Phosphorylation of serine 523 on 5-lipoxygenase in human B lymphocytes.

The key enzyme in leukotriene (LT) biosynthesis is 5-lipoxygenase (5-LO), which is expressed in myeloid cells and in B lymphocytes. There are three phosphorylation sites on 5-LO (Ser271, Ser523 and Ser663). Protein kinase A (PKA) phosphorylates 5-LO on Ser523. In this report, we demonstrate by immunoblotting that native 5-LO in mantle B cell lymphoma (MCL) cells (Granta519, JEKO1, and Rec1) and in primary chronic B lymphocytic leukemia cells (B-CLL) is phosphorylated on Ser523. In contrast, we could not detect phosphorylation of 5-LO on Ser523 in human granulocytes or monocytes. Phosphorylated 5-LO was purified from Rec1 cells, using an ATP-agarose column, and the partially purified enzyme could be dephosphorylated with alkaline phosphatase. Incubation of Rec1 cells with 8-Br-cAMP or prostaglandin E2 stimulated phosphorylation at Ser523. Furthermore, FLAG-5LO was expressed in Rec1 cells, and the cells were cultivated in the presence of 8-Br-cAMP. The 5-LO protein from these cells was immunoprecipitated, first with anti-FLAG, followed by anti-pSer523-5-LO. The presence of 5-LO protein in the final precipitate further supported the finding that the protein recognized by the pSer523 antibody was 5-LO. Taken together, this study shows that 5-LO in B cells is phosphorylated on Ser523 and demonstrates for the first time a chemical difference between 5-LO in myeloid cells and B cells.