Novel processing of Notch 1 within its intracellular domain by a cysteine protease.
In order to study N1 processing, we expressed human N1 (hN1) in HEK293 cells (293-hN1). Following Western blot analysis of 293-hN1 extracts, we detected, in addition to full-length hN1 and the N1 extracellular domain truncated form (N1-TM), a novel extracellular domain truncated form of hN1 with a COOH-terminal deletion, designated hN1-TMdeltaCT. Treatment of cells with the gamma-secretase inhibitor L-685,458 resulted in an accumulation of hN1-TMdeltaCT suggesting that this fragment is a gamma-secretase substrate. To identify the proteolytic activity(ies) that generates hN1-TMdeltaCT, we treated 293-hN1 cells with inhibitors of proteasome, calpains, caspases, serine and cysteine proteases. Despite the presence of a caspase-3 cleavage site within hN1 intracellular domain, none of the caspase inhibitors inhibited hN1-TMdeltaCT production. The proteasomal inhibitors used had also no effect. Incubation of cells with the cysteine protease inhibitor E64d resulted in the accumulation of hN1-TM and the inhibition of hN1-TMdeltaCT production suggesting a precursor-product relationship and that a cysteine protease is involved. Similarly, treatment of cells expressing amyloid precursor protein or E-cadherin with E-64d resulted in the accumulation of COOH-terminal fragments suggesting that these proteins are also processed within their intracellular domain by a cysteine protease. Processing towards hN1-TMdeltaCT requires maturation and transport of hN1 to the cell surface since treatment with brefeldin A inhibited its production and resulted in accumulation of hN1. Processing of hN1 within its intracellular domain could generate fragments that can exert novel functions and/or interfere with the function of hN1 intracellular domain.
Fassa
Angeliki
A
Division of Animal and Human Physiology, Department of Biology, University of Athens, Athens, Greece.
Parisiadou
Loukia
L
Robakis
Nikolaos K
NK
Efthimiopoulos
Spiros
S
D002219:Carbamates / Q000494:pharmacology; D002461:Cell Line, Transformed; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004151:Dipeptides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D009154:Mutation / Q000502:physiology; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D051881:Receptor, Notch1 / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D002219:Carbamates; D004151:Dipeptides; D004791:Enzyme Inhibitors; C410009:L 685458; D051881:Receptor, Notch1; D003546:Cysteine Endopeptidases