletion of the N-terminal third of p12 or all of p12 enhanced the transformation of both pre-B cells and NIH 3T3 cells. In contrast, deletions in MA or a deletion removing all of Gag except the first 34 amino acids important for myristoylation highly compromised the ability to transform either cell type. Although all of the mutant proteins retained kinase activity, those defective in transformation were reduced in their ability to activate Erk, suggesting a role for Gag sequences in v-Abl signaling. Immunofluorescence analysis revealed that a v-Abl protein retaining only the first 34 amino acids of Gag localized to the nucleus. These data indicate that Gag sequences are important for normal v-Abl signaling and that they suppress nuclear localization of the molecule.     

SH2-containing inositol 5'-phosphatase inhibits transformation of Abelson murine leukemia virus

v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) transforms pre-B cells. Transformation requires the phosphatidylinositol 3-kinase (PI3K) pathway. This pathway is antagonized by SH2-containing inositol 5'-phosphatase (SHIP), raising the possibility that v-Abl modulates PI3K signaling through SHIP. Consistent with this, we show that v-Abl expression reduces levels of full-length p145 SHIP in a v-Abl kinase activity-dependent fashion. This event requires signals from the Abl SH2 domain but not the carboxyl terminus. Forced expression of full-length SHIP significantly reduces Ab-MLV pre-B-cell transformation. Therefore, reduction of SHIP protein by v-Abl is a critical component in Ab-MLV transformation.     

Absence of p53 complements defects in Abelson murine leukemia virus signaling

The v-Abl protein encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation of pre-B cells via a two-stage process. An initial proliferative phase during which cells with limited tumorigenic potential expand is followed by a crisis period marked by high levels of apoptosis and erratic growth. Transformants that survive this phase emerge as fully malignant cells and usually contain mutations that disable the p53 tumor suppressor pathway. Consistent with the importance of p53 in this process, pre-B cells from p53 null animals bypass crisis. Thus, the transformation process reflects a balance between signals from the v-Abl protein that drive transformation and those coming from the cellular response to inappropriate growth. One prediction of this hypothesis is that Ab-MLV mutants that are compromised in their ability to transform cells may be less equipped to overcome the effects of p53. To test this idea, we examined the ability of the P120/R273K mutant to transform pre-B cells from wild-type, p53 null, and Ink4a/Arf null mice. The SH2 domain of the v-Abl protein encoded by this mutant contains a substitution that affects the phosphotyrosine-binding pocket, and this mutant is compromised in its ability to transform NIH 3T3 and pre-B cells, especially at 39.5 degrees C. Our data reveal that loss of p53 or Ink4a/Arf locus products complements the transforming defect of the P120/R273K mutant, but it does not completely restore wild-type function. These results indicate that one important transforming function of v-Abl proteins is overcoming the effects of a functional p53 pathway.     

Active Akt and functional p53 modulate apoptosis in Abelson virus-transformed pre-B cells

Suppression of apoptosis is an important feature of the Abelson murine leukemia virus (Ab-MLV) transformation process. During multistep transformation, Ab-MLV-infected pre-B cells undergo p53-dependent apoptosis during the crisis phase of transformation. Even once cells are fully transformed, an active v-Abl protein tyrosine kinase is required to suppress apoptosis because cells transformed by temperature-sensitive (ts) kinase mutants undergo rapid apoptosis after a shift to the nonpermissive temperature. However, inactivation of the v-Abl protein by a temperature shift interrupts signals transmitted via multiple pathways, making it difficult to identify those that are critically important for the suppression of apoptosis. To begin to dissect these pathways, we tested the ability of an SH2 domain Ab-MLV mutant, P120/R273K, to rescue aspects of the ts phenotype of pre-B cells transformed by the conditional kinase domain mutant. The P120/R273K mutant suppressed apoptosis at the nonpermissive temperature, a phenotype correlated with its ability to activate Akt. Apoptosis also was suppressed at the nonpermissive temperature by constitutively active Akt and in p53-null pre-B cells transformed with the ts kinase domain mutant. These data indicate that an intact Src homology 2 (SH2) domain is not critical for apoptosis suppression and suggest that signals transmitted through Akt and p53 play an important role in the response.     

