The localization and activity of sphingosine kinase 1 are coordinately regulated with actin cytoskeletal dynamics in macrophages.
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.
Kusner
David J
DJ
Inflammation Program, Division of Infectious Diseases, Department of Internal Medicine, University of Iowa Carver College of Medicine and Veterans Affairs Medical Center, Iowa City, Iowa 52245, USA. david-kusner@uiowa.edu
Thompson
Christopher R
CR
Melrose
Natalie A
NA
Pitson
Stuart M
SM
Obeid
Lina M
LM
Iyer
Shankar S
SS
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D010588:Phagosomes / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000502:physiology*; D021381:Protein Transport; D000099157:Sphingosine Kinase
D000199:Actins; D049452:Green Fluorescent Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D000099157:Sphingosine Kinase