Mahshid Yilmaz, Markoutsa Stavroula, Dincbas-Renqvist Vildan, Sürün Duran, Christensson Birger, Sander Birgitta, Björkholm Magnus, Sorg Bernd L, Rådmark Olof, Claesson Hans-Erik
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden.
Institute of Pharmaceutical Chemistry/ZAFES, Goethe-University, 60438 Frankfurt am Main, Germany.
Prostaglandins Leukot Essent Fatty Acids. 2015 Sep;100:33-40. doi: 10.1016/j.plefa.2015.06.003. Epub 2015 Jun 24.
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.
白三烯（LT）生物合成中的关键酶是5-脂氧合酶（5-LO），它在髓样细胞和B淋巴细胞中表达。5-LO上有三个磷酸化位点（Ser271、Ser523和Ser663）。蛋白激酶A（PKA）使5-LO的Ser523位点磷酸化。在本报告中，我们通过免疫印迹证明，套细胞淋巴瘤（MCL）细胞（Granta519、JEKO1和Rec1）以及原发性慢性B淋巴细胞白血病细胞（B-CLL）中的天然5-LO在Ser523位点被磷酸化。相比之下，我们在人类粒细胞或单核细胞中未检测到5-LO的Ser523位点磷酸化。使用ATP-琼脂糖柱从Rec1细胞中纯化出磷酸化的5-LO，部分纯化的酶可用碱性磷酸酶去磷酸化。用8-溴-cAMP或前列腺素E2孵育Rec1细胞可刺激Ser523位点的磷酸化。此外，在Rec1细胞中表达FLAG-5LO，并在8-溴-cAMP存在下培养这些细胞。首先用抗FLAG抗体，然后用抗-pSer523-5-LO抗体对这些细胞的5-LO蛋白进行免疫沉淀。最终沉淀物中5-LO蛋白的存在进一步支持了pSer523抗体识别的蛋白是5-LO这一发现。综上所述，本研究表明B细胞中的5-LO在Ser523位点被磷酸化，并首次证明了髓样细胞和B细胞中5-LO的化学差异。
