Ectodomain shedding of membrane-anchored heparin-binding EGF like growth factor and subcellular localization of the C-terminal fragment in the cell cycle.
Heparin-binding EGF-like growth factor (HB-EGF) is initially synthesized as a type I transmembrane protein (proHB-EGF). The proHB-EGF is shed by specific metalloproteases, releasing the N-terminal fragment into the extracellular space as a soluble growth factor (HB-EGF) and the C-terminal fragment (HB-EGF-C) into the intracellular space, where it prevents transcriptional repression by the promyelocytic leukemia zinc finger protein (PLZF). The goal of the present study was to characterize regulation of proHB-EGF shedding and study its temporal variations in HB-EGF-C localization throughout the cell cycle. Quantitative combination analyses of cell surface proHB-EGF and HB-EGF in conditioned medium showed that proHB-EGF shedding occurred during the G(1) cell cycle phase. Laser scanning cytometry (LSC) revealed that HB-EGF-C was internalized into the cytoplasm during the late G1 phase and accumulated in the nucleus beginning in the S phase. Subsequent nuclear export of PLZF occurred during the late S phase. Further, HB-EGF-C was localized around the centrosome following breakdown of the nuclear envelope and was localized to the interzonal space with chromosome segregation in the late M phase. Temporal variations in HB-EGF localization throughout the cell cycle were also characterized by time-lapse imaging of cells expressing YFP-tagged proHB-EGF, and these results were consistent with those obtained in cytometry studies. These results indicate that proHB-EGF shedding and subsequent HB-EGF-C signaling are related with progression of the cell cycle and may provide a clue to understand the unique biological significance of non-receptor-mediated signaling of proHB-EGF in cell growth.
Toki
Fujio
F
Department of Molecular and Cellular Biology, Division of Biochemistry and Molecular Genetics, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime, Japan.
Nanba
Daisuke
D
Matsuura
Nariaki
N
Higashiyama
Shigeki
S
D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors; D008856:Microscopy, Fluorescence / Q000379:methods; D010446:Peptide Fragments / Q000378:metabolism*; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry*; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D051741:Kruppel-Like Transcription Factors; D010446:Peptide Fragments; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C092037:ZBTB16 protein, human; D004815:Epidermal Growth Factor