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J. Biol. Chem., Vol. 282, Issue 13, 10047-10056, March 30, 2007

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Neutral Sphingomyelinase 2 Is Palmitoylated on Multiple Cysteine Residues

ROLE OF PALMITOYLATION IN SUBCELLULAR LOCALIZATION^*

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425

The neutral sphingomyelinases (nSMases) are considered major candidates for mediating the stress-induced production of ceramide. nSMase2, which has two hydrophobic segments near the NH₂-terminal region, has been reported to be located at the plasma membrane and play important roles in ceramide-mediated signaling. In this study, we found that nSMase2 is palmitoylated on multiple cysteine residues via thioester bonds. Site-directed mutagenesis of cysteine residues to alanine indicated that two cysteine clusters of the enzyme are multiply palmitoylated; one cluster is located between the two hydrophobic segments, and the second one is located in the middle of the catalytic region of the protein. When overexpressed in the confluent phase of MCF-7 cells, wild-type nSMase2 was strictly localized in the plasma membranes, and the cysteine mutants of each palmitoylated cysteine cluster were seen not only at the plasma membrane but also in some punctate structures. Furthermore, mutation of all potential palmitoylation sites resulted in a dramatic reduction in the plasma membrane distribution and an increase in the punctate structures. The palmitoylation-deficient mutant was directed to lysosomes and rapidly degraded. Palmitoylation had no effect on enzyme activity but affected membrane-association properties of the protein. Finally, the catalytic region of nSMase2 where palmitoylation occurs was found to be localized at the inner leaflet of the plasma membrane. In summary, the results from this study reveal for the first time the palmitoylation of nSMase2 via thioester bonds and its importance in the subcellular localization and stability of this protein.

Received for publication, December 7, 2006 , and in revised form, January 22, 2007.

^* This work was supported in part by National Institutes of Health Grant GM43825 and the Japan Society for the Promotion of Science. 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 Table 1.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave., P. O. Box 250509, Charleston, SC 29425. Tel.: 843-792-4321; Fax: 843-792-4322; E-mail: hannun{at}musc.edu.

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