Disruption of the Shc/Grb2 Complex during Abelson Virus Transformation Affects Proliferation, but Not Apoptosis

ABSTRACT 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.

p16Ink4a Interferes with Abelson Virus Transformation by Enhancing Apoptosis

ABSTRACT Pre-B-cell transformation by Abelson virus (Ab-MLV) is a multistep process in which primary transformants are stimulated to proliferate but subsequently undergo crisis, a period of erratic growth marked by high levels of apoptosis. Inactivation of the p53 tumor suppressor pathway is an important step in this process and can be accomplished by mutation of p53 or down-modulation of p19Arf, a p53 regulatory protein. Consistent with these data, pre-B cells from either p53 or Ink4a/Arf null mice bypass crisis. However, the Ink4a/Arf locus encodes both p19Arf and a second tumor suppressor, p16Ink4a, that blocks cell cycle progression by inhibiting Cdk4/6. To determine if p16Ink4a plays a role in Ab-MLV transformation, primary transformants derived from Arf −/− and p16 Ink4a−/− mice were compared. A fraction of those derived from Arf −/− animals underwent crisis, and even though all p16 Ink4a−/− primary transformants experienced crisis, these cells became established more readily than cells derived from +/+ mice. Analyses of Ink4a/Arf −/− cells infected with a virus that expresses both v-Abl and p16Ink4a revealed that p16Ink4a expression does not alter cell cycle profiles but does increase the level of apoptosis in primary transformants. These results indicate that both products of the Ink4a/Arf locus influence Ab-MLV transformation and reveal that in addition to its well-recognized effects on the cell cycle, p16Ink4a can suppress transformation by inducing apoptosis.

Temperature-Sensitive Transformation by an Abelson Virus Mutant Encoding an Altered SH2 Domain

ABSTRACT 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 βB5 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°C, P120/R273K transformed about 10-fold fewer cells than wild-type virus of equivalent titer; at 39.5°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.

Loss of Heterozygosity at the Ink4a/ArfLocus Facilitates Abelson Virus Transformation of Pre-B Cells

ABSTRACT In many tumor systems, analysis of cells for loss of heterozygosity (LOH) has helped to clarify the role of tumor suppressor genes in oncogenesis. Two important tumor suppressor genes, p53 and the Ink4a/Arf locus, play central roles in the multistep process of Abelson murine leukemia virus (Ab-MLV) transformation. p53 and the p53 regulatory protein, p19Arf, are required for the apoptotic crisis that characterizes the progression of primary transformed pre-B cells to fully malignant cell lines. To search for other tumor suppressor genes which may be involved in the Ab-MLV transformation process, we used endogenous proviral markers and simple-sequence length polymorphism analysis to screen Abelson virus-transformed pre-B cells for evidence of LOH. Our survey reinforces the role of the p53-p19 regulatory pathway in transformation; 6 of 58 cell lines tested had lost sequences on mouse chromosome 4, including theInk4a/Arf locus. Consistent with this pattern, a high frequency of primary pre-B-cell transformants derived fromInk4a/Arf +/− mice became established cell lines. In addition, half of them retained the single copy of the locus when the transformation process was complete. These data demonstrate that a single copy of the Ink4a/Arf locus is not sufficient to fully mediate the effects of these genes on transformation.

A Protein Binding Specifically to the IgG2b Switch Region

The Abelson-virus-transformed mouse pre-B-cell line 18-81 switches almost exclusively from μ to γ2b. From nuclear extracts of this cell line, we have isolated a factor that specifically binds to Sγ2b. After an eight-step purification scheme, in which different types of DNA-affinity chromatography were used as key elements, we obtained a preparation with two narrowly spaced bands at approximately 69 kD on a silver-stained SDS gel. Binding specificity of main-peak fractions of affinity-purified proteins was analyzed by gel shift assays in which Sγ2b, but not Sμ, competes. The results are consistent with this factor being part of the switch recombinase.