Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

Itö, Tatsuo; Kitamura, Haruka; Uwatoko, Chisana; Azumano, Makiko; Itoh, Kohji; Kuwahara, Jun

doi:10.1016/j.bbrc.2010.10.036

Cited by 27 publications

(20 citation statements)

References 28 publications

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“…Using importin ␣5 deletion mutants, we found that broad regions of importin ␣5 associated with Snail but that the Arm repeat was predominant in the recognition of Snail (Fig. 3A), consistent with a previous report showing that the zinc finger-type transcription factor, SP1, has a similar binding property (34). Furthermore, the N-terminal half of importin ␤1, which contains the Ran binding domain, was mapped as the Snail-binding region (supplemental Fig.…”

Section: Discussionsupporting

confidence: 90%

Importin α Protein Acts as a Negative Regulator for Snail Protein Nuclear Import

Sekimoto¹,

Miyamoto

Arai

et al. 2011

Journal of Biological Chemistry

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Snail, a zinc finger-containing transcriptional regulator, migrates into the nucleus where it controls gene expression. We demonstrated previously that importin ␤1 directly recognizes the zinc finger domain of Snail and transports it into the nucleus. Here, using in vitro and in vivo assays, we show that importin ␣, an adaptor protein for importin ␤1, negatively regulates the nuclear import of Snail mediated by importin ␤1. In vitro binding assays indicated that importin ␣ interacted with the zinc finger domain of Snail to compete with the binding of importin ␤1 and that Snail did not form a ternary complex with importin ␣/importin ␤1. Overexpression of importin ␣ in A549 cells reduced the endogenous Snail protein level, which was restored by inhibitors of the proteasome and glycogen synthase kinase 3␤. Furthermore, knockdown of importin ␣ by siRNA treatment increased the endogenous Snail protein level in several cancer cell lines. This study provides a novel regulatory mechanism of the nuclear protein import process by importin ␣ and gives an implication to control Snail activity by inhibiting its nuclear localization. The epithelial mesenchymal transition (EMT)2 functions as a developmental switch that changes the morphology and behavior of epithelial cells to the highly motile mesenchymal phenotype. The hallmark of EMT is a decrease in E-cadherin expression (1). E-cadherin is a cell-cell adhesion molecule that forms stable epithelial adherent junctions and maintains the epithelial phenotype. Down-regulation of E-cadherin is mediated by several transcription factors such as Snail, Slug, ZEB, or Twist which repress E-cadherin transcription via interaction with the E-cadherin promoter (2). The loss of epithelial characteristics and the acquisition of migratory properties in EMT have recently been proposed to be the initial step of tumor metastasis. Indeed, the E-cadherin expression level is often inversely correlated to tumor grade (3). Although the loss of E-cadherin is one of the important markers of EMT, it is noteworthy that additional genes should be regulated to diminish the epithelial characteristics and to provoke the mesenchymal transition (4).Snail is one of the prominent transcriptional regulators that contributes to EMT by suppressing E-cadherin expression (5, 6) and participates in EMT concerning the mesoderm and neural crest formation during embryonic development. In fact, Snail knockout mice represent early embryonic lethality (7). Emerging evidence indicates that Snail is also a key factor in the tumor progression and metastasis in melanoma, bladder, colorectal, and pancreatic carcinomas (5,6,8,9). In these tumors, the down-regulation of E-cadherin was observed to correlate with the high expression of Snail. Signaling pathways such as TGF␤ and Wnt regulate Snail gene expression at the transcriptional level (10).On the other hand, it has been shown that Snail is a fragile protein and that phosphorylation of Snail is critical for its posttranslational regulation, which induces the degradation of th...

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Section: Discussionsupporting

confidence: 90%

Importin α Protein Acts as a Negative Regulator for Snail Protein Nuclear Import

Sekimoto¹,

Miyamoto

Arai

et al. 2011

Journal of Biological Chemistry

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“…In addition, the finger plays a role of the interface with the regulatory protein(s) [61]. SP1 is a well-characterized member of the SP/KLF family, and all three zinc fingers are essential for binding to importin alpha and contribute to nuclear localization [62]. Here, we identified a novel frameshift mutation caused by a 2-bp insertion in the Sp6 coding sequence, resulting in disruption of the third zinc finger domain with an addition of an unrelated 11 amino acids (Figure 1B,C).…”

