Identification and characterization of novel isoforms of human DP-1: DP-1{alpha} regulates the transcriptional activity of E2F1 as well as cell cycle progression in a dominant-negative manner.
The cell cycle-regulating transcription factors DP-1 and E2F form a heterodimeric complex and play a central role in cell cycle progression. Two different DP subunits (DP-1 and DP-2) exist in humans. In this study, we identified two novel DP-1 isoforms (DP-1alpha and DP-1beta) and characterized their structure and function. DP-1alpha is composed of 278 amino acids and lacks a portion of the C-terminal heterodimerization domain, whereas DP-1beta is composed of 357 amino acids with a frameshift that causes truncation of the C-terminal domain. Yeast two-hybrid and immunoprecipitation assays demonstrated that DP-1alpha binding to E2F1 was significantly reduced as compared with that of wild-type DP-1 or DP-1beta. Immunofluorescence analysis revealed that the subcellular localization of both DP-1 isoforms changed from the cytoplasm to the nucleus in HEK 293 cells cotransfected with E2F1 and wild-type DP-1 or DP-1beta. However, such a translocation for DP-1alpha was barely observed. Reverse transcription-PCR results showed that the three DP-1 isoforms are expressed ubiquitously at equal levels in several normal human tissues. We also demonstrated the expression of these isoforms at the protein level by Western blotting. Interestingly, we observed a significant decrease in transcriptional activity, a marked delay of cell cycle progression, and an inhibition of cell proliferation in DP-1alpha-transfected HEK 293 cells. Together, the results of the present study suggest that DP-1alpha is a novel isoform of DP-1 that acts as a dominant-negative regulator of cell cycle progression.
Ishida
Hironori
H
Division of Oral Infectious Diseases and Immunology, Faculty of Dental Science, Kyushu University, Higashiku, Fukuoka 812-8582, Japan.
Masuhiro
Yoshikazu
Y
Fukushima
Akie
A
Argueta
Jose Guillermo Martinez
JG
Yamaguchi
Noboru
N
Shiota
Susumu
S
Hanazawa
Shigemasa
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D016368:Frameshift Mutation; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014018:Tissue Distribution; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D020798:Two-Hybrid System Techniques
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D020033:Protein Isoforms; C493735:TFDP1 protein, human; D050680:Transcription Factor DP1; D014157:Transcription Factors; D008156:Luciferases