Analysis of AP-1 function in cellular transformation pathways

Abstract

To understand the role of endogenous AP-1 activity in cellular transformation induced by oncogenes, we have made use of a fos mutant (supfos-1) and a jun mutant (supjun-1), either of which can function as a transdominant inhibitor of AP-1-mediated transcriptional regulation. Chicken embryo fibroblasts (CEF) infected with a series of transforming retroviruses were doubly infected with retrovirus carrying supfos-1 or supjun-1, and suppression of cellular transformation was monitored in terms of reversion to normal cellular morphology or acquisition of anchorage-dependent growth. Cellular transformation induced by several exogenously expressed transforming genes of thefos orjun family was efficiently suppressed, as expected. CEF transformed by v-src, v-yes, v-.fps, c-Ha-ras, and N-terminally truncated c-rafwere also induced to revert to the normal phenotype by these transdominant mutants, suggesting that functional transcription factor AP-1 activity is essential for the cellular transformation induced by these oncogenes. The suppression is not attributable to nonspecific inhibition of cellular proliferation, because CEF transformed by v-ros or v-myc were not induced to revert to the normal phenotype. We next analyzed changes in all known components of chicken AP-1 induced by v-src, c-Ha-ras, or activated c-raf transformation. The levels of both Fra-2 and c-Jun expression were elevated two-to fourfold, and hyperphosphorylation of Fra-2 was also observed. We further showed that Fra-2-c-Jun heterodimer is mainly responsible for the elevated AP-1 DNA-binding activity in these transformed cells, and we propose that this heterodimer play a crucial role in the transformation induced by these oncogenes.

Nerve Growth Factor, But Not Epidermal Growth Factor, Increases Fra-2 Expression and Alters Fra-2/JunD Binding to AP-1 and CREB Binding Elements in Pheochromocytoma (PC12) Cells

“…Western blots were performed to identify NGF-specific changes in expression or size of the Fra-2 or JunD proteins. In agreement with previous reports, Fra-2 was represented by two or more bands ranging from 40 -46 kDa, with the larger bands representing phosphorylated forms of the protein Gruda et al, 1994;Suzuki et al, 1994). The exact number Figure 5.…”

“…In chick embryo fibroblasts Fra-2/c-Jun diminishes transcription, but Fra-2/JunD enhances transcriptional activity (Suzuki et al, 1991). The Fra-2/c-Jun complex is crucial to transformation in these cells (Nishina et al, 1990;Suzuki et al, 1994). In other cells, Fra-1, but not Fra-2, complexes with c-Jun to mediate transformation (Mechta et al, 1997).…”

Differentiation modulates the balance of positive and negative Jun/AP-1 DNA binding activities to regulate cellular proliferative potential: different effects in nontransformed and transformed cells.

“…Numerous viral oncogenes and carcinogenic agents have been shown to activate AP-1 DNA binding activity. Exampies include v-src, v-mos, polyoma middle T antigen, neu,dimethylbenzanthracene,quinolineoxide, ionizing radiation, ultraviolet light, and asbestos (Sherman et al, 1990;Angel and Karin, 1991;Heintz et al, 1993;Suzuki et al, 1994). Among these, ionizing radiation and asbestos were reported to specifically induce c-jun expression (Sherman et al, 1990;Heintz et al, 1993).…”

Adrenocorticotropic hormone stimulates CYP11B1 gene transcription through a mechanism involving AP‐1 factors

“…DNA fragments carrying the sequence from position Ϫ317 to Ϫ136 and from Ϫ199 to ϩ20 used as probe were prepared by PCR with appropriate primer sets [6,11]. The following antibodies obtained from Santa Cruz Biotechnology were used in both gelmobility shift assays and immunoblot analyses: Anti-Jun family proteins [33], anti-c-Jun [33], anti-JunB [34], anti-JunD [34], anti-Fos family proteins [35], anti-c-Fos [36], anti-FosB [37], anti-Fra-1 [38], and anti-Fra-2 [39]. For measurement of radioactive intensities of DNA-protein complexes, dried polyacrylamide gels were exposed to a phosphor screen, and the signals were quantified using a Molecular Imager GS-535 (BioRad) within the range of linear response.…”