Section: Resultsmentioning

confidence: 99%

Novel genetic linkage of rat Sp6 mutation to Amelogenesis imperfecta

Muto

Miyoshi

Horiguchi

et al. 2012

Orphanet J Rare Dis

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BackgroundAmelogenesis imperfecta (AI) is an inherited disorder characterized by abnormal formation of tooth enamel. Although several genes responsible for AI have been reported, not all causative genes for human AI have been identified to date. AMI rat has been reported as an autosomal recessive mutant with hypoplastic AI isolated from a colony of stroke-prone spontaneously hypertensive rat strain, but the causative gene has not yet been clarified. Through a genetic screen, we identified the causative gene of autosomal recessive AI in AMI and analyzed its role in amelogenesis.MethodscDNA sequencing of possible AI-candidate genes so far identified using total RNA of day 6 AMI rat molars identified a novel responsible mutation in specificity protein 6 (Sp6). Genetic linkage analysis was performed between Sp6 and AI phenotype in AMI. To understand a role of SP6 in AI, we generated the transgenic rats harboring Sp6 transgene in AMI (Ami/Ami + Tg). Histological analyses were performed using the thin sections of control rats, AMI, and Ami/Ami + Tg incisors in maxillae, respectively.ResultsWe found the novel genetic linkage between a 2-bp insertional mutation of Sp6 gene and the AI phenotype in AMI rats. The position of mutation was located in the coding region of Sp6, which caused frameshift mutation and disruption of the third zinc finger domain of SP6 with 11 cryptic amino acid residues and a stop codon. Transfection studies showed that the mutant protein can be translated and localized in the nucleus in the same manner as the wild-type SP6 protein. When we introduced the CMV promoter-driven wild-type Sp6 transgene into AMI rats, the SP6 protein was ectopically expressed in the maturation stage of ameloblasts associated with the extended maturation stage and the shortened reduced stage without any other phenotypical changes.ConclusionWe propose the addition of Sp6 mutation as a new molecular diagnostic criterion for the autosomal recessive AI patients. Our findings expand the spectrum of genetic causes of autosomal recessive AI and sheds light on the molecular diagnosis for the classification of AI. Furthermore, tight regulation of the temporospatial expression of SP6 may have critical roles in completing amelogenesis.

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“…It has been suggested that virtually all cellular Cu is tightly bound, and intracellular Cu trafficking is carried out by Cu chaperones to different cellular compartments, i.e, CCS to superoxide dismutase in the cytoplasm [12], Cox17 to mitochondrial cytochrome C oxidase, and Atox1 to two trans -Golgi P1B-type ATPases (ATP7A and ATP7B) [13]. It has been reported that ZF domain is involved in nuclear targeting of Sp1 [14]. However, our results from a cell-free system demonstrating that free Cu can affect Sp1-DNA binding suggest that regulation of Sp1-mediated transcriptional regulation by Cu may not need a carrier protein.…”

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confidence: 99%

Effects of Cu(II) and cisplatin on the stability of Specific protein 1 (Sp1)-DNA binding: Insights into the regulation of copper homeostasis and platinum drug transport

Dong

Aiba

Chen

et al. 2016

Journal of Inorganic Biochemistry

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The human high-affinity copper transporter 1 (hCtr1) transports both Cu(I) and cisplatin (cDDP). Because Cu deficiency is lethal yet Cu overload is poisonous, hCtr1 expression is transcriptionally upregulated in response to Cu deficiency but is downregulated under Cu replete conditions in controlling Cu homeostasis. The up- and down-regulation of hCtr1 is regulated by Specific protein 1 (Sp1), which itself is also correspondingly regulated under these Cu conditions. hCtr1 expression is also upregulated by cDDP via upregulation of Sp1. The underlying mechanisms of these regulations are unknown. Using gel-electrophoretic mobility shift assays, we demonstrated here that Sp1-DNA binding affinity is reduced under Cu replete conditions but increased under reduced Cu conditions. Similarly, Sp1-DNA binding affinity is increased by cDDP treatment. This in vitro system demonstrated, for the first time, that regulation of Sp1/hCtr1 expression by these agents is modulated by the stability of Sp1-DNA binding, the first step in the Sp1-mediated transcriptional regulation process.

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Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

Cited by 27 publications

References 28 publications

Importin α Protein Acts as a Negative Regulator for Snail Protein Nuclear Import

Importin α Protein Acts as a Negative Regulator for Snail Protein Nuclear Import

Novel genetic linkage of rat Sp6 mutation to Amelogenesis imperfecta

Effects of Cu(II) and cisplatin on the stability of Specific protein 1 (Sp1)-DNA binding: Insights into the regulation of copper homeostasis and platinum drug transport

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