Coordinate regulation of sphingosine kinase and actin dynamics.
The activation of lipid-modifying enzymes generally involves a physical change in their interactions with the membrane substrate. For sphingosine kinase, a predominantly cytosolic enzyme in resting cells, activation is accompanied by translocation to specific subsets of cellular membranes where catalysis occurs. As all eukaryotic membranes have a tightly associated filamentous actin skeleton, we investigated potential regulatory interactions between sphingosine kinase and actin. Sphingosine kinase 1 (SK1) exhibited constitutive- and stimulus-enhanced association with actin filaments and F-actin-enriched membrane fractions in both intact macrophages and an in vitro reconstitution assay, whereas SK1 bound G-actin only under stimulated conditions. Actin inhibitors disrupted SK1 localization and increased its enzymatic activity. Both the localization and the activity of SK1 were coordinately regulated with the actin cytoskeleton. The association of enzymes with the actin cytoskeleton and how this regulates their activities and functions are subjects of intense interest. Here, we describe the approach we used to investigate regulation of SK1. This provides general methods that can be used to examine the role of actin in regulating enzyme activity in macrophages and other myeloid cells.
Iyer
Shankar S
SS
Physiology and Biophysics, and the Graduate Program in Immunology, University of Iowa Carver College of Medicine and Iowa City Veterans Affairs Medical Centre, Iowa City, IA, USA.
Kusner
David J
DJ
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D003850:Deoxyribonuclease I / Q000378:metabolism; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D018613:Microscopy, Confocal; D008967:Molecular Biology / Q000379:methods*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099157:Sphingosine Kinase
D000199:Actins; D003850:Deoxyribonuclease I; D017853:Phosphotransferases (Alcohol Group Acceptor); D013755:Tetradecanoylphorbol Acetate; D000099157:Sphingosine Kinase