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

• Published in: The Journal of biological chemistry Vol. 280; no. 26; p. 24642
• Main Authors: Ishida, Hironori, Masuhiro, Yoshikazu, Fukushima, Akie, Argueta, Jose Guillermo Martinez, Yamaguchi, Noboru, Shiota, Susumu, Hanazawa, Shigemasa
• Format: Journal Article
• Language: English
• Published: United States 01-07-2005
• Subjects: Amino Acid Sequence; Base Sequence; Blotting, Western; Cell Cycle; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - metabolism; Cell Cycle Proteins - physiology; Cell Line; Cell Proliferation; Cloning, Molecular; Cytoplasm - metabolism; Dimerization; DNA Primers - chemistry; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; E2F Transcription Factors; E2F1 Transcription Factor; Exons; Fibroblasts - metabolism; Flow Cytometry; Frameshift Mutation; Gene Expression Regulation; Genes, Dominant; Humans; Immunoprecipitation; Kinetics; Luciferases - metabolism; Microscopy, Fluorescence; Molecular Sequence Data; Plasmids - metabolism; Protein Binding; Protein Isoforms; Protein Structure, Tertiary; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tissue Distribution; Transcription Factor DP1; Transcription Factors - chemistry; Transcription Factors - metabolism; Transcription Factors - physiology; Transcription, Genetic; Transfection; Two-Hybrid System Techniques
• ISSN: 0021-9258

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.