Abstract
The nuclear import of 5-lipoxygenase modulates its capacity to produce leukotrienes from arachidonic acid. However, the molecular determinants of its nuclear import are unknown. Recently, we used structural and functional criteria to identify a novel import sequence at Arg(518) on human 5-lipoxygenase (Jones, S. M., Luo, M., Healy, A. M., Peters-Golden, M., and Brock, T. G. (2002) J. Biol. Chem. 277, 38550-38556). However, this analysis also indicated that other import sequences must exist. Here, we identify two additional sites, at Arg(112) and Lys(158), as nuclear import sequences. Both sites were found to be common to 5-lipoxygenases from different species but not found on other lipoxygenases. Both sites also appeared to be a part of structures that were predominantly random loops. Peptide sequences at these sites were sufficient to direct nuclear import of green fluorescent protein. Mutation of basic residues in these sites impaired nuclear import and combinations of mutations at different sites were additive in effect. Mutations in all three sites were required to disable nuclear accumulation of 5-lipoxygenase in all cells. Significantly, mutation in these sites did not inhibit catalytic function. Taken together, these results indicate that nuclear import of 5-lipoxygenase may reflect the combined functional effects of three discrete import sequences. Mutation of individual sites can, by itself, impair nuclear import, which in turn could impact arachidonic acid metabolism.
Highlights
Leukotrienes (LTs)1 are lipid mediators derived from arachidonic acid
Since the BR518 nuclear import sequence (NIS) was identified using structural criteria noted above, these criteria were applied to other candidate sites
Previous work indicated that an NIS in the amino-terminal region of 5-LO, potentially at BR68, might function as an NIS
Summary
Two complementary primers (125 ng each) containing the desired mutation and 20 ng of template in 1ϫ reaction buffer were denatured at 95 °C for 30 s and annealed at 55 °C for 30s, and DNA synthesis was carried out by Pfu polymerase at 68 °C for 14 min This cycle was repeated 12–18 times, depending on the number of bases substituted, according to the manufacturer’s directions. LTB4 determinations were standardized for transfection efficiency: cells were washed following stimulation, harvested by scraping, sonicated on ice, assayed by immunoblot analysis (using 10 g of protein/sample) for expression using anti-GFP, with expression quantitated by densitometry. Lysates were centrifuged (5000 rpm, 8 min, 4 °C) to remove cell debris and protein expression for each construct was confirmed first by Western blot using anti-GFP. Pairs of group means were analyzed using the TukeyKramer post-test
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