Phosphorylation of Lysophosphatidylcholine Acyltransferase 2 at Ser34 Enhances Platelet-activating Factor Production in Endotoxin-stimulated Macrophages*

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions under physiological and pathological conditions. We have recently identified two enzymes involved in PAF production: lysophosphatidylcholine acyltransferase-1 (LPCAT1) and LPCAT2. We found that LPCAT2 is highly expressed in inflammatory cells and is activated by lipopolysaccharide (LPS) treatment through Toll-like receptor 4. However, the molecular mechanism for the activation remains elusive. In this study, Phos-tag SDS-PAGE revealed the LPS-induced phosphorylation of LPCAT2. Furthermore, mass spectrometry and mutagenesis analyses identified Ser34 of LPCAT2 as the phosphorylation site to enhance the catalytic activities. The experiments using inhibitors and siRNA against MAPK cascades demonstrated that LPCAT2 phosphorylation through LPS-TLR4 signaling may directly depend on MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2). These findings develop a further understanding of both PAF production and phospholipid remodeling triggered by inflammatory stimuli. Specific inhibition of the PAF biosynthetic activity by phosphorylated LPCAT2 will provide a novel target for the regulation of inflammatory disorders.

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions under physiological and pathological conditions. We have recently identified two enzymes involved in PAF production: lysophosphatidylcholine acyltransferase-1 (LPCAT1) and LPCAT2. We found that LPCAT2 is highly expressed in inflammatory cells and is activated by lipopolysaccharide (LPS) treatment through Toll-like receptor 4. However, the molecular mechanism for the activation remains elusive. In this study, Phos-tag SDS-PAGE revealed the LPS-induced phosphorylation of LPCAT2. Furthermore, mass spectrometry and mutagenesis analyses identified Ser 34 of LPCAT2 as the phosphorylation site to enhance the catalytic activities. The experiments using inhibitors and siRNA against MAPK cascades demonstrated that LPCAT2 phosphorylation through LPS-TLR4 signaling may directly depend on MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2). These findings develop a further understanding of both PAF production and phospholipid remodeling triggered by inflammatory stimuli. Specific inhibition of the PAF biosynthetic activity by phosphorylated LPCAT2 will provide a novel target for the regulation of inflammatory disorders.
LPCAT2 also possesses LPCAT activity to produce the major membrane phospholipid, PC, which mainly contains polyunsaturated fatty acids (PUFAs) at the sn-2 position. This biosynthetic pathway of phospholipids, known as Lands' cycle or remodeling pathway, is responsible for generating the membrane diversity (16). PUFAs in phospholipids may affect membrane curvature and fluidity and store lipid mediator precursors that are converted to eicosanoids, such as prostaglandins, leukotrienes, and lipoxins (1). PC plays an important role as a precursor of both eicosanoids and PAF.
By mass spectrometry and mutagenesis studies, we demonstrated that LPCAT2 is activated by Ser 34 phosphorylation in mouse peritoneal macrophages and RAW264.7 cells with LPS treatment. Consensus sequence and experiments with an MK2 inhibitor and siRNA suggested that MK2 might directly phosphorylate and activate LPCAT2. These findings contribute to a better understanding of the regulatory mechanisms of PAF biosynthesis in inflammatory cells.

EXPERIMENTAL PROCEDURES
Materials-PC from frozen egg yolk, LPS from Salmonella minnesota, and anti-FLAG M2 antibody were from Sigma.
Mice-C57BL/6J mice were obtained from Clea Japan, Inc. (Tokyo, Japan). Mice were maintained in a light-dark cycle with lights on from 0800 -2000 h at 22°C. Mice were fed with a standard laboratory diet and water ad libitum. All animal studies were conducted in accordance with the guidelines for Animal Research at the University of Tokyo and were approved by the University of Tokyo Ethics Committee for Animal Experiments.
Isolation of Mouse Peritoneal Macrophages-Mouse peritoneal macrophages were isolated as previously described (6). Cells were cultured for 16 h before stimulation.
For primary cultured mouse peritoneal macrophages, the resultant supernatant at 9,000 ϫ g was centrifuged at 100,000 ϫ g for 1 h at 4°C. The resultant pellet was resuspended with ice-cold buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM sodium orthovanadate, 5 mM 2-mercaptoethanol, 1ϫ EDTAfree Complete. The concentration of each protein was measured by the Bradford method (17), using protein assay solution (Bio-Rad). Bovine serum albumin (fraction V, fatty acid-free; Sigma) served as a standard.
