The Recessive Phenotype Displayed by a Dominant Negative Microphthalmia-Associated Transcription Factor Mutant Is a Result of Impaired Nuclear Localization Potential

作者：Kimiko Takebayashi, Kazuhiro Chida, Ikuyo Tsukamoto, Eiichi Morii, Hiroshi Munakata, Heinz Arnheiter, Toshio Kuroki, Yukihiko Kitamura, Shintaro Nomura · 发表于：Molecular and Cellular Biology · 年份：1996 · DOI：10.1128/mcb.16.3.1203 · 被引用次数：118 · 研究领域：melanin and skin pigmentation、Garlic and Onion Studies、RNA regulation and disease

In the DNA binding domain of microphthalmia-associated transcription factor (MITF), four mutations are reported: mi, Mi wh, mi ew, and mi or. MITFs encoded by the mi, Mi wh, mi ew, and Mi or mutant alleles (mi-MITF, Mi wh-MITF, Mi ew-MITF, and Mi or-MITF, respectively) interfered with the DNA binding of wild-type MITF, TFE3, and another basic helix-loop-helix leucine zipper protein in vitro. Polyclonal antibody against MITF was produced and used for investigating the subcellular localization of mutant MITFs. Immunocytochemistry and immunoblotting revealed that more than 99% of wild-type MITF and Mi wh-MITF located in nuclei of transfected NIH 3T3 and 293T cells. In contrast, mi-MITF predominantly located in the cytoplasm of cells transfected with the corresponding plasmid. When the immunoglobulin G (IgG)-conjugated peptides representing a part of the DNA binding domain containing mi and Mi wh mutations were microinjected into the cytoplasm of NRK49F cells, wild-type peptide and Mi wh-type peptide-IgG conjugate localized in nuclei but mi-type peptide-IgG conjugate was detectable only in the cytoplasm. It was also demonstrated that the nuclear translocation potential of Mi or-MITF was normal but that Mi ew-MITF was impaired as well as mi-MITF. In cotransfection assay, a strong dominant negative effect of Mi wh-MITF against wild-type MITF-dependent transactivation system on tyrosinase promoter was observed, but mi-MITF had a small effect. However, by the conjugation of simian vi...