Abstract
Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser(36) as the major p70S6k phosphorylation site, along with a low frequency site at Thr(40), using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 μmol/min/mg) of the activity of WT enzyme (95 μmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser(36) is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser(36) upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg(104) in the adjacent subunit. Because Arg(104) is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser(36) inhibits the catalytic function of LTC4S by interference with the catalytic machinery.
Highlights
Leukotriene (LT)2 C4 synthase (LTC4S) catalyzes the formation of leukotriene C4 (LTC4) by conjugating the unstable allylic epoxide inter
LTC4 and its metabolites LTD4 and LTE4 are known as cysteinyl leukotrienes, which are involved in bronchial asthma and allergic inflammatory disorders [1,2,3]
Leukotriene C4 synthase (LTC4S) is an integral membrane protein that belongs to the MAPEG superfamily whose six human members share structural similarity and form homotrimeric enzymes involved in arachidonic acid metabolism and detoxification [12]
Summary
Leukotriene (LT) C4 synthase (LTC4S) catalyzes the formation of LTC4 by conjugating the unstable allylic epoxide inter-. Additional G-protein-coupled receptors that recognize cys-LTs have been identified, in particular gpr and CysLT3 [5, 6]. LTC4S is yet another enzyme in the 5-lipoxygenase pathway that is regulated by intracellular phosphorylation [10, 11]. For 5-lipoxygenase-activating protein, LTC4S, and microsomal prostaglandin E synthase-1, crystal structures have been determined [13,14,15,16]. Phosphoregulation of LTC4S was first recognized when protein kinase C (PKC) activation of leukocytes was found to down-regulate LTC4S enzyme activity and attenuate cys-LT production [10]. The aim of our study was to identify the site(s) on LTC4S that is phosphorylated by p70S6k and investigate the molecular mechanism for suppression of enzyme activity. To obtain a mechanistic explanation for our experimental results, we analyzed phosphorylated and unphosphorylated forms of LTC4S by comparative molecular dynamics (MD) and determined the crystal structure of the phosphomimetic S36E mutant
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