Temperature-sensitive transformation by an Abelson virus mutant encoding an altered SH2 domain

Abelson murine leukemia virus (Ab-MLV) encodes the v-Abl protein tyrosine kinase and induces transformation of immortalized fibroblast lines and pre-B cells. Temperature-sensitive mutations affecting the kinase domain of the protein have demonstrated that the kinase activity is absolutely required for transformation. Despite this requirement, mutations affecting other regions of v-Abl modulate transformation activity. The SH2 domain and the highly conserved FLVRES motif within it form a phosphotyrosine-binding pocket that is required for interactions between the kinase and cellular substrates. To understand the impact of SH2 alterations on Ab-MLV-mediated transformation, we studied the Ab-MLV mutant P120/R273K. This mutant encodes a v-Abl protein in which the beta B5 arginine at the base of the phosphotyrosine-binding pocket has been replaced by a lysine. Unexpectedly, infection of NIH 3T3 or pre-B cells with P120/R273K revealed a temperature-dependent transformation phenotype. At 34 degrees C, P120/R273K transformed about 10-fold fewer cells than wild-type virus of equivalent titer; at 39.5 degrees C, 300-fold fewer NIH 3T3 cells were transformed and pre-B cells were refractory to transformation. Temperature-dependent transformation was accompanied by decreased phosphorylation of Shc, a protein that interacts with the v-Abl SH2 and links the protein to Ras, and decreased induction of c-Myc expression. These data suggest that alteration of the FLVRES pocket affects the ability of v-Abl to interact with at least some of its substrates in a temperature-dependent fashion and identify a novel type of temperature-sensitive Abelson virus.     

Growth, differentiation, and malignant transformation of pre-B cells mediated by inducible activation of v-Abl oncogene

The nonreceptor tyrosine kinase, encoded by the v-Abl oncogene of Abelson murine leukemia virus induces transformation of progenitor B cells. The v-Abl oncogene promotes cell cycle progression and inhibits pre-B cell differentiation. The temperature-sensitive form of Abelson murine leukemia virus offers a reversible model to study the role of v-Abl in regulating growth and differentiation. Inactivation of v-Abl elevates p27 and Foxo3a levels and activates NF-kappaB/Rel, which leads to G1 arrest and induction of Ig L chain gene rearrangement, respectively. In turn, v-Abl reactivation reduces p27 and Foxo3a levels, thus permitting G1-arrested cells to reenter the cell cycle. However, the cell lines derived from SCID mice that are defective in the catalytic subunit of DNA-dependent protein kinase retain elevated levels of p27 and Foxo3a proteins despite reactivation of v-Abl. Consequently, these cells are locked in the G1 phase for an extended period of time. The few cells that manage to bypass the G1 arrest become tumorigenic and fail to undergo pre-B cell differentiation induced by v-Abl inactivation. Deregulation of p27, Foxo3a, c-myc, and NF-kappaB/Rel was found to be associated with the malignant transformation of SCID temperature-sensitive form of Abelson murine leukemia virus pre-B cells.     

Disruption of the Shc/Grb2 complex during abelson virus transformation affects proliferation, but not apoptosis

The v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces pre-B-cell transformation. Signals emanating from the SH2 domain of the protein are required for transformation, and several proteins bind this region of v-Abl. One such protein is the adaptor molecule Shc, a protein that complexes with Grb2/Sos and facilitates Ras activation, an event associated with Ab-MLV transformation. To test the role this interaction plays in growth and survival of infected pre-B cells, dominant-negative (DN) Shc proteins were coexpressed with v-Abl and transformation was examined. Expression of DN Shc reduced Ab-MLV pre-B-cell transformation and decreased the ability of v-Abl to stimulate Ras activation and Erk phosphorylation in a Raf-dependent but Rac-independent fashion. Further analysis revealed that Shc is required for v-Abl-mediated Raf tyrosine 340 and 341 phosphorylation, an event associated with Erk phosphorylation. In contrast to effects on proliferation, survival of the cells and activation of Akt were not affected by expression of DN Shc. Together, these data reveal that v-Abl-Shc interactions are a critical part of the growth stimulatory signals delivered during transformation but that they do not affect antiapoptotic pathways. Furthermore, these data highlight a novel role for Shc in signaling from v-Abl to Raf.     

Abelson murine leukemia virus variants with increased oncogenic potential.

