The SV40 small-t antigen enhances transformation by viral large-T-T antigen and this requires the functions of at least two domains of small-t. The first falls within amino acids 97-103 and affects the ability of small-t to inhibit protein phosphatase 2A. PP2A inhibition correlates with the ability of small-t to activate a variety of cellular kinases (MAPK, MEK, JNK1) and to stimulate AP1 DNA binding activity. The increased AP1 activity results in transactivation of constructs that contain a cyclin D1 promoter by small-t. Proposed experiments will determine the mechanisms for increased AP1 activity and the role of small-t in regulating cyclin D1 expression in natural infections (aim 1). The second domain, the 42-47 region domain, is also required for transformation in small-t dependent systems. Preliminary results have shown that this domain is required for small-t to transactivate the cellular cyclin. A promoter in transient assays and to enhance expression of the endogenous cyclin A gene. The 42- 47 region will be probed genetically to determine whether transformation and transactivation functions can be separated and whether small-t and large-T antigens contribute equally in providing the 42-47 region function (aim 2). Proposed experiments will explore sequences in the cyclin A promoter that are required for small-t transactivation, and define segments of the promoter that are sufficient for small-t responsiveness when placed into minimal reporter constructs (aim 3). Based in part on results of studies of the cyclin A promoter, several possible mechanisms through which small-t may act will be tested (aim 4). The potential effect of small-t on cyclin A is of even greater interest given recent reports that, under restrictive growth conditions such as the absence of anchorage, cells may fail to grow because of a block to cyclin A expression. Thus, a final goal is to explore the effects of small -t antigen on cyclin A expression. Thus, a final goal is to explore the effects of small-t antigen on cyclin A levels in cells in which cyclin A expression is restricted. Results of these studies may explain why a requirement for small-t antigen has most frequently been observed in growth-arrested cells and in assays that require the anchorage-independent growth of transformed cells.
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