title: Presenilin/gamma-secretase-mediated cleavage of the voltage-gated sodium channel beta2-subunit regulates cell adhesion and migration.
abstract: The voltage-gated sodium channel beta2-subunit (beta2) is a member of the IgCAM superfamily and serves as both an adhesion molecule and an auxiliary subunit of the voltage-gated sodium channel. Here we found that beta2 undergoes ectodomain shedding followed by presenilin (PS)-dependent gamma-secretase-mediated cleavage. 12-O-Tetradecanoylphorbol-13-acetate treatment or expression of an alpha-secretase enzyme, ADAM10, resulted in ectodomain cleavage of beta2 in Chinese hamster ovary cells. Subsequent cleavage of the remaining 15-kDa C-terminal fragment (beta2-CTF) was independently inhibited by three specific gamma-secretase inhibitors, expression of the dominant negative form of PS1, and in PS1/PS2 knock-out cells. gamma-Secretase inhibitor treatment also increased endogenous beta2-CTF levels in neuroblastoma cells and mouse primary neuronal cultures. In a cell-free gamma-secretase assay, we detected gamma-secretase activity-dependent generation of a 12 kDa beta2 intracellular domain (ICD), which was loosely associated with the membrane fraction. To assess the functional role of beta2 processing by gamma-secretase, we tested whether N-[N-(3,5-difluorophenylacetyl-l-alanyl)]-S-phenylglycine t-butylester (DAPT), a specific gamma-secretase inhibitor, would alter beta2-mediated cell adhesion and migration. We found that DAPT inhibited cell-cell aggregation and migration in a wound healing assay carried out with Chinese hamster ovary cells expressing beta2. DAPT also reduced migration of neuroblastoma cells in a modified Boyden chamber assay. Since DAPT treatment resulted in increased beta2-CTF levels, we also tested whether beta2-CTFs or beta2-ICDs would directly affect cell migration by overexpressing recombinant proteins. Interestingly, elevated levels of beta2-CTFs, but not ICDs, also blocked cell migration by 81 to 93%. Together, our findings show for the first time that beta2 is a PS/gamma-secretase substrate and gamma-secretase mediated cleavage of beta2-CTF is required for cell-cell adhesion and migration of beta2-expressing cells.
authors.affiliation: Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, 02129, USA.
authors.forename: Doo Yeon
authors.initials: DY
authors.lastname: Kim
authors.forename: Laura A Mackenzie
authors.initials: LA
authors.lastname: Ingano
authors.forename: Bryce W
authors.initials: BW
authors.lastname: Carey
authors.forename: Warren H
authors.initials: WH
authors.lastname: Pettingell
authors.forename: Dora M
authors.initials: DM
authors.lastname: Kovacs
mesh_terms: D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D015153:Blotting, Western; D016466:CHO Cells; D002448:Cell Adhesion; D002450:Cell Communication; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002474:Cell-Free System; D002540:Cerebral Cortex / Q000196:embryology; D006224:Cricetinae; D010450:Endopeptidases / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000378:metabolism; D053764:Presenilin-1; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry; D014280:Triglycerides / Q000494:pharmacology; D062548:Voltage-Gated Sodium Channel beta-2 Subunit; D014945:Wound Healing; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D021122:Protein Subunits; D011994:Recombinant Proteins; C117620:SCN2B protein, human; D015222:Sodium Channels; D014280:Triglycerides; D062548:Voltage-Gated Sodium Channel beta-2 Subunit; D005680:gamma-Aminobutyric Acid; C057077:1,2-dilinolenoyl-3-(4-aminobutyryl)propane-1,2,3-triol; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse; D013755:Tetradecanoylphorbol Acetate