Phosphorylation by protein kinase a inhibits nuclear import of 5-lipoxygenase.
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.
Luo
Ming
M
Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Health System, Ann Arbor, Michigan 48109, USA.
Jones
Sandra M
SM
Flamand
Nicolas
N
Aronoff
David M
DM
Peters-Golden
Marc
M
Brock
Thomas G
TG
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000409:Alanine; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002467:Cell Nucleus / Q000201:enzymology*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D015232:Dinoprostone / Q000494:pharmacology; D015870:Gene Expression; D018698:Glutamic Acid; D049452:Green Fluorescent Proteins / Q000235:genetics; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D018698:Glutamic Acid; D012694:Serine; D000242:Cyclic AMP; D001094:Arachidonate 5-Lipoxygenase; D017868:Cyclic AMP-Dependent Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone; D000409:Alanine