Site-directed Mutagenesis of LPCAT2-Mouse LPCAT2 mutants (S34A and S34D) were constructed by overlap extension PCR. The amplified PCR products were cloned into the pCXN2.1 vector, and the sequence was confirmed. The primer sets utilized were S34A (forward, CGC CAG GCG GCC TTC TTC CCG CCG C; reverse, GCG GCG GGA AGA AGG CCG CCT GGC G); and S34D (forward, CGC CAG GCG GAC TTC TTC CCG CCG C; reverse, GCG GCG GGA AGA AGT CCG CCT GGC G).
Transfection into RAW264.7 Cells-RAW264.7 cells (5 ϫ 10 6 cells), 100 l of Nucleofector solution V, and 5 g of each DNA of vector, FLAG-mLPCAT2, S34A, or S34D, were mixed. The mixture in the cuvette was set onto the Amaxa Nucleofector and electroporated with the program D-032. Then cells were seeded onto 6-cm dishes. Twenty-four hours after transfection, cells were stimulated with 100 ng/ml LPS for 30 min. The siRNA transfection was performed similarly. The mixture in the cuvette contained 120 pmol of siRNA.
Production of Anti-LPCAT2 and Anti-phospho-LPCAT2 Antibodies-Anti-LPCAT2 antiserum was generated at Immuno-Biological Laboratories (Gunma, Japan). The C-terminal peptide, SNKVSPESQEEGTSDKKVD, was used to immunize rabbits. Anti-LPCAT2 antibody was purified from the anti-LP-CAT2 antiserum using activated thiol-Sepharose 4B binding to the LPCAT2 epitope. Anti-phospho-LPCAT2 antibody was generated by SCRUM (Tokyo, Japan) using a phosphopeptide, RQApSFFPPP (where pS represents phosphoserine) at the N terminus of LPCAT2.
Western Blot Analysis-Western blot analyses were performed as described previously (18). To detect the band shift, which represents phosphorylated protein, an SDS-polyacrylamide gel containing 50 M Phos-tag acrylamide with 100 M Mn 2ϩ was used.
Quantitative Real-time PCR-Total RNAs were prepared using the RNeasy Mini Kit (Qiagen), and first strand cDNA was subsequently synthesized using Superscript III (Invitrogen). The PCRs were performed using Fast Start DNA Master SYBR Green I (Roche Applied Science). The primers for MK2 designed to amplify a 185-bp fragment were as follows: forward, GGA TCT TCG ACA AGA GAA CCC AG; reverse, GAG ACA CTC CAT GAC AAT CAG CA).

Phosphorylation of LPCAT2 by LPS Stimulation-To exam-
ine the different characteristics of the two lyso-PAFATs (LPCAT1 and LPCAT2), FLAG-tagged LPCAT1 and LPCAT2 were transiently transfected into the mouse macrophage cell line RAW264.7 using the Amaxa Nucleofector transfection kit V. Because RAW264.7 cells express TLR4 signaling molecules, cells were stimulated with LPS for 30 min, and the lyso-PAFAT activity was examined using the supernatant at 9,000 ϫ g for 10 min. The lyso-PAFAT activities of LPCAT1 and LPCAT2 were measured by radioisotope assays. Although the LPCAT1 activity was unchanged after LPS stimulation, the LPCAT2 activity was enhanced 4-fold compared with non-stimulated LPCAT2 (Fig. 1A). Lyso-PAFAT activity in the vector-transfected cells was slightly increased by LPS stimulation, possibly due to the presence of endogenously expressed LPCAT2 in RAW264.7 cells.
The mechanism of LPCAT2 activation was investigated using Phos-tag acrylamide gel electrophoresis. Phos-tag makes a complex with two Mn 2ϩ ions and acts as a phosphate-binding molecule (19). The complex is used for phosphate affinity SDS-PAGE, which results in the mobility shift of the phosphorylated proteins. A shifted band of FLAG-LPCAT2, but not FLAG-LPCAT1, was observed after LPS stimulation (Fig. 1B). The upper band may represent the phosphorylated form of LPCAT2. This result suggests that LPCAT2 is phosphorylated and activated by extracellular stimuli.
