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Association with E2F-1 governs intracellular trafficking and polyubiquitination of DP-1

Oncogene volume 18, pages 593–605 (1999)Cite this article

Abstract

The cell cycle-regulated transcription factor E2F is a family of heterodimers composed of E2F and DP protein subunits. While DP proteins stabilize DNA binding of E2F proteins, and influence the entry of E2F-4 and E2F-5 into the nucleus, the role of DP proteins in E2F-dependent gene expression is not well understood. Using immunolocalization, immunoprecipitation, and cell fractionation experiments, here we show association with E2F subunits governs intracellular trafficking and ubiquitination of DP-1. In transient transfection experiments, DP-1 polypeptides that stably bound E2F-1 entered the nucleus. DP-1 proteins that failed to associate with E2F subunits accumulated in the cell cytoplasm as polyubiquitinated DP-1. A Chinese hamster cell line that conditionally expresses HA-DP-1 was used to examine the effect of DP-1 on cell cycle progression. In serum response experiments, moderate increases in HA-DP-1 led to a threefold increase in E2F DNA binding activity in vitro, a corresponding increase in dhfr gene expression during transition of G1, and higher rates of S phase entry. However, flow cytometry showed cells expressing very high levels of HA-DP-1 failed to enter the S phase. Inhibition of cell cycle progression by high levels of HA-DP-1 was associated with the accumulation of other ubiquitinated cellular proteins, including c-jun and the cyclin-dependent kinase inhibitor p21, indicating that degradation of ubiquitinated proteins is required for progression from G0 to S phase even in the presence of activated E2F. Under similar conditions, expression of E2F-1 reduced the levels of ubiquitinated cellular proteins and accelerated cell cycle progression. Our studies indicate association with E2F subunits prevents ubiquitin-dependent degradation of DP-1 in the cytoplasm by promoting nuclear entry of E2F/DP heterodimers.

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Acknowledgements

We are indebted to K Helin for assisting with the construction of the pUHD expression plasmids and E1-12 and D1-38 cell lines, C-L Wu and E Harlow for plasmids and antibodies, L Prakash for the affinity purified anti-ubiquitin antibody; L Guarino for monoclonal antibody 4F3 and suggestions for the detection of ubiquitin in Western blots; D. Bohmann for HA-ubiquitin and His6-ubiquitin expression plasmids; H Bujard for pUHD15-1 and pUHD10-3; S van den Heuvel for pUHD15-1-neo; C Charland for assistance with flow cytometry; and N Dyson and other members of the Dyson/Harlow lab for critical discussions. We especially thank E Harlow, who supported a portion of this work during a mini-sabbatical by NHH at MGH Cancer Center. This work was supported by an American Cancer Research Faculty Award to NHH, the Vermont Cancer Center, the Lake Champlain Cancer Research Organization, NIH grant #GM54726, and a Center of Excellence grant to YM from STA, Japan.

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Authors and Affiliations

  1. Department of Pathology, University of Vermont College of Medicine, Burlington, 05405, Vermont, USA

    Junji Magae, Sharon Illenye & Nicholas H Heintz

  2. National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, 305, Japan

    Junji Magae, Young-Chae Chang & Youji Mitsui

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Magae, J., Illenye, S., Chang, YC. et al. Association with E2F-1 governs intracellular trafficking and polyubiquitination of DP-1. Oncogene 18, 593–605 (1999). https://doi.org/10.1038/sj.onc.1202345

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