Changes in p19Arf Localization Accompany Apoptotic Crisis during Pre-B-Cell Transformation by Abelson Murine Leukemia Virus

ABSTRACT Transformation by Abelson murine leukemia virus (Ab-MLV) is a multistep process in which growth-stimulatory signals from the v-Abl oncoprotein and growth-suppressive signals from the p19Arf-p53 tumor suppressor pathway oppose each other and influence the outcome of infection. The process involves a proliferative phase during which highly viable primary transformants expand, followed by a period of marked apoptosis (called “crisis”) that is dependent on the presence of p19Arf and p53; rare cells that survive this phase emerge as fully transformed and malignant. To understand the way in which v-Abl expression affects p19Arf expression, we examined changes in expression of Arf during all stages of Ab-MLV transformation process. As is consistent with the ability of v-Abl to stimulate Myc, a transcription factor known to induce p19Arf, Myc and Arf are induced soon after infection and p19Arf is expressed. At these early time points, the infected cells remain highly viable. The onset of crisis is marked by an increase in p19Arf expression and a change in localization of the protein from the nucleoplasm to the nucleolus. These data together suggest that the localization and expression levels of p19Arf modulate the effects of the protein during oncogenesis and reveal that the p19Arf-mediated response is subject to multiple layers of regulation that influence its function during Ab-MLV-mediated transformation.

Mutations Affecting the MA Portion of the v-Abl Protein Reveal a Conserved Role of Gag in Abelson Murine Leukemia Virus (MLV) and Moloney MLV

ABSTRACT Abelson murine leukemia virus (Ab-MLV) arose from a recombination between gag sequences in Moloney MLV (Mo-MLV) and the c-abl proto-oncogene. The v-Abl oncoprotein encoded by Ab-MLV contains MA, p12, and a portion of CA sequences derived from the gag gene of Mo-MLV. Previous studies indicated that alteration of MA sequences affects the biology of Mo-MLV and Ab-MLV. To understand the role of these sequences in Ab-MLV transformation more fully, alanine substitution mutants that affect Mo-MLV replication were examined in the context of Ab-MLV. Mutations affecting Mo-MLV replication decreased transformation, while alanine mutations in residues dispensable for Mo-MLV replication did not. The altered v-Abl proteins displayed aberrant subcellular localization that correlated to transformation defects. Immunofluorescent analyses suggested that aberrant trafficking of the altered proteins and improper interaction with components of the cytoskeleton were involved in the phenotype. Similar defects in localization were observed when the Gag moiety containing these mutations was expressed in the absence of abl-derived sequences. These results indicate that MA sequences within the Gag moiety of the v-Abl protein contribute to proper localization by playing a dominant role in trafficking of the v-Abl molecule.

Gag Influences Transformation by Abelson Murine Leukemia Virus and Suppresses Nuclear Localization of the v-Abl Protein

ABSTRACT 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.