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 G1 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. © 2004 Wiley‐Liss, Inc.

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