Identification of a bipartite nuclear localization sequence necessary for nuclear import of 5-lipoxygenase.
5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. This enzyme can reside either in the cytoplasm or the nucleus; its subcellular distribution is influenced by extracellular factors, and its nuclear import correlates with changes in leukotriene synthetic capacity. To identify sequences responsible for the nuclear import of 5-lipoxygenase, we transfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors encoding various 5-lipoxygenase constructs fused to green fluorescent protein. Overexpression of wild type 5-lipoxygenase with or without fusion to green fluorescent protein resulted in a predominantly intranuclear pattern of fluorescence, similar to the distribution of native 5-lipoxygenase in primary alveolar macrophages. Within the 5-lipoxygenase protein is a sequence (Arg(638)-Lys(655)) that closely resembles a bipartite nuclear localization signal. Studies using deletion mutants indicated that this region was necessary for nuclear import of 5-lipoxygenase. Analysis of mutants containing specific amino acid substitutions within this sequence confirmed that it was this sequence that was necessary for nuclear import of 5-lipoxygenase and that a specific arginine residue was critical for this function. As nuclear import of 5-lipoxygenase may regulate leukotriene production, natural or induced mutations in this bipartite nuclear localization sequence may also be important in affecting leukotriene synthesis.
Healy
A M
AM
Division of Pulmonary Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109-0642, USA.
Peters-Golden
M
M
Yao
J P
JP
Brock
T G
TG
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology
D017931:DNA Primers; D019913:Nuclear Localization Signals; D001094:Arachidonate 5-Lipoxygenase