The p110α Isoform of Phosphatidylinositol 3-Kinase Is Essential for Polyomavirus Middle T Antigen-Mediated Transformation

Abstract

Middle T antigen (MT) of polyomavirus is known to play an important role in virus-mediated cellular transformation. While MT has been extensively examined in spontaneously immortalized rodent fibroblasts, its interactions with cells of other types and species are less well understood. We have undertaken a cross-species and cross-cell-type comparison of MT-induced transformation in cells with genetically defined backgrounds. We tested the transforming abilities of a panel of MT mutants, Y250F, Y315F, and Y322F, that are selectively mutated in the binding sites for the principal effectors of MT-Src homology 2 domain-containing transforming protein, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-␥, respectively-in fibroblasts and epithelial cells of murine or human origin. We found that the Y315F mutation disabled the ability of MT to induce transformation in all cell types and species tested. While Y315F also failed to activate the PI3K pathway in these cells, genetic evidence has indicated Y315 may make other contributions to transformation. To confirm the role of PI3K, the PIK3CA gene, encoding p110␣, the prime effector of PI3K signaling downstream from activated growth factor receptors, was genetically ablated. This abolished the transforming activity of MT, demonstrating the essential role for this PI3K isoform in MT-mediated transformation. The Y250F mutant was able to transform the human, but not the murine, cells that were examined. Interestingly, this mutant fully activates the PI3K pathway in human cells but activated PI3K signaling poorly in the murine cells used in the study. This again points to the importance of PI3K activation for transformation and suggests that the mechanism by which MT activates the PI3K pathway differs in different species.Polyomavirus (PyV) is a small, double-stranded, closed-circular-DNA virus with an approximately 5-kb genome divided into two roughly equal regions. The late transcripts produce the viral capsid proteins, whereas the early region encodes three so-called tumor (T) antigens (46) that are important for both productive infection and transformation. The 785-aminoacid large T antigen (LT) is a nuclear protein with originspecific DNA binding properties and an ATPase activity essential for viral replication (46). LT cannot transform cells in culture but has the ability to immortalize primary cells (16,32). Small t antigen (st) is a 195-amino-acid protein found both in the nucleus and in the cytoplasm (46). It is known to bind and thereby inhibit protein phosphatase 2A (PP2A) (39, 52). Middle T antigen (MT), a 421-amino-acid protein associated with membranes and underlying cytoskeletal elements, has been shown to be essential for transformation of rodent fibroblasts in tissue cultures (20,34,47). Mammary epithelial cell-specific expression of MT in transgenic mice results in the induction of multifocal mammary tumors with 100% penetrance (21).MT functions by providing a platform for the assembly of cellular signaling proteins (11,26). Like st, it binds to the ...

Lessons in Signaling and Tumorigenesis from Polyomavirus Middle T Antigen

“…In human mammary epithelial cells and fibroblasts, mutation of Y250 to F has no effect on transformation (303). Even in a single cell type, rat F111 fibroblasts, different clones respond differently to Y250F mutations (214).…”

“…The mutations found in p110␣ in human tumors have been shown to be activating (145,154,248,347). Knockout of p110␣ is sufficient to block transformation in standard mouse embryo fibroblasts systems (303). Conditional knockout of p110␣ (346) or heterozygous knock-in of a kinase dead allele of p110␣ (107) shows that p110␣ is also critical to RTK signaling.…”

“…MT is phosphorylated at tyrosine 250 in the MT-Src family complexes (141). Mutation of tyrosine 250 (185,214,303) has a dramatic effect on transforming ability in many systems. When the MT gene is expressed as a transgene in the mammary gland, there is a dramatic decrease in tumorigenesis compared to that seen with the wild type (322).…”

“…A number of nuclear oncogenes, such as those encoding PyLT (167), E1A (244), and Myc (167), complement. Mutant p53 (303) and ST (194,216), which acts on the p53 pathway, complement as well. MT can also be sufficient to cause tumors in animals when introduced through a viral vector or a transgene (1,12,92,126,170,234,286).…”

A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling

“…The mutated p110␣ found in human tumors has been shown to be activating (44)(45)(46)(47). Knockout of p110␣ is sufficient to block MT transformation in mouse embryo fibroblasts (48). The defects we observed for E349K MT are sufficient to explain the transformation defect, but raise new questions about how PI3K signaling is regulated.…”