A number of strains of Abelson murine leukemia virus (A-MuLV) with various abilities to transform cells have been identified. Among these is the A-MuLV-P90 strain, a mutant derived from A-MuLV-P120 that encodes an A-MuLV protein missing sequences that are normally present at the extreme carboxy terminus of P120 (N. Rosenberg and O. N. Witte, J. Virol. 33:340-348, 1980). This virus transforms NIH 3T3 cells efficiently but does not transform a high frequency of lymphoid cells in vitro or in vivo. In this communication, we show that of the relatively few tumors induced by A-MuLV-P90 nearly all contained new variant viruses that stably expressed either larger or smaller A-MuLV proteins. Strains that expressed larger A-MuLV proteins behaved like A-MuLV-P120 in transformation assays, whereas those expressing smaller A-MuLV proteins induced a high frequency of tumors after a short latent period in vivo but failed to transform large numbers of lymphoid cells in vitro. Thus, these latter viruses separated the requirements for in vitro transformation of lymphoid cells from those for tumor induction. All of the variants differed from A-MuLV-P90 in the carboxy-terminal region of the A-MuLV protein, suggesting that sequences in this region play a key role in the ability of the virus to interact with hematopoietic cells in vivo and in vitro.     

Dok1 and SHIP Act as Negative Regulators of v-Abl-Induced Pre-B Cell Transformation,Proliferation and Ras/Erk Activation

The v-Abl tyrosine kinase activates several signaling pathways during transformation of bonemarrow cells in mice. Because the SH2-containing inositol 5’-phosphatase (SHIP) andDownstream of tyrosine kinase 1 (Dok1) have been shown interact with Abl, the effect ofSHIP and Dok1 deficiency on v-Abl transformation was investigated. Bone marrow cellsfrom either Dok1- or SHIP-deficient mice are more susceptible to transformation by v-Abl.v-Abl-transformed pre-B cells from these knockout mice show Abl kinase-dependenthyperproliferation and moderate resistance to apoptosis. Elevated activation of Ras, Raf-1,and Erk, but not of Akt, was observed in either SHIP (-/-) or Dok1 (-/-) v-Abl-transformedcells. This activation is sensitive to treatment with STI571. Furthermore, treatment of thesecells with either a farnesyltransferase inhibitor or a MEK1/2 inhibitor abrogates the increasedproliferation of SHIP (-/-) or Dok1 (-/-) cells in a dose-dependent manner. Complementationof SHIP (-/-) or Dok1 (-/-) cells abrogates their hyperproliferation and intracellular Erkactivation. These data indicate that both SHIP and Dok1 functionally regulate the activationof Ras-Erk pathway by v-Abl and affect the mitogenic activity of v-Abl transformed bonemarrow cells.     

v-Abl signaling disrupts SOCS-1 function in transformed pre-B cells

The v-Abl oncogene activates Jak-Stat signaling during transformation of pre-B cells in mice. Disrupting Jak activation by deleting the Jak binding domain of v-Abl or by expressing a dominant-negative Jak1 decreases v-Abl transformation efficiency. As SOCS-1 is a known potent inhibitor of Jak kinases, the mechanism by which v-Abl bypasses SOCS-1 regulation to constitutively activate Jak kinases was investigated. SOCS-1 is expressed in v-Abl-transformed cells but is unable to inhibit v-Abl-mediated Jak-Stat signaling. In v-Abl transformants, SOCS-1 can inhibit cytokine signals, but it is more efficient at doing so when the cells are treated with STI571, an Abl kinase inhibitor. Downstream effects of v-Abl signaling include phosphorylation of SOCS-1 on nontyrosine residues, disruption of the interaction between SOCS-1 and the Elongin BC complex, and inhibition of SOCS-1-mediated proteasomal targeting of activated Jaks. These findings reveal a mechanism by which Jak-dependent oncogenes may bypass SOCS-1 inhibition.     

