MTF-1 (metal-responsive transcription factor 1) is an essential mammalian protein for embryonic development and modulates the expression of genes involving in zinc homoeostasis and responding to oxidative stress. We report in the present paper that PTEN (phosphatase and tensin homologue deleted on chromosome 10) associates with MTF-1 in the cells. These two proteins interact via the acidic domain of MTF-1 and the phosphatase/C2 domain of PTEN. Depletion of PTEN reduced MT (metallothionein) gene expression and increased cellular sensitivity to cadmium toxicity. PTEN did not alter the nuclear translocation, protein stability or DNA-binding activity of MTF-1. Zinc increased MTF-1-PTEN interaction in a dose-dependent manner. The interaction elevated within 2 h of zinc addition and declined afterwards in the cells. The enhanced binding activity occurred mainly in the cytoplasm and reduced after translocating the MTF-1 into the nucleus. Blocking signalling through the PI3K (phosphoinositide 3-kinase) pathway did not alter the zinc-induced MT expression. Analysis of enzymatically inactive PTEN mutants demonstrated that protein but not lipid phosphatase activity of PTEN was involved in the regulation of MTF-1 activity. The same regulatory role of PTEN was also noted in the regulation of ZnT1 (zinc transporter 1), another target gene of MTF-1.

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Metal-responsive transcription factor 1 (MTF-1) 2 is a zinc finger protein that regulates gene expressions in response to various stresses, such as heavy metal exposure (1), oxidative stress (2), and hypoxia (3). MTF-1 genes have been identified in many species from insects to mammals, and encode proteins with high amino acid identities (4 -6). The N-terminal region of MTF-1 has six Cys 2 -His 2 -type zinc finger motifs that specifically bind to metal responsive elements (MREs) at the promoters of target genes. Three other distinct domains (an acidic, a proline-rich, and a serine/threonine-rich) on MTF-1 are responsible for the transactivation activity of the protein (7,8).Metallothioneins (MTs) are the most extensively studied target genes of MTF-1 (7). MTs are low molecular weight, cysteine-rich proteins that play important roles in zinc homeostasis and heavy metal detoxification (9). MTF-1 is responsible for the basal and metal-induced expression of MT genes (10). In addition, MTF-1 regulates the expression of zinc transporter-1 (11), ␥-glutamyl-cysteine synthetase heavy chain (12), and placental growth factor (13). It also plays a critical role in embryonic development because disruption of the Mtf-1 gene in mouse leads to oxidative damage-derived impairment of hepatocytic development and fetal lethality (12).MTF-1 resides mainly in the cytoplasm. Upon metal exposure or stress induction, MTF-1 translocates into the nucleus and binds to MREs (14,15). Several factors, such as USF-1, USF-2, NF-B, and HIF-1␣, can modulate the transcriptional activity of . Histone acetyltransferase p300/ CBP and transcription factor Sp1 can rapidly form a complex with MTF-1 upon zinc treatment and stimulate gene transcription (20). Alternatively, zinc and cadmium activate MTF-1 by phosphorylating MTF-1 through different kinase cascades. The exact mechanism of the modifications remains unknown (21-23).Post-translational modification of proteins by small ubiquitin-like modifier (SUMO) has emerged as an important regulatory mechanism of cellular processes (24). Among the three major SUMO isoforms identified in mammalian cells, SUMO-1 is the best characterized isoform. SUMO-1 and ubiquitin have only 18% sequence identity but share similar structural folds (25). Comparable with ubiquitination, SUMO conjugates covalently to substrates via a specific enzymatic cascade, including the heterodimeric SUMO-activating enzyme SAE1/SAE2 (E1), the SUMO-conjugating enzyme Ubc9 (E2), and in some cases, the SUMO E3 ligases (26). Sumoylation of proteins occurs mostly on lysine residues with a ⌿KXE (where ⌿ is a bulky hydrophobic residue and X is any amino acid) sequence and the modification can be dynamically reversed by the sentrin/SUMO-specific proteases (SENPs) (27). In contrast to the ubiquitination of proteins for degradation, the functional consequences of protein sumoylation are diverse. Sumoylation * This work was supported by Grant NSC98-2311-B-007-015-MY3 from the National Science Council, Taiwan, Republic of China. □ S The on-line version...

Metal-responsive transcription factor 1 (MTF-1) regulates a variety of genes involving in metal homeostasis and oxidative stress. We have shown that MTF-1 can be conjugated by small ubiquitin-like modifier (SUMO) and forms complexes with cellular factor(s) in a SUMO-interacting motif (SIM)-dependent manner. To investigate whether the interaction of MTF-1 and its SUMO conjugate occurs, we expressed and isolated MTF-1 and sumoylated MTF-1 (S-MTF-1) for functional studies. Various conditions were examined to optimize the expressions of MTF-1 and S-MTF-1. Results from affinity column chromatography demonstrated that the unmodified MTF-1 consistently co-eluted with the S-MTF-1. Mutations at the SIM did not reduce the level of MTF-1 sumoylation but the sumoylated product can then be purified to homogeneity. The presence of MTF-1 cross-interaction was further supported by in vitro pull-down assays. The ability of the purified proteins in binding metal-responsive element (MRE) was assessed with electrophoretic mobility shift assay. Noticeably, MTF-1 required the presence of cell extracts to render the binding activity. However, S-MTF-1 binds MRE in void of other cellular factors. The same characteristic was found for MTF-1 with SUMO fusion at the carboxyl terminus. These results indicate that the presence of SUMO moiety allows the protein to interact directly with MRE.