Binding of Src family kinases to membrane-associated polyoma virus middle T-antigen (PyMT) can result in the phosphorylation of PyMT tyrosine 250, which serves as a docking site for the binding of Shc and subsequent activation of the Raf-MEK-ERK (MAP) kinase cascade. In a screen for PyMT variants that could not activate the ARF tumor suppressor, we isolated a cytoplasmic nontransforming mutant (MTA) that encoded a C-terminal truncated form of the PyMT protein. Surprisingly, MTA was able to strongly activate the MAP kinase pathway in the absence of Src family kinase and Shc binding. Interestingly, the polyoma small T-antigen (PyST), which shares with MTA both partial amino acid sequence homology and cellular location, also activates the MAP kinase cascade. Activation of the MAP kinase cascade by both MTA and PyST has been demonstrated to be PP2A-dependent. Neither MTA nor PyST activate the phosphorylation of AKT. The SV40 small T-antigen, which is similar to PyST in containing a J domain and in binding to the PP2A AC dimer, does not activate the MAP kinase cascade, but does stimulate phosphorylation of AKT in a PP2A-dependent manner. These findings highlight a novel role of PP2A in stimulating the MAP kinase cascade and indicate that the similar polyoma and SV40 small T-antigens influence PP2A to activate discrete cellular signaling pathways involved in growth control.AKT ͉ MAP kinase cascade ͉ polyoma virus T-antigens ͉ SV40 small T-antigen A ctivating oncogenes overcome normal cell regulatory control systems and stimulate growth signaling pathways leading to unregulated cell division. The polyoma virus (Py) early region specifies three proteins, large T-antigen (PyLT), middle T-antigen (PyMT), and small T-antigen (PyST), by differential splicing of mRNA derived from the same genomic DNA sequence (1). The membrane-bound PyMT is a potent activating oncogene, which has no intrinsic enzyme activity but acts as a scaffolding protein and binds and modulates the activities of a number of important cellular signaling proteins (1, 2). Studies with PyMT have provided great insight into a number of critical cellular signaling pathways involved in growth control (3).Membrane-bound PyMT binds and activates the cellular Src tyrosine kinase family resulting in phosphorylation of a number of important PyMT tyrosine residues (1, 2) ( Figs. 1 and 2). Phosphorylation of PyMT tyrosine 315 (Y315) results in the binding of the PI3-kinase regulatory p85 subunit and subsequent activation of PI3-kinase activity, resulting in the phosphorylation of a number of cellular targets, including AKT ( Figs. 1 and 2). Phosphorylation of PyMT tyrosine 250 (Y250) results in the binding of the Shc family of cellular proteins. Bound Shc associates with the adapter molecule Grb2, which binds to the Sos guanine nucleotide exchange factor, resulting in the activation of Ras. Ras activation leads to the activation of the Raf-MAP kinase pathway resulting in the phosphorylation of MEK and ERK (Fig. 1). Phosphorylation of PyMT tyrosine 322 (Y322) results i...

Activation of the ARF-p53 tumor suppressor pathway is one of the cell's major defense mechanisms against cancer induced by oncogenes. The ARF-p53 pathway is dysfunctional in a high proportion of human cancers. The regulation of the ARF-p53 signaling pathway has not yet been well characterized. In this study polyoma virus (Py) is used as a tool to better define the ARF-p53 signaling pathway. Py middle T-antigen (PyMT) induces ARF, which consequently up-regulates p53. We show that Py small T-antigen (PyST) blocks ARF-mediated activation of p53. This inhibition requires the small T-antigen PP2A-interacting domain. Our results reveal a previously unrecognized role of PP2A in the modulation of the ARF-p53 tumor suppressor pathway. The ARF-p53 tumor suppressor pathway is one of the cell's major defenses against stimulation of uncontrolled cell division induced by activated cellular and viral oncogenes (1-5). ARF and͞or p53 are mutated in Ͼ70% of human cancers. The inappropriate activation of growth promoting cellular signaling pathways by oncogenes can result in the induction of ARF. Expression of ARF can activate p53 leading to apoptotic cell death or cell-cycle arrest. The mechanisms by which the ARFp53 pathway is modulated remain to be precisely elucidated.The small DNA viruses have provided great insight into a number of the cellular signaling pathways that they modulate. During their infectious cycle they overcome cell regulatory control systems and activate signaling pathways required for viral DNA synthesis and virus assembly. Unscheduled activation of these cellular growth signals activates ARF and, consequently, p53, leading to abortive infection. To counteract this outcome, most DNA viruses encode proteins that inhibit p53 function. Most of these viral proteins target p53 directly. For example, the SV40 large T-antigen binds to p53 and inhibits its transcriptionfactor activity (6-8). Adenovirus E1B 55K and HPV E6 bind to p53 and, in coordination with other proteins, block its function and cause its degradation (9-15).Polyoma virus (Py) is unusual in that none of its early region proteins, large T-antigen (PyLT), middle T-antigen (PyMT), and small T-antigen (PyST), bind to p53 (16). We have previously shown that the Py oncogene PyMT activates ARF and p53 and therefore will not transform primary mouse cells and REF52 cells that contain an intact ARF-p53 pathway (17). PyMT transforms these cell types when either p53 or ARF is inactivated (17, 18). PyMT will also transform primary mouse and REF52 cells in which PyLT and PyST are also expressed (19,20). In these transformed cells, wild-type ARF is abundantly expressed, but the level of wild-type p53 is low (17,18). This finding suggests that PyLT and͞or PyST prevent ARF from activating p53. In this paper, we identify and define which Py protein and function is involved in abrogating this critical regulatory step. Materials and MethodsCell Culture and Transfection. REF52 and Rat-1 are established lines of rat embryo fibroblasts and are described in refs. 17, 18, a...

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