Conservation of the biochemical mechanisms of signal transduction among mammalian Notch family members.
Mouse Notch1, which plays an important role in cell fate determination in development, is proteolytically processed within its transmembrane domain by unidentified gamma-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 DeltaE). The cytoplasmic portion of Notch1 DeltaE was released from the membrane upon incubation at 37 degrees C, which was inhibited by the specific gamma-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 DeltaE 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.
Mizutani
T
T
Department of Medical Chemistry and Department of Neurosurgery, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Taniguchi
Y
Y
Aoki
T
T
Hashimoto
N
N
Honjo
T
T
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides; D053764:Presenilin-1; D051880:Receptors, Notch; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D004791:Enzyme Inhibitors; C432344:MW167; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D010455:Peptides; D053764:Presenilin-1; D051880:Receptors, Notch; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse