Requirement of nuclear localization and transcriptional activity of p53 for its targeting to the yolk syncytial layer (YSL) nuclei in zebrafish embryo and its use for apoptosis assay.
We expressed zebrafish p53 protein fused to GFP by a neuron-specific HuC promoter in zebrafish embryos. Instead of displaying neuronal expression patterns, p53-GFP was targeted to zebrafish YSL nuclei. This YSL targeting is p53 sequence-specific because GFP fusion proteins of p63 and p73 displayed neuronal-specific patterns. To dissect the underlying mechanisms, various constructs encoding a series of p53 mutant proteins under the control of different promoters were generated. Our results showed that expression of p53, in early zebrafish embryo, is preferentially targeted to the nuclei of YSL, which is mediated by importin. Similarly, this targeting is abrogated when p53 nuclear localization signal is disrupted. In addition, the transcriptional activity of p53 is required for this targeting. We further showed that fusion of pro-apoptotic BAD protein to p53-GFP led to apoptosis of YSL cells, and subsequent imperfect microtubule formation and abnormal blastomere movements.
Chen
Gen-Der
GD
Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan.
Chou
Chih-Ming
CM
Hwang
Sheng-Ping L
SP
Wang
Fung-Fang
FF
Chen
Yi-Chung
YC
Hung
Chin-Chun
CC
Chen
Jeou-Yuan
JY
Huang
Chang-Jen
CJ
D000818:Animals; D017209:Apoptosis; D001681:Biological Assay; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D051959:ELAV Proteins / Q000235:genetics; D000067778:ELAV-Like Protein 3; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D028884:Karyopherins / Q000378:metabolism; D009154:Mutation; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015017:Yolk Sac / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D051959:ELAV Proteins; D000067778:ELAV-Like Protein 3; D028884:Karyopherins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D029961:Zebrafish Proteins; C494818:badb protein, zebrafish; D051025:bcl-Associated Death Protein; C088635:elavl3 protein, zebrafish; D049452:Green Fluorescent Proteins