To identify the phosphorylated amino acid residue(s) of LPCAT2, RAW264.7 cells stably overexpressing FLAG-LPCAT2 were established using Fugene HD in the presence of Geneticin. The cells were stimulated with LPS for 30 min, and the pellet at 100,000 ϫ g for 1 h was analyzed by Phos-tag SDS-PAGE. The position corresponding to the shifted band in the Phos-tag Western blot was cut and subjected to in-gel trypsin digestion (20). After immobilized metal affinity chromatography enrichment of phosphopeptides (21), only one phospho-LPCAT2 peptide candidate ( 32 QApSFFPPPVPNPF-VQQTTISASR 54 ) was detected by liquid chromatographymass spectrometry (LTQ, Thermo Electron, San Jose, CA) (data not shown). Peptides containing unphosphorylated Ser 34 were not detected in the phosphopeptide-enriched fraction. The flow-through fraction of immobilized metal affinity chromatography contained several other unphosphorylated peptides derived from LPCAT2. Although the Mascot score was 38, which is not significant, these results suggest that Ser 34 of LPCAT2 is a candidate residue of the phosphorylation induced by LPS stimulation. Ser 34 of mouse LPCAT2 is well conserved among mammals, such as human, bovine, dog, and rat (Fig. 1C).
Site-directed Mutagenesis of LPCAT2-To confirm Ser 34 as the target of phosphorylation, site-directed mutagenesis of LPCAT2 was performed. Ser 34 was substituted for alanine (S34A) and aspartate (S34D). These constructs were transiently transfected into RAW264.7 cells using Amaxa, and the cells were stimulated with LPS for 30 min. In the Phos-tag Western blot analysis using the M2 anti-FLAG antibody, a mobility shift was detected in wild-type (WT) LPCAT2 but not in the S34A or S34D mutant ( Fig. 2A).
Next, we examined the effect of phosphorylation on the dual activities of LPCAT2 (lyso-PAFAT and LPCAT). Both activities of mutants were measured by radioisotope assays. Lyso-PAFAT and LPCAT activities were enhanced in WT LPCAT2 with LPS stimulation (Fig. 2, B and C). The enzyme activity of S34A was similar to WT but was not increased by LPS stimulation. In contrast, S34D exhibited a higher enzyme activity than WT, but no further stimulation was observed (Fig. 2B). The expression level of each mutant was similar to that of WT ( Fig. 2A). These results indicate that both lyso-PAFAT and LPCAT activities were enhanced by the Ser 34 phosphorylation of LPCAT2.
Signaling Pathway for LPCAT2 Phosphorylation-To investigate the time course of LPCAT2 phosphorylation, thioglycolate-induced murine peritoneal macrophages were stimulated with LPS for varying times (0 -120 min). Each microsomal protein (pellet at 100,000 ϫ g for 1 h) was analyzed by Western blot using anti-LPCAT2 and anti-phospho-LPCAT2 antibodies. The amount of total LPCAT2 was nearly equal among the samples. The most intense phospho-LPCAT2 signal was detected at 15-30 min and decreased as the incubation continued until 120 min (Fig. 3). This is consistent with lyso-PAFAT activation in our previous study (6). Similarly, MK2 phosphorylation reached a peak at 15-30 min. The consensus phosphorylation sequence (HydXRXXS; where Hyd represents a hydrophobic residue) of MK2 substrates (22) is conserved around Ser 34 (VPRQAS) in LPCAT2 (Fig. 1C). These results suggest that LPCAT2 is one of the protein substrates of MK2. Murine MK2 has two splice variant proteins (23), and thus MK2 appeared at the positions of 45 and 55 kDa by the Western blot.
We also performed enzymatic assays and examined the effect of MK2 siRNA on LPCAT2 activation. Both lyso-PAFAT and LPCAT activities were enhanced by LPS stimulation in the NC cells; however, both activations were abolished in MK2-KD cells (Fig. 5, C and  D). These results are consistent with the effect of the MK2 inhibitor on LPCAT2 phosphorylation (Fig. 4C) and thus indicate the MK2-dependent phosphorylation of LPCAT2.

DISCUSSION
Here, we present the activation mechanism of PAF biosynthetic enzyme by endotoxin stimulation. In response to inflammatory stimuli, LPCAT2 was phosphorylated and activated in mouse peritoneal macrophages and RAW264.7 cells. Mass spectrometry and mutagenesis analyses identified Ser 34 of LPCAT2 as the phosphorylation site to enhance the enzymatic activities. MK2 inhibitor and siRNA suppressed LPCAT2 phosphorylation, suggesting that LPCAT2 might be directly phosphorylated by MK2 to promote PAF and PC biosynthesis (Fig. 6).
