Presenilin/gamma-secretase-mediated cleavage of the voltage-gated sodium channel beta2-subunit regulates cell adhesion and migration.
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.
Kim
Doo Yeon
DY
Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, 02129, USA.
Ingano
Laura A Mackenzie
LA
Carey
Bryce W
BW
Pettingell
Warren H
WH
Kovacs
Dora M
DM
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
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