Induction of interleukin-2 receptor-alpha gene expression is regulated by post-translational activation of kappa B specific DNA binding proteins

T cell mitogens induce the expression of specific trans-acting DNA binding proteins that in turn regulate the expression of the interleukin-2 receptor-alpha (IL-2R alpha) gene. To investigate whether de novo protein synthesis is required for the activation of these transacting factors and the induced expression of this receptor gene, Jurkat T cells were incubated with various inhibitors of protein synthesis prior to stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PMA). Despite the presence of cycloheximide or anisomycin at concentrations sufficient to block greater than 97% of cellular protein synthesis, phytohemagglutinin and phorbol 12-myristate 13-acetate effectively induced the expression of the IL-2R alpha gene as measured at the mRNA level. Similarly, gel retardation, DNA footprinting, and DNA-protein cross-linking studies revealed that these mitogens induced the activation of two predominant DNA binding proteins (50-55 and 80-90 kDa) in the presence or absence of cycloheximide and anisomycin. Both of these proteins specifically interacted with a kappa B-like binding site present in the IL-2R alpha promoter (-267 to -256) that is requisite for mitogen-induced expression of this receptor gene. These findings support a post-translational mechanism of induction of pre-existing, but inactive, DNA binding proteins which in turn bind to and activate the IL-2R alpha gene.     

Phorbol ester- and protein kinase C-mediated phosphorylation of the cellular Kirsten ras gene product

The effect of phorbol 12-myristate 13-acetate on the phosphorylation of the ras p21 protein was studied by metabolically labeling cultured cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. Phorbol 12-myristate 13-acetate (100 nM) induced phosphorylation of cKi-ras p21 in a mouse adrenocortical cell line (Yl) expressing high levels of cKi-ras with exon 4B. Phosphorylation was detected at 10 min and was maximal at 2 h. The ras protein was not phosphorylated in response to phorbol 12-myristate 13-acetate in NIH 3T3 cells expressing activated cHa-ras or vHa-ras. In vitro, protein kinase C phosphorylated cKi-ras in a phosphatidylserine and diolein-dependent manner. Both in intact cells and in vitro the amino acid phosphorylated was serine. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of cKi-ras. Phosphorylation affected neither the binding nor the GTPase activity of the ras protein. We conclude that cKi-ras is a substrate for protein kinase C and that the site of phosphorylation is likely to be serine 181 encoded by exon 4B.     

The nfkb1 promoter is controlled by proteins of the Ets family.

The gene encoding NFKB1 is autoregulated, responding to NF-kappa B/Rel activation through NF-kappa B binding sites in its promoter, which also contains putative sites for Ets proteins. One of the Ets sites, which we refer to as EBS4, is located next to an NF-kappa B/Rel binding site, kB3, which is absolutely required for activity of the promoter in Jurkat T cells in response to activation by phorbol 12-myristate 13-acetate (PMA), PMA/ionomycin, or the Tax protein from human T cell leukemia virus type I. We show that EBS4 is, required for the full response of the nfkb1 promoter to PMA or PMA/ionomycin in Jurkat cells. EBS4 is bound by Ets-1, Elf-1, and other species. Overexpression of Ets-1 augments the response to PMA/ionomycin and this is reduced by mutation of EBS4. Elf-1 has less effect in conjunction with PMA/ionomycin, but by itself activates the promoter 12-fold. This activation is only partly affected by mutation of EBS4, and a mutant promoter that binds Ets-1, but not Elf-1, at the EBS4 site responds to PMA/ionomycin as efficiently as the wild-type. Ets proteins may be responsible for fine-tuning the activity of the nfkb1 gene in a cell-type-specific manner.     

Dynamic regulation of cyclooxygenase-2 promoter activity by isoforms of CCAAT/enhancer-binding proteins.

To elucidate the mechanism by which isoforms of CCAAT/enhancer-binding proteins regulate cyclooxygenase-2 expression, we determined by a novel technique binding of six isoforms of this transactivator to two sequence-specific CCAAT/enhancer-binding protein (-132/-125) and cyclic AMP (-59/-53) regulatory elements in human foreskin fibroblasts treated with phorbol 12-myristate 13-acetate for 4 h. The delta isoform bound to these two elements at basal state, which was displaced by full-length as well as two truncated beta isoforms, a 41-kDa liver-enriched activating protein and a 16-kDa liver-enriched inhibitory protein, after phorbol ester stimulation. Kinetic analysis shows time-dependent changes in beta and delta binding that were concordant with time-dependent increase in cyclooxygenase-2 induction. Overexpression of the 16-kDa beta isoform blocked the promoter activity and protein level induced by phorbol ester. Paradoxically, it increased binding of beta isoforms to the sequence-specific promoter DNA but suppressed cyclooxygenase-2 promoter activation by p300 cotransfection. These findings provide new insight into the regulation of cyclooxygenase-2 promoter by an interplay between two opposite beta isoforms and p300 co-activator.     

Neural differentiation of amphibian gastrula ectoderm exposed to phorbol ester

Summary Ectoderm from early gastrula stages of amphibians was isolated and treated with phorbol 12-myristate 13-acetate. The ectoderm formed neural tissue and in a few cases also mesenchyme and melanophores. The control explants formed atypical epidermis. In explants treated with phorbol 12-myristate 13-acetate the mitotic rate was increased.     

Inhibition of denuded mouse oocyte meiotic maturation by tumor-promoting phorbol esters and its reversal by retinoids

Two tumor-promoting phorbol esters, phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA), when added to the culture medium of denuded mouse oocytes prevent their spontaneous meiotic maturation, whereas phorbol 13-acetate, which is inactive as a tumor promoter, does not inhibit this process. Retinoids appear to antagonize this inhibitory action of tumor promoters. However, the inhibitory effect of forskolin on meiotic maturation is not prevented, but is potentiated by retinal. These data indirectly suggest a role for calcium and/or phospholipids in the regulation of meiotic maturation. They also suggest that forskolin and phorbol esters mediate their effects through different pathways.