Middle T antigen activation of signal transduction pathways does not overcome p53-mediated growth arrest

Abstract

Polyomavirus middle T antigen does not overcome p53-mediated G(1) arrest in mouse embryo fibroblasts. Middle T antigen still associates with the signaling molecules phosphatidylinositol 3-kinase and SHC and activates the transcriptional activity of c-Myc and AP1 in p53-arrested cells. Examination of cell cycle regulatory proteins indicated that p53 does not interfere with these mitogenic signals but acts later in the G(1) phase of the cell cycle.

FIG. 1

MT associates with a tyrosine kinase and interacts with PI3K and SHC in p53 growth-arrested cells. (A) Extracts were prepared from M/puro, M/tsp53, and M/tsp53/MT cells cultured at 37 and 32°C. MT was immunoprecipitated and incubated with [γ-³²P]ATP to detect associated kinase activity. The autoradiographs displayed in the upper panel show the MT-associated kinase from designated cells cultured at indicated temperatures (°C). The lower panel shows Western blots of total-cell lysates probed with antibodies to MT and antibodies to phosphotyrosine (Y-P). (B) Extracts were prepared from M/tsp53 and M/tsp53/MT cells cultured at 37 or 32°C. Immunoblots of MT immune complexes (IP MT) were probed with antibodies to PI3K or SHC (left panels). As a control, Western blots of total-cell lysate were probed to detect cellular levels of PI3K and SHC at each temperature (°C) (right panels). Numbers at right of each panel indicate molecular masses in kilodaltons.

FIG. 2

Transcriptional activity driven by c-Myc and AP1 is greater in p53 growth-arrested cells expressing MT. M/tsp53 and M/tsp53/MT cells were transfected with luciferase reporter vectors containing multiple ATF, c-myc, or AP1 binding sites upstream of a minimal promoter. The luciferase reporter plasmid with the minimal promoter was also transfected (Vector). A reporter plasmid containing the rat β-actin promoter driving expression of β-galactosidase was cotransfected and used as a control for transfection efficiency. Following transfection, cells were cultured in Dulbecco modified Eagle medium plus 0.5% serum at 37°C for 24 h and then at 32°C for 48 h. Cell extracts were harvested, and luciferase and β-galactosidase activities were measured. The graph compares luciferase activity in M/tsp53/MT cells to that in M/tsp53 cells. Each transfection was done in duplicate. The data represent the averages of four independent experiments. The error bars represent standard deviations between experiments.

FIG. 3

p53 arrests cells in late G₁ phase, after expression of cyclin D. M/tsp53 and M/tsp53/MT cells were grown at 32°C for 24 h to arrest growth (0), followed by culture at 37°C for the times (hours) indicated above the panels. Cell extract was made, and protein levels were analyzed by Western blotting. Blots were probed with antibodies to cyclin D, p21/WAF1, and pRB.