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J. Biol. Chem., Vol. 280, Issue 24, 23251-23261, June 17, 2005

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Presenilin/-Secretase-mediated Cleavage of the Voltage-gated Sodium Channel 2-Subunit Regulates Cell Adhesion and Migration^*

Doo Yeon Kim, Laura A. MacKenzie Ingano, Bryce W. Carey, Warren H. Pettingell, and Dora M. Kovacs

From the Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

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-O-Tetradecanoylphorbol-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 C-terminal 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.

Received for publication, November 16, 2004 , and in revised form, March 29, 2005.

^* This work was supported by grants from the National Institutes of Health/NIA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

These authors contributed equally to this work.

To whom correspondence should be addressed. E-mail: dora_kovacs{at}hms.harvard.edu.

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