Conservation of the Biochemical Mechanisms of Signal Transduction among Mammalian Notch Family Members

• Published in: Proceedings of the National Academy of Sciences - PNAS 2001-07, Vol.98 (16), p.9026-9031
• Main Authors: Mizutani, Tomohiko, Taniguchi, Yoshihito, Aoki, Tomokazu, Hashimoto, Nobuo, Honjo, Tasuku
• Format: Article
• Language: English
• Subjects: Alzheimer's disease; Alzheimers disease; Amino Acid Sequence; Amino acids; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Biochemistry; Biological Sciences; Cell Line; Cell lines; Cell membranes; Cells; Centrifugation; Endopeptidases - metabolism; Enzyme Inhibitors - pharmacology; Family members; Humans; Hydrolysis; Mammals; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Membranes; Mice; Molecular Sequence Data; Neurons; Peptides; Plasmids; Presenilin-1; Proteins; Receptors, Notch; Sequence Homology, Amino Acid; Signal Transduction; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.161269998

Mouse Notch1, which plays an important role in cell fate determination in development, is proteolytically processed within its transmembrane domain by unidentified γ-secretase-like activity that depends on presenilin. To study this proteolytic event, we established a cell-free Notch cleavage assay system using the membrane fraction of fibroblast transfectants of various Notch constructs with deletion of the extracellular portion (Notch ΔE). The cytoplasmic portion of Notch1 ΔE was released from the membrane upon incubation at 37°C, which was inhibited by the specific γ-secretase inhibitor, MW167, or by overexpression of dominant negative presenilin1. Likewise, other members of mouse Notch family were proteolytically cleaved in a presenilin-dependent, MW167-sensitive manner in vivo as well as in the cell-free Notch ΔE cleavage assay system. All four members of the mouse Notch family migrated to the nucleus and activated the transcription from the promoter carrying the RBP-J consensus sequences after they were released from the membrane. These results demonstrate the conserved biochemical mechanism of signal transduction among mammalian Notch family members.