Analysis of 5‐lipoxygenase phosphorylation on molecular level by MALDI‐MS

Abstract

The enzyme 5-lipoxygenase (5-LO) catalyzes the first reactions in the biosynthesis of leukotrienes, powerful lipid mediators that are involved in several physiological and pathological processes. 5-LO activity is tightly regulated by several factors, including post translational modifications (PTMs). Phosphorylations of 5-LO by the kinases extracellular signal-regulated kinase 2 (Erk2), mitogen-activated protein kinase activated protein kinase 2 (MK2) and protein kinase A (PKA) have been described to regulate 5-LO activity. Furthermore, 5-LO phosphorylation is considered a determinant of drug candidate potency. However, no evidence on a molecular level, as can be provided by MS, has as yet been presented for these PTMs. Here, we employ a workflow including different proteolytic cleavages and phosphopeptide enrichment for detection of 5-LO phosphorylation by MALDI-MS. Proof for the known phosphorylation sites of MK2 (Ser271) and PKA (Ser523) was provided by MS after in vitro phosphorylation, but not for the postulated Erk2 site (Ser663). Detection limits have been determined for all three sites. Moreover, we identified novel tyrosine kinase target sites within 5-LO using in silico and in vitro methods. Tyr42, Tyr53 and either Tyr94 or Tyr445 were phosphorylated by the Src kinases Fgr, hematopoietic cell kinase (HCK) and Yes. To analyze the phosphorylation state in the cellular context, we created stably 5-LO-transduced Mono Mac 6 cells. Here, we only detected phospho-Ser271 by MS, whereas immunoblot analysis indicated tyrosine phosphorylation, phospho-Ser271 and phospho-Ser663. Unexpectedly, phospho-Ser271 occurred independent of cell stimulation. Taken together, we describe a method for the molecular analysis of 5-LO phosphorylation, provide insights regarding the occurrence of known phosphorylation sites partly in contrast to earlier studies and present first evidence on novel phosphosites.

Progesterone rapidly down-regulates the biosynthesis of 5-lipoxygenase products in human primary monocytes

“…Although we have previously identified 5-LO phosphorylation at Ser271 and Ser663 in primary leukocytes [7][8][9][10]29], we were unable here to detect phospho-Ser523 by the use of the phosphospecific 5-LO Phospho-Ser523 polyclonal antibody (Cayman Chemical, Ann Arbor, MI) in unstimulated, progesterone-, PGE 2 -or 8-BrcAMP-activated monocytes at various time-points (not shown), in agreement with Markoutsa et al who failed to detected phosphoSer523 5-LO in intact monocytes by mass spectrometry [30].…”

Eicosanoids and cancer

“…Additionally, 5-LOX is phosphorylated at residues Ser271, Ser663 and Ser523 by the activity of MAPKAP2 (Ser271), ERK2 (Ser663) and protein kinase A (PKA) (Ser523). Several additional phosphorylation sites, at Tyr42, Tyr53 and either Tyr94 or Tyr445, were recently identified by Markoutsa et al 89 . Once bound to Ca 2+ , 5-LOX translocates to the nuclear envelope.…”

Product formation controlled by substrate dynamics in leukotriene A4 hydrolase

“…The activity of 5LO is also regulated by phosphorylation at different sites [48]. Certain phosphorylation upregulates the activity of 5LO, other down regulate the activity and some affect localisation [53]. Phosphorylation at Serine 523 is regulated by protein kinase A (PKA).…”

“…Phosphorylation on Serine 271 and 663 upregulates the enzymatic activity and the translocation of 5LO to the membrane. The former one is regulated by p38 kinase-dependent MAPKAP kinases [57,58] and the later one by an extracellular signal-regulated kinase 2dependent kinase (ERK2) [53,59].…”