Mechanism of metal-activated gene expression

Elevated concentrations of many transition metals elicit the rapid induction of metallothionein (MT) transcription in a variety of animal tissues and cultured cells. Regulation of MT transcription is mediated by metal response element (MRE)-specific DNA binding proteins. A protein, designated MTF-1 that binds to MRE sequences and regulates metal-inducible MT transcription has been isolated from puffer fish, mice, and humans. MTF-1 contains a six-zinc finger DNA-binding domain and several putative trans-activation domains. Deletion analysis of MTF-1 indicates that zinc-induced MT transcription may be the result of interactions among all of the domains. Current models do not adequately explain how metals other than zinc activate MTF-1-mediated MT transcription. Here we investigate the hypothesis that metals activate signal transduction cascades, which cause a change in the level of MTF-1-phosphorylation ultimately affecting its ability to activate MT transcription. COS-7 cells were transiently transfected with an MTF-1-myc-his expression vector. Transfected cells were exposed to either CdCl₂ or ZnCl₂ for 1–4h. 32P-labeled orthophosphate was added to transfected cells and non transfected control cells. The fusion protein was subsequently purified by either metal affinity chromatography or immunoprecipitation. Proteins were then resolved by SDS-PAGE and transferred to PVDF membranes. The amount of 32P incorporated into the MTF-1-fusion protein was assessed by PhosphorImager analysis. Western immunoblot analysis, using both anti-myc and anti-poly-histidine antibodies, confirmed that metal-exposed and non-exposed transfected cells expressed high levels of the MTF-1-fusion protein. A 32P-labeled protein, corresponding to the MTF-1-fusion, was observed m all metal exposed and non-exposed transfected cells. This protein was not present in non-transfected control cells. Cadmium did not appear to affect the level of MTF-1 phosphorylation, whereas zinc exposure resulted in an increase in phosphorylation. The level of phosphorylation of the MTF-1-myc-his fusion protein was also assessed using antiphosphoserine, antiphosphothreonine and antiphosphotyrosine antibodies. Phosphorylation occurred on serine and tyrosine residues. Inhibition studies suggest that the mitogen activated protein kinase (MAPK) signaling pathways ERK-1/2, JNK/SAPK and p38 contribute to the regulation of MT transcription. Inhibition of Protein Kinase C leads to an increase in MTF-1 phosphorylation, and total inhibition of metal activated MT transcription. These results support a model in which MTF-1 is phosphorylated in vivo, and regulates MT transcription through changes in its phosphorylation status.