Presenilin/γ-Secretase-mediated Cleavage of the Voltage-gated Sodium Channel β2-Subunit Regulates Cell Adhesion and Migration

Abstract

The voltage-gated sodium channel ␤2-subunit (␤2) 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 ␤2 undergoes ectodomain shedding followed by presenilin (PS)-dependent ␥-secretase-mediated cleavage. 12-OTetradecanoylphorbol-13-acetate treatment or expression of an ␣-secretase enzyme, ADAM10, resulted in ectodomain cleavage of ␤2 in Chinese hamster ovary cells. Subsequent cleavage of the remaining 15-kDa Cterminal fragment (␤2-CTF) was independently inhibited by three specific ␥-secretase inhibitors, expression of the dominant negative form of PS1, and in PS1/PS2 knock-out cells. ␥-Secretase inhibitor treatment also increased endogenous ␤2-CTF levels in neuroblastoma cells and mouse primary neuronal cultures. In a cell-free ␥-secretase assay, we detected ␥-secretase activity-dependent generation of a 12 kDa ␤2 intracellular domain (ICD), which was loosely associated with the membrane fraction. To assess the functional role of ␤2 processing by ␥-secretase, we tested whether N-[N-(3,5-difluorophenylacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT), a specific ␥-secretase inhibitor, would alter ␤2-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 ␤2. DAPT also reduced migration of neuroblastoma cells in a modified Boyden chamber assay. Since DAPT treatment resulted in increased ␤2-CTF levels, we also tested whether ␤2-CTFs or ␤2-ICDs would directly affect cell migration by overexpressing recombinant proteins. Interestingly, elevated levels of ␤2-CTFs, but not ICDs, also blocked cell migration by 81 to 93%. Together, our findings show for the first time that ␤2 is a PS/␥-secretase substrate and ␥-secretase mediated cleavage of ␤2-CTF is required for cell-cell adhesion and migration of ␤2-expressing cells.A major pathologic hallmark of Alzheimer disease is the deposition of amyloid ␤-peptide (A␤) 1 into senile plaques. A␤ is produced from ␤-amyloid precursor protein (APP) by sequential ␤-and ␥-secretase-mediated cleavages. An alternative to the ␤-secretase pathway is a cleavage mediated by ␣-secretase, which cuts in the middle of the A␤ region of APP (1). APP has been shown to serially undergo either ␤-␥ and ␣-␥ cleavages. Both A␤ and the ␤-secretase-mediated C-terminal cleavage product of APP, C99 or CTF␤, have been shown to contribute to neurodegeneration in Alzheimer disease (2, 3). ␥-Secretase is a multisubunit aspartyl protease complex, harboring presenilin 1 (PS1) or 2 (PS2), nicastrin, aph-1, pen2, and likely additional factors (4). The presenilins are polytopic membrane proteins that constitute either catalytic subunits or necessary cofactors of the ␥-secretase complex (5, 6). Besides APP, PS/␥-secretase is required for the intramembraneous cleavages of at least 15 additional proteins, including Notch, nectin-1␣, and recently NRADD (4, 7-11). All the known substrates...

Notch Signaling Regulates the Extent of Hair Cell Regeneration in the Zebrafish Lateral Line

“…DAPT treatment phenocopies mind bomb mutants, which inactivate an E3 ubiquitin ligase necessary for efficient Notch signaling (Itoh et al, 2003) and is reversed by expression of processed Notch (Geling et al, 2002). Although DAPT blocks processing of other ␥-secretase targets such as ErbB4 (Sardi et al, 2006), amyloid precursor protein (Dovey et al, 2001), or sodium channel ␤ subunit (Kim et al, 2005), the similarities in its effect on HC production after Notch loss of function leads us to conclude that DAPT is most likely affecting this pathway. Although DAPT appears to have no differential specificity to different forms of presenilin (Zhao et al, 2008), there may be other ␥-secretases still functioning to modulate lateral line development after DAPT treatment.…”

Sodium channel β1 subunit mutations associated with Brugada syndrome and cardiac conduction disease in humans

“…The processed C-terminal fragment of β2 and β4 has been reported to be associated with cell adhesion, migration, and morphogenesis in neuronal cells as well as regulation of the expression level of the neuronal sodium channel Na V 1.1 (44)(45)(46). Thus, p.Trp179X β1B may result in absence of functions depending on the generation of a β subunit C-terminal fragment by BACE1.…”

Ion channel regulation by β-secretase BACE1 – enzymatic and non-enzymatic effects beyond Alzheimer's disease

“…51 Mutations in both a-or b-subunits have been associated, among other disorders, with epilepsy. 52 46,55 Five years later, BACE1 cleavage sites were mapped in the b2 subunit, which plays an important role in hippocampus and cortex. 56 Our group then showed that proteolysis of b2 by BACE1 caused a leftward shift in the voltage dependence of reconstituted Na v 1.2 current, demonstrating that the cleavage bears significance for channel gating.…”