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Analysis of 5‐lipoxygenase phosphorylation on molecular level by MALDI ‐ MS
Author(s) -
Markoutsa Stavroula,
Sürün Duran,
Karas Michael,
Hofmann Bettina,
Steinhilber Dieter,
Sorg Bernd L.
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12759
Subject(s) - phosphorylation , kinase , chemistry , biochemistry , protein phosphorylation , phosphopeptide , protein kinase a , microbiology and biotechnology , biology
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 ( PTM s). Phosphorylations of 5‐ LO by the kinases extracellular signal‐regulated kinase 2 (Erk2), mitogen‐activated protein kinase activated protein kinase 2 ( MK 2) 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 PTM s. 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 MK 2 (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. Structured digital abstractYes phosphorylates 5-LO by enzymatic study ( 1 , 2 ) PKA phosphorylates 5-LO by enzymatic study ( 1 , 2 ) CaMK II phosphorylates 5-LO by enzymatic study ( View interaction ) Fgr phosphorylates 5-LO by enzymatic study ( 1 , 2 ) MK2 phosphorylates 5-LO by enzymatic study ( View interaction ) HCK phosphorylates 5-LO by enzymatic study ( 1 , 2 )