Caveolin-1 and a 29-kDa caveolin-associated protein are phosphorylated on tyrosine in cells expressing a temperature-sensitive v-Abl kinase

Caveolin-1 was originally identified as a tyrosine-phosphorylated protein in v-Src-transformed cells and it was suggested that phosphorylation of this protein could mediate transformation by the tyrosine kinase class of oncogenes (J. R. Glenney, 1989, J. Biol. Chem. 264, 20163--20166). We found that caveolin-1 is also phosphorylated on tyrosine in v-Abl-transformed cells. In fact, caveolin-1 and a caveolin-associated protein of 29 kDa are among the strongest phosphotyrosine signals detected in the Abl-expressing cells. In addition, v-Abl shows a preferential phosphorylation of caveolin-1 and the 29-kDa caveolin-associated protein over other proteins in the caveolin-enriched Triton-resistant cell fraction. These data indicate that caveolin-1 and the 29-kDa caveolin-associated protein may be preferred substrates of the Abl kinase. Caveolin-1 is phosphorylated at tyrosine 14 in v-Abl-expressing cells as has been observed previously in v-Src-expressing cells. However, using a temperature-sensitive allele of v-Abl (ts120 v-Abl) we provide evidence that caveolin-1 phosphorylation is not sufficient to mediate the loss of caveolin expression or loss of cell adhesion induced by v-Abl.     

Structural domains of the avian erythroblastosis virus erbB protein required for fibroblast transformation: dissection by in-frame insertional mutagenesis.

Avian erythroblastosis virus (AEV) induces erythroblastosis and fibrosarcomas. The viral erbB protein is required for AEV-mediated oncogenesis. To explore the structural aspects of the v-erbB polypeptide necessary for its oncogenic function, we created a series of small in-frame insertions in different domains of the v-erbB oncogene. AEV genomes bearing lesions within the v-erbB kinase domain demonstrated a drastically decreased ability to transform avian fibroblasts, establishing a functional role for this structurally conserved oncogene domain. In contrast, mutations in the extracellular domain, between the transmembrane region and the kinase domain, or at the extreme C terminus of the v-erbB protein had no effect on AEV-mediated fibroblast transformation. One lesion within the v-erbB kinase domain, a 10-amino acid insertion, produced a temperature-sensitive mutant capable of fibroblast transformation at 36 degrees C but not at 41 degrees C, suggesting that small in-frame insertions have general utility for the in vitro creation of conditional mutants.     

Site-directed deletions of Abelson murine leukemia virus define 3'' sequences essential for transformation and lethality.

Abelson murine leukemia virus (A-MuLV) encodes a single protein with tyrosine kinase activity that can transform fibroblast cell lines in vitro and lymphoid target cells in vitro and in vivo. Expression of kinase-active A-MuLV protein can result in a deleterious effect on transformed fibroblast populations, leading to cell death or selection for nonlethal mutants of the virus. These mutants retain expression of the kinase activity but have lost large portions of the carboxy terminus of the Abelson protein. To more precisely map the sequences involved in this lethal effect, we have isolated a series of site-directed deletions from a DNA clone of the P160 wild-type strain of A-MuLV. In addition, a number of unexpected, spontaneous deletions occurring during transfection of NIH 3T3 cells were isolated. These deletions result in expression of carboxy-terminal truncated forms of the A-MuLV protein ranging from 130,000 to 84,000 in molecular weight. Analysis of the transforming and lethal activities of each mutant recovered in its RNA viral form shows that the transformation-essential and lethal-essential sequences do not overlap. These data and our previous work suggest that a function carried by the carboxy-terminal region of the A-MuLV protein acts in cis with the kinase-essential region to mediate the lethal effect.     

Nonmyristoylated Abl proteins transform a factor-dependent hematopoietic cell line.

N-terminal myristoylation can promote the association of proteins with the plasma membrane, a property that is required for oncogenic variants of Src and Abl to transform fibroblastic cell types. The P210bcr/abl protein of chronic myelogenous leukemia cells is not myristoylated and does not stably transform NIH 3T3 fibroblasts; however, it will transform lymphoid and myeloid cell types in vitro and in vivo, suggesting that myristoylation is not required for Abl variants to transform hematopoietic cells. To test this hypothesis, we introduced point mutations that disrupt myristoylation into two activated Abl proteins, v-Abl and a deletion mutant of c-Abl (delta XB), and examined their ability to transform an interleukin-3-dependent lymphoblastoid cell line, Ba/F3. Neither of the nonmyristoylated Abl proteins transformed NIH 3T3 fibroblasts, but like P210bcr/abl, both were capable of transforming the Ba/F3 cells to factor independence and tumorigenicity. Nonmyristoylated Abl variants did not associate with the plasma membrane in the transformed Ba/F3 cells. These results demonstrate that Abl proteins can transform hematopoietic cells in the absence of membrane association and suggest that distinct functions of Abl are required for transformation of fibroblast and hematopoietic cell types.     

Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene.

A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.