Active nuclear import and export pathways regulate E2F-5 subcellular localization.
Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.
Apostolova
Margarita D
MD
Department of Physiology, Child Health Research Institute and Lawson Health Research Institute, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Ivanova
Iordanka A
IA
Dagnino
Carla
C
D'Souza
Sudhir J A
SJ
Dagnino
Lina
L
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D050703:E2F2 Transcription Factor; D050704:E2F4 Transcription Factor; D050700:E2F5 Transcription Factor; D006801:Humans; D028884:Karyopherins; D015603:Keratinocytes / Q000737:chemistry; D008969:Molecular Sequence Data; D018160:Receptors, Cytoplasmic and Nuclear; D016160:Retinoblastoma Protein / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000097606:Exportin 1 Protein
D004268:DNA-Binding Proteins; D050703:E2F2 Transcription Factor; D050704:E2F4 Transcription Factor; D050700:E2F5 Transcription Factor; D028884:Karyopherins; D018160:Receptors, Cytoplasmic and Nuclear; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D000097606:Exportin 1 Protein; C092558:E2F5 protein, human; C094103:TFDP2 protein, human