Mapping of polyomavirus middle T domain that is responsible for AP-1 activation

Abstract

Cell transformation by Polyomavirus middle T (MT) oncoprotein involves binding and activation of several cytoplasmic proteins that participate in growth factorsinduced mitogenic signal transduction to the nucleus. We have previously reported that the AP-1 transcriptional complex is a target for MT during cell transformation. To analyse the interactions between MT and cellular proteins that are required for constitutive AP-1 activation, we compared wild type and transformation-defective MT mutant cell lines. High AP-1 activity, assessed by gel mobility shift assays, displayed by MT-overexpressing cells, is dependent on MT binding to phosphatidylinositol-3 kinase (P13K). Treatment with wortmannin (a speci®c P13K inhibitor) leads to decreased AP-1 activity. Supershift and Western blot analysis with speci®c antisera, indicate that JunB and cJun, but not cFos or FosB are present in the AP-1 complex. The results con®rm the AP-1 complex as a downstream MT target and indicate that AP-1 activation may not be su cient for cell transformation, since two transformation-defective MT mutants (250phe and MT322) display high AP-1 activity.

Natural Biology of Polyomavirus Middle T Antigen

“…This AP-1 composition, which contrasts with earlier findings indicating that MTinduced transformation was dependent on activation of the c-fos promoter, could be a result of either the cell type used (BALB-3T3) or the separation of MT from other components of Py, such as ST, which is important for the activation of the fos promoter (237,246). The connection between the tyrosine 315 phosphate site and AP-1 has been postulated to be due to the activation of Akt by the PI 3-kinase pathway, which leads to phosphorylation and resultant inactivation of glycogen synthase kinase-3␤ (GSK-3) (246,348). GSK-3 is a negative regulator of jun family members and phosphorylates residues in their DNA binding domains (80,242).…”

“…These observations may be a result of the cell type used or alternative pathways for activating JNK. The AP-1 expression induced by the combined activation of PI 3-kinase and Shc in 3T3 cells infected with a recombinant retrovirus containing a cDNA encoding MT (or mutant forms of MT) was made up of c-jun and junB, not c-fos or fosB, although the presence of other fos (Fra1, Fra2) and jun (junD) proteins has not been determined (246). This AP-1 composition, which contrasts with earlier findings indicating that MTinduced transformation was dependent on activation of the c-fos promoter, could be a result of either the cell type used (BALB-3T3) or the separation of MT from other components of Py, such as ST, which is important for the activation of the fos promoter (237,246).…”

“…The AP-1 expression induced by the combined activation of PI 3-kinase and Shc in 3T3 cells infected with a recombinant retrovirus containing a cDNA encoding MT (or mutant forms of MT) was made up of c-jun and junB, not c-fos or fosB, although the presence of other fos (Fra1, Fra2) and jun (junD) proteins has not been determined (246). This AP-1 composition, which contrasts with earlier findings indicating that MTinduced transformation was dependent on activation of the c-fos promoter, could be a result of either the cell type used (BALB-3T3) or the separation of MT from other components of Py, such as ST, which is important for the activation of the fos promoter (237,246). The connection between the tyrosine 315 phosphate site and AP-1 has been postulated to be due to the activation of Akt by the PI 3-kinase pathway, which leads to phosphorylation and resultant inactivation of glycogen synthase kinase-3␤ (GSK-3) (246,348).…”

Functional analysis of newly discovered growth control genes: experimental approaches

“…Plasmids and viral vectors (among which are retrovirus and adenovirus) have been used to transduce gene sequences to mammalian cells (2)(3)(4)(5). Overexpression is a powerful approach to study genes associated with activation of cell proliferation, cell cycle and oncogenesis.…”

Suppression of AP‐1 constitutive activity interferes with polyomavirus MT antigen transformation ability

“…Association of MT antigen with these cellular proteins occurs, in general, through SH2 or PTB domains present in these proteins and MT phosphotyrosine residues. Generation of a large number of MT mutants [Carmichael et al, 1984; Morgan et al, 1988; Druker and Roberts, 1991; Druker et al, 1992] was important for identification of the MT domains that are responsible for binding to these cellular proteins and determination of their importance in MT‐induced cell transformation [Druker et al, 1990; Glenn and Eckhart, 1993; Campbell et al, 1994, 1995; Oliveira et al, 1998].…”