In 1980, the lyso-PAFAT activity as the PAF biosynthetic enzyme was reported (3). Since then, several groups have attempted to characterize the enzyme. Lyso-PAFAT is rapidly activated in response to extracellular stimuli, such as calcium ionophore (4), acid stress (7), and LPS (16). However, neither the lyso-PAFAT cDNA sequence nor the mechanism of lyso-PAFAT activation had been elucidated. Recently, we identified two types of lyso-PAFATs: LPCAT2, which is an inducible lyso-PAFAT (9), and LPCAT1, which has constitutive lyso-PAFAT activity (8). LPCAT2 mRNA in macrophages is also up-regulated by LPS treatment for 16 h (9). The difference between LPCAT1 and LPCAT2 resembles that of cyclooxygenase-1 and -2 to produce prostaglandins (25,26). In mouse peritoneal macrophages, LPCAT2 is activated within 30 min by LPS stim-  The results of the present study indicate that LPCAT2 phosphorylation under LPS stimulation depends on the MyD88 (myeloid differentiation primary response gene 88), TAK1, p38␣, and MK2 signaling pathway. Ser 34 is the only phosphorylated site of LPCAT2 that enhances its catalytic activities. ER, endoplasmic reticulum. ulation (Fig. 3), consistent with the characteristics of endogenous lyso-PAFAT (6,9).
In this study, phosphorylated LPCAT2 was detected with the Phos-tag Western blot by mobility shift (Figs. 1B and 2A). Through mass spectrometric analysis of the phosphorylated enzyme, Ser 34 was identified as a phosphorylation site. Both the band shift and the activation were observed in WT LPCAT2, whereas the S34A mutant displayed neither characteristic (Fig.  2). Because mutagenesis at Ser 34 did not abolish the basal activities, it is proposed that Ser 34 is located in a regulatory region of LPCAT2. Moreover, the mutagenesis study indicated that Ser 34 was the only target of the phosphorylation that led to the enzymatic activation of LPCAT2. Furthermore, Ser 34 phosphorylation enhanced both the lyso-PAFAT and LPCAT activities of LPCAT2 (Fig. 2).
The activation of LPCAT2 in LPS-stimulated RAW264.7 cells was dependent on MK2 located downstream of p38 MAPK. Both p38␣ and p38␦ are mainly expressed in macrophages (27), and p38␣ and p38␤ signals are inhibited by SB203580. Thus, Figs. 3 and 4 suggest an LPCAT2 phosphorylation mediated by p38␣-MK2 axis. MK2 induces the phosphorylation of its substrates with the consensus sequence (HydXRXXS) (12,22). Near the N terminus of LPCAT2, 29 VPRQAS 34 was detected as corresponding to the consensus sequence. Thus, MK2 may directly phosphorylate LPCAT2, although it is possible that other kinases are present to link the two proteins. Future determination of the three-dimensional structure of LPCAT2 should definitively clarify this activation mechanism.
LPCAT2 has lyso-PAFAT and LPCAT activities, both of which are enhanced by LPS stimulation. The endogenous LPCAT activity in RAW264.7 cells was much higher than its lyso-PAFAT activity (Fig. 2, B and C). Thus, activated LPCAT2 may function as a lyso-PAFAT to produce PAF. It is also possible that the LPCAT activity of LPCAT2 plays an important role in the storage of phospholipid precursors of PAF and eicosanoids (1). LPCAT2 catalyzes the membrane biogenesis (LPCAT activity) of inflammatory cells while producing PAF (lyso-PAFAT activity) in response to external stimuli. Further studies are needed to elucidate the physiological and pathological importance of these dual activities.
This is the first report on the posttranslational modification of lysophospholipid acyltransferases functioning in Lands' cycle. Our results showed that LPCAT2, a member of the lysophospholipid acyltransferases, can produce lipid mediator and may contribute to membrane dynamics in response to extracellular stimuli.
This study will aid in the development of new anti-inflammatory drugs that inhibit PAF production by exogenous insults while maintaining the constitutive levels of the mediator. Because of the physiologically important roles of PAF, PAF receptor antagonists have encountered several adverse effects during drug development. Inhibition of inducible PAF production by phospho-LPCAT2, but not unphospho-LPCAT2 or LPCAT1 (constitutive lyso-PAFAT), could serve as a potential target of medical interventions. These findings improve our understanding of both inflammatory responses and membrane biogenesis.