Identification of nuclear localization and nuclear export signals in Ets2, and the transcriptional regulation of Ets2 and CTP:phosphocholine cytidylyltransferase alpha in tetradecanoyl-13-acetate or macrophage-colony stimulating factor stimulated RAW264 cells.
PC is made via the CDP-choline pathway, in which CTP:phosphocholine cytidylyltransferase alpha (CTalpha), encoded by Pcyt1a, is the rate-limiting enzyme whose mRNA expression is strictly regulated. Previously, we reported that Ets1 enhanced and Net repressed CTalpha transcription by binding at the Ets binding site (-49/-47) in the Pcyt1a promoter. In this study, we asked if an Ets1 analogue, Ets2, also regulates CTalpha transcription and investigated the importance of its nuclear localization signal (NLS) and nuclear export signal (NES). Ets2 is primarily detected in the nucleus. Various mutated Ets2 proteins fused with enhanced green fluorescent protein were constructed to identify the NLS and NES in Ets2. Mutation of Ets2 at amino acids 404-410 results in a protein that is evenly distributed in the cell. Interestingly, an Ets2 protein deleted at the C-terminus (amino acids 1-392 present) was localized to the cytoplasm and site-specific mutation in the region 364-372 of this construct resulted in cytoplasmic and nuclear distribution. These results suggest that the NLS in Ets2 is between amino acids 404 and 410, and that the NES is between amino acids 364 and 372. Ets2 enhanced, but the mutant forms of Ets2 had little effects on the transcription of a CTalpha-reporter construct. When RAW264 cells, murine macrophage cell-line, were stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) or macrophage-colony stimulating factor, the transcription of CTalpha was enhanced accompanied by increased mRNA of Ets2. These results suggest that the induction of Ets2 is important for CTalpha transcription by TPA and macrophage-colony stimulating factor.
Okamura
Koichi
K
Department of Biochemistry, Dokkyo Medical University School of Medicine, Mibu 321-0293, Japan.
Yamashita
Satoko
S
Ando
Hiromi
H
Horibata
Yasuhiro
Y
Aoyama
Chieko
C
Takagishi
Kenji
K
Izumi
Takashi
T
Vance
Dennis E
DE
Sugimoto
Hiroyuki
H
D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D008156:Luciferases / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D051379:Mice; D009154:Mutation / Q000235:genetics; D049790:Nuclear Export Signals / Q000235:genetics*; D019913:Nuclear Localization Signals / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D050784:Proto-Oncogene Protein c-ets-1 / Q000235:genetics / Q000378:metabolism; D050787:Proto-Oncogene Protein c-ets-2 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic; D014162:Transfection
D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D050784:Proto-Oncogene Protein c-ets-1; D050787:Proto-Oncogene Protein c-ets-2; D012333:RNA, Messenger; D016173:Macrophage Colony-Stimulating Factor; D008156:Luciferases; D019916:Choline-Phosphate Cytidylyltransferase; C500611:Pcyt1a protein, mouse; D013755:Tetradecanoylphorbol Acetate