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J. Biol. Chem., Vol. 280, Issue 49, 40609-40616, December 9, 2005

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Phosphorylation by Protein Kinase A Inhibits Nuclear Import of 5-Lipoxygenase^*

Ming Luo, Sandra M. Jones, Nicolas Flamand, David M. Aronoff, Marc Peters-Golden, and Thomas G. Brock1

From the Department of Internal Medicine, Divisions of Pulmonary and Critical Care Medicine and Infectious Diseases, University of Michigan Health System, Ann Arbor, Michigan 48109

The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Protein kinase A phosphorylates 5-lipoxygenase on Ser⁵²³, and this reduces its activity. We report here that phosphorylation of Ser⁵²³ 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 , by pharmacological activators of protein kinase A, and by prostaglandin E₂. Mimicking phosphorylation by replacing Ser⁵²³ with glutamic acid caused cytoplasmic localization; replacement of Ser⁵²³ with alanine prevented phosphorylation and redistribution in response to protein kinase A activation. Because Ser⁵²³ 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 Ser⁵²³ with glutamic acid within the peptide impaired nuclear accumulation; overexpression of the protein kinase A catalytic subunit and pharmacological activation of protein kinase caused phosphorylation of the fusion protein at Ser⁵²³, and the phosphorylated protein was found chiefly in the cytoplasm. Taken together, these results indicate that phosphorylation of Ser⁵²³ 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 Ser⁵²³ serves to inhibit leukotriene production by both impairing catalytic activity and by placing the enzyme in a site that is unfavorable for action.

Received for publication, June 28, 2005 , and in revised form, September 21, 2005.

^* This work was supported by Grants RO1 AI43955, HL50496, and HL078727 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Internal Medicine, University of Michigan, 6301 MSRB III, Ann Arbor, MI 48109-0642. Tel.: 734-763-9077; E-mail: brocko{at}umich.edu.

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