Differential localization of 5- and 15-lipoxygenases to the nuclear envelope in RAW macrophages.
Leukotriene formation is initiated in myeloid cells by an increase in intracellular calcium and translocation of 5-lipoxygenase from the cytoplasm to the nuclear envelope where it can utilize arachidonic acid. Monocyte- macrophages and eosinophils also express 15-lipoxygenase, which converts arachidonic acid to 15(S)-hydroxyeicosatetraenoic acid. Enhanced green fluorescent 5-lipoxygenase (5-LO) and 15-lipoxygenase (15-LO) fusion proteins were expressed in the cytoplasm of RAW 264.7 macrophages. Only 5-lipoxygenase translocated to the nuclear envelope after cell stimulation, suggesting that differential subcellular compartmentalization can regulate the generation of leukotrienes versus 15(S)-hydroxyeicosatetraenoic acid in cells that possess both lipoxygenases. A series of truncation mutants of 5-LO were created to identify putative targeting domains; none of these mutants localized to the nuclear envelope. The lack of targeting of 15-LO was then exploited to search for specific targeting motifs in 5-LO, by creating 5-LO/15-LO chimeric molecules. The only chimera that could sustain nuclear envelope translocation was one which involved replacement of the N-terminal 237 amino acids with the corresponding segment of 15-LO. Significantly, no discrete targeting domain could be identified in 5-LO, suggesting that sequences throughout the molecule are required for nuclear envelope localization.
Christmas
P
P
Arthritis Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA.
Fox
J W
JW
Ursino
S R
SR
Soberman
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D015289:Leukotrienes / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology* / Q000648:ultrastructure; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D015289:Leukotrienes; D011993:Recombinant Fusion Proteins; D001093:Arachidonate 15-Lipoxygenase; D001094:Arachidonate 5-Lipoxygenase