The Role of Small Ubiquitin-like Modifier-interacting Motif in the Assembly and Regulation of Metal-responsive Transcription Factor 1

Abstract

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...

Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development

“…The new MTF-1 transcript is 2,773 nucleotides in length and encodes a 722 amino acid protein containing the Zn-finger DNA binding domain, all three transactivation domains and the highly conserved cysteine-rich motif (Supplementary Figure 1). In addition, the last exon of the zebrafish MTF-1 gene also contains the more recently characterized SUMO-interacting motif (SIM), 601 VIIIKQEE 608 , that immediately precedes the cysteine motif and is also conserved in other vertebrate species [7]. …”

“…In addition, the C-terminal region of MTF-1 contains a cysteine-rich motif (C-X-C-X-C-X-C) believed to be conserved in vertebrates and has been shown to be necessary for full metal induction [8]. Recent studies have also identified a conserved SUMO-interacting motif ((V/I-X-(V/I)-(V/I)) that immediately precedes the cysteine-rich domain and appears to play important roles in regulating the subcellular localization and transcriptional activity of MTF-1 [7, 43]. A truncated MTF-1 homologue in zebrafish lacking the cysteine-rich motif typically found in vertebrates has previously been reported [5].…”

“…Recent studies have demonstrated that sumoylation of MTF-1 suppresses its transcriptional activity [7] even though it does not alter the Zn-induced nuclear translocation or MRE-binding activity of MTF-1 [7, 43]. Although the exact function of sumoylated MTF-1 is not known, it is speculated that the SUMO moiety may interact with either transcriptional suppressors or activators to regulate DNA-binding activity [43].…”

“…Although the exact function of sumoylated MTF-1 is not known, it is speculated that the SUMO moiety may interact with either transcriptional suppressors or activators to regulate DNA-binding activity [43]. While a mouse MTF-1 K627R mutant was no longer able to undergo sumoylation, it did not display any reduction in transcriptional activity compared to the wild-type MTF-1 [7]. Thus it is likely that the significant reduction in transcriptional activation associated with the zebrafish MTF-1ΔCys is due to the lack of the cysteine motif.…”

“…MTF-1 also contains three different transactivation domains, an acidic domain, a proline-rich domain and a serine/threonine-rich domain [6], all of which contribute to transcriptional activation. Another highly conserved region in vertebrate MTF-1 orthologues is a short 32-amino acid section found immediately after the serine-threonine-rich domain that contains a sumoylation motif [7] and a highly conserved cysteine-rich motif (Cys-X-Cys-Cys-X-Cys) that is required for the proper induction of MT genes [8]. …”