Biphasic effect of Max on Myc cotransformation activity and dependence on amino- and carboxy-terminal Max functions.

Abstract

In Ras cotransformation assays, Max exhibited a biphasic effect on Myc transformation activity. Cotransfection of low levels of Max expression plasmid stimulated Myc transformation activity, but cotransfection of high levels suppressed it. Mutations in the functionally undefined Max amino-and carboxy-terminal regions outside of the B/HLH/LZ motif partly separated these activities, suggesting various modes of Max regulation. We demonstrate that the Max protein is a nuclear protein in vivo and identify a carboxy-terminal region similar to nuclear localization signals whose integrity is necessary for efficient localization. Two mutants that delete amino-or carboxy-terminal consensus signals for casein kinase II (CKII) exhibited altered gel mobility and DNA-binding potential in vitro and showed modified transforming potential in the Ras cotransformation assay, suggesting that CKII or a CKII-related enzyme may regulate Max function in vivo. Our data suggest that both the ratio of Myc/Max hetero-oligomers to Max homo-oligomers and Max-specific regulation can contribute to determining the biological activity of Myc in vivo.

Discrimination between different E-box-binding proteins at an endogenous target gene of c-myc.

“…We show here that, although synergy does not occur on proximal E-boxes, Myc and Max do synergize to trans-activate the prothymosin-~ reporter. This is in agreement with the synergy observed for Myc and Max in transformation (Prendergast et al 1992). It is still unclear why Myc and Max only synergize to activate from distal sites and not from proximal sites.…”

“…In contrast to transformation assays, we observed little to no inhibition of Myc-mediated trans-activation by high concentrations of Max expression plasmids; biochemically, this might reflect the higher affinity of Max in forming heterodimers with Myc rather than homodimers. We suggest, therefore, that the apparent inhibition of focus formation observed with high concentrations of Max (Prendergast et al 1992) may be attributable to increased apoptosis and not to inhibition of transformation, and thus, may reflect increased, rather than decreased, transactivation of target genes in the transformed cells.…”

“…Myc and Max have been reported to cooperate in cell transformation (Prendergast et al 1992), but no or only low levels of synergy have been observed in transient trans-activation assays (Kretzner et al 1992;Amin et al 1993;Gaubatz et al 1994;Lee et al 1995). To address this issue, expression plasmids encoding Myc and Max were cotransfected into HeLa cells together with the prothymosin-~ reporter plasmid.…”

Mechanisms of apoptosis by c-Myc

“…Phosphorylation and alternate splicing proximal to the basic region inhibit the ability of Max homodimers to associate with DNA in cells (Berberich and Cole, 1992;Prochownik and Van Antwerp, 1993;Zhang et al, 1997). Max association and DNA binding are required for transcriptional activation of target genes by c-Myc as well as its ability to drive proliferation, malignant cell transformation, and apoptosis (Amati et al, 1992;Kretzner et al, 1992a,b;MaÈ kelaÈ et al, 1992;Mukherjee et al, 1992;Prendergast et al, 1992;Amati et al, 1993a,b;Gu et al, 1993). Access to CACGTG binding sites in target genes may be regulated by CpG methylation of the central dinucleotide .…”

Bin1 functionally interacts with Myc and inhibits cell proliferation via multiple mechanisms

“…Di erentiation of C2C12 was induced by removing shifting cells at *70% con¯uence to DMEM supplemented with 5% horse serum and antibiotics for 5 days, when myotube formation was maximal. Secondary passage rat embryo ®broblasts (REFs) were obtained from Whittaker Bioproducts and cultured and transfected as described (Prendergast et al, 1992). For transformation assays, secondary passage REFs seeded in 10 cm dishes were transfected overnight by a calcium phosphate coprecipitation method (Chen and Okayama, 1987) with 5 mg each of oncogenic Ras plus Myc, E1A, or mutant p53 expression plasmids and 10 mg of Bin1 plasmid or empty vector.…”