Arachidonic acid promotes phosphorylation of 5-lipoxygenase at Ser-271 by MAPK-activated protein kinase 2 (MK2).
We demonstrated previously 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 with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/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 Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation.
Werz
Oliver
O
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Szellas
Dagmar
D
Steinhilber
Dieter
D
Rådmark
Olof
O
D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016718:Arachidonic Acid / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D021381:Protein Transport; D012694:Serine / Q000378:metabolism*; D000099190:MAP-Kinase-Activated Kinase 2
D001094:Arachidonate 5-Lipoxygenase; D016718:Arachidonic Acid; D002118:Calcium; D047908:Intracellular Signaling Peptides and Proteins; D020928:Mitogen-Activated Protein Kinases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D012694:Serine; D000099190:MAP-Kinase-Activated Kinase 2