Abstract
The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Protein kinase A phosphorylates 5-lipoxygenase on Ser(523), and this reduces its activity. We report here that phosphorylation of Ser(523) also shifts the subcellular distribution of 5-lipoxygenase from the nucleus to the cytoplasm. Phosphorylation and redistribution of 5-lipoxygenase could be produced by overexpression of the protein kinase A catalytic subunit alpha, by pharmacological activators of protein kinase A, and by prostaglandin E(2). Mimicking phosphorylation by replacing Ser(523) with glutamic acid caused cytoplasmic localization; replacement of Ser(523) with alanine prevented phosphorylation and redistribution in response to protein kinase A activation. Because Ser(523) is positioned within the nuclear localization sequence-518 of 5-lipoxygenase, the ability of protein kinase A to phosphorylate and alter the localization of green fluorescent protein fused to the nuclear localization sequence-518 peptide was also tested. Site-directed replacement of Ser(523) with glutamic acid within the peptide impaired nuclear accumulation; overexpression of the protein kinase A catalytic subunit alpha and pharmacological activation of protein kinase caused phosphorylation of the fusion protein at Ser(523), and the phosphorylated protein was found chiefly in the cytoplasm. Taken together, these results indicate that phosphorylation of Ser(523) inhibits the nuclear import function of a nuclear localization sequence, resulting in the accumulation of 5-lipoxygenase enzyme in the cytoplasm. As cytoplasmic localization can be associated with reduced leukotriene synthetic capacity, phosphorylation of Ser(523) serves to inhibit leukotriene production by both impairing catalytic activity and by placing the enzyme in a site that is unfavorable for action.
Highlights
Previous studies have shown that the subcellular localization of soluble 5-LO before cell stimulation can affect the amount of LT secreted following cell stimulation
Staining for phospho 5-LO (p5-LO) matched the fluorescence pattern of green fluorescent protein (GFP)/5-LO almost exactly (Fig. 1E). These results demonstrated that the overexpression of the active catalytic subunit of protein kinase A (PKA) with GFP/5-LO resulted in the accumulation of 5-LO in the cytoplasm, rather than in the nucleus
As 5-LO serves the key function of initiating LT synthesis from arachidonic acid, it represents a primary point for regulating the generation of these potent proinflammatory mediators
Summary
Previous studies have shown that the subcellular localization of soluble 5-LO before cell stimulation can affect the amount of LT secreted following cell stimulation. To clearly evaluate the effects of phosphorylation of Ser523 on the localization of 5-LO, we co-transfected NIH 3T3 cells with plasmids encoding GFP/5-LO with or without C␣. Cells overexpressing GFP/5-LO with the S523A mutation showed moderate to strong nuclear fluorescence that was not affected by PKA activation (data not shown).
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