Primate Monocytes - CD14, CD16 - Ziegler-Heitbrock

Arachidonic acid promotes phosphorylation of 5-lipoxygenase at Ser-271 by MAPK-activated protein kinase 2 (MK2)

Abstract

We demonstrated before that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation, and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared to heat shock protein 27 (Hsp27), 5-LO was a weak substrate for MK2. However, addition of unsaturated fatty acids (i.e. arachidonate 1-50 M) upregulated phosphorylation of 5-LO, but not of Hsp27, by active MK2 in vitro, resulting in similar phosphorylation as for Hsp27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca2+/calmodulin dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca2+-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower, as compared to wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus which does not prominently increase intracellular Ca2+, and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation.