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J. Biol. Chem., Vol. 282, Issue 32, 23147-23162, August 10, 2007

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The Localization and Activity of Sphingosine Kinase 1 Are Coordinately Regulated with Actin Cytoskeletal Dynamics in Macrophages^*

David J. Kusner^¶||**1, Christopher R. Thompson², Natalie A. Melrose^**3, Stuart M. Pitson, Lina M. Obeid, and Shankar S. Iyer

From the Inflammation Program, Division of Infectious Diseases, Departments of Internal Medicine, ^¶Physiology and Biophysics, and the Graduate Programs in ^||Immunology and ^**Molecular Biology, University of Iowa Carver College of Medicine and Veterans Affairs Medical Center, Iowa City, Iowa 52245, the Hanson Institute, Division of Human Immunology, Institute of Medical and Veterinary Science, School of Molecular and Biomedical Science, University of Adelaide, Australia, and the Departments of Medicine, Biochemistry, and Molecular Biology, Medical University of South Carolina, and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina

The physiologic and pathologic functions of sphingosine kinase (SK) require translocation to specific membrane compartments. We tested the hypothesis that interactions with actin filaments regulate the localization of SK1 to membrane surfaces, including the plasma membrane and phagosome. Macrophage activation is accompanied by a marked increase in association of SK1 with actin filaments. Catalytically-inactive (CI)- and phosphorylation-defective (PD)-SK1 mutants exhibited reductions in plasma membrane translocation, colocalization with cortical actin filaments, membrane ruffling, and lamellipodia formation, compared with wild-type (WT)-SK1. However, translocation of CI- and PD-SK1 to phagosomes were equivalent to WT-SK1. SK1 exhibited constitutive- and stimulus-enhanced association with actin filaments and F-actin-enriched membrane fractions in both intact macrophages and a novel in vitro assay. In contrast, SK1 bound G-actin only under stimulated conditions. Actin inhibitors disrupted SK1 localization and modulated its activity. Conversely, reduction of SK1 levels or activity via RNA interference or specific chemical inhibition resulted in dysregulation of actin filaments. Thus, the localization and activity of SK1 are coordinately regulated with actin dynamics during macrophage activation.

Received for publication, January 8, 2007 , and in revised form, May 14, 2007.

^* This work was supported in part by National Institutes of Health RO1 Grants GM62302 and AI055916, P01 Grant AI44642-08, a Veterans Affairs Merit Review (to D. J. K.), and R01 GM062887 (to L. M. O.). 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 Movies 1–4.

² Supported by a T32 Infectious Diseases Training Grant from NIAID, National Institutes of Health. Current address: Dept. of Biology, Loyola College, 4501 N. Charles St., Baltimore, MD 21210.

³ Supported by a T32 Training Grant in Molecular and Cellular Biology. NIGMS, National Institutes of Health.

¹ To whom correspondence should be addressed: 200 Hawkins Dr., SW54-8 GH, IA City, IA 52245. Tel.: 319-353-6525; Fax: 319-335-4194; E-mail: david-kusner{at}uiowa.edu.

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