A 23-amino acid motif spanning the basic domain targets zebrafish myogenic regulatory factor Myf5 into nucleolus
A 23-amino acid motif spanning the basic domain targets zebrafish myogenic regulatory factor Myf5 into nucleolus
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DOI：
10.1089/dna.2005.24.651
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发表时间：
2005-10-01
影响因子：
3.1
通讯作者：
Tsai, HJ
中科院分区：
文献类型：
--
作者：
Wang, YH;Chen, YH;Tsai, HJ
Myf5是一种核蛋白，是基本的螺旋-环-螺旋（bHLH）成肌因子之一，在肌肉规范和分化中起重要作用。导致Myf5核易位的基序是未知的。通过在线监测EGFP（增强型绿色荧光蛋白）标记的斑马鱼Myf5易位，我们发现Myf5 -EGFP蛋白存在于斑马鱼成纤维细胞系（ZEM2S和ZF4）、哺乳动物非肌肉细胞系（COS1）和肌肉细胞系（RD和C2C12）的核质和核仁中。相比之下，斑马鱼MyoD - EGFP定位于细胞核，而不是在核仁中凝聚。通过间接免疫荧光染色，我们确定斑马鱼Myf5与核仁蛋白（nucleophosmin/B23）共定位。缺失分析显示Myf5的60 ~ 82 (（60)KRKASTVDRRRAATMRERRRLKK(82)）氨基酸残基对于核仁靶向是足够和必需的。GST下拉实验和Western分析表明，GST - Myf5能特异性地下拉核仁蛋白/C23，而GST - MyoD不能。基于这些发现，我们提出Myf5和MyoD的不同功能可能是由于它们与核仁蛋白/C23的不同结合亲和力。
Myf5 is a nuclear protein and one of the basic helix-loop-helix ( bHLH) myogenic factors that play an important role in muscle specification and differentiation. The motif responsible for the nuclear translocation of Myf5 was unknown. Using on-line monitoring of EGFP ( enhanced green fluorescent protein)-tagged zebrafish Myf5 translocation, we demonstrated that Myf5 -EGFP protein resided in the nucleoplasm and nucleolus of zebrafish fibroblast cell lines (ZEM2S and ZF4), mammalian nonmuscle cell line (COS1), and muscle cell lines (RD and C2C12). In contrast, zebrafish MyoD - EGFP was localized in the nucleus but did not condense in the nucleolus. Using indirect immunofluorescent staining, we determined that zebrafish Myf5 was colocalized with nucleophosmin/B23, a nucleolus protein. Deletion analysis revealed that amino acid residues 60 to 82 ((60)KRKASTVDRRRAATMRERRRLKK(82)) of Myf5 were sufficient and necessary for nucleolus targeting. A GST pulldown assay followed by Western analysis showed that nucleolin/C23 could be pulled down specifically by GST - Myf5, but not by GST - MyoD. Based on these findings, we propose that the distinct functions of Myf5 and MyoD may result from their differential binding affinity to nucleolin/C23.