MAPPING OF POLYOMAVIRUS MIDDLE T-DOMAIN THAT IS RESPONSIBLE FOR AP-1 ACTIVATION

Cell transformation by Polyomavirus middle T (MT) oncoprotein involves binding and activation of several cytoplasmic proteins that participa te in growth factors-induced mitogenic signal transduction to the nucl eus. We have previously reported that the AP-I transcriptional complex is a target for MT during cell transformation. To analyse the interac tions between MT and cellular proteins that are required for constitut ive AP-1 activation, we compared wild type and transformation-defectiv e MT mutant cell lines. High AP-1 activity, assessed by gel mobility s hift assays, displayed by MT-overexpressing cells, is dependent on MT binding to phosphatidylinositol-3 kinase (P13K). Treatment with wortma nnin (a specific P13K inhibitor) leads to decreased AP-I activity. Sup ershift and Western blot analysis with specific antisera, indicate tha t JunB and cJun, but not cFos or FosB are present in the AP-1 complex. The results confirm the AP-1 complex as a downstream MT target and in dicate that AP-1 activation may not be sufficient for cell transformat ion, since two transformation-defective MT mutants (250phe and MT322) display high AP-1 activity.