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Biochem J. Jun 1, 2005; 388(Pt 2): 527–535.
Published online May 24, 2005. Prepublished online Feb 2, 2005. doi:  10.1042/BJ20041614
PMCID: PMC1138960

Roles of Rac and p38 kinase in the activation of cytosolic phospholipase A2 in response to PMA

Abstract

The roles of Rac and p38 kinase in the activation of cPLA2 (cytosolic PLA2) in Rat-2 fibroblasts were investigated. In the present study, we found that PMA activates cPLA2 by a Rac-p38 kinase-dependent pathway. Consistent with this, Rac, if activated, was shown to stimulate cPLA2 in a p38 kinase-dependent manner. In another experiment to understand the signalling mechanism by which the Rac-p38 kinase cascade mediates cPLA2 activation in response to PMA, we observed that PMA-induced cPLA2 translocation to the perinuclear region is completely inhibited by the expression of Rac1N17 or treatment with SB203580 (inhibitor of p38 kinase), suggesting that Rac-p38 kinase cascade acts in this instance by mediating the translocation of cPLA2. The mediatory role of p38 kinase in cPLA2 activation was further demonstrated after a treatment with anisomycin, a very effective activator of p38 kinase. Consistent with the mediatory role of p38 kinase in stimulating cPLA2, anisomycin induced the translocation and activation of cPLA2 in a p38 kinase-dependent manner.

Keywords: anisomycin, arachidonic acid, cPLA2, p38 kinase, PMA, Rac
Abbreviations: AA, arachidonic acid; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; CHOP, C/EBP homologous protein-10; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GST, glutathione S-transferase; LTB4, leukotriene B4; MAPK, mitogen-activated protein kinase; PAK, p21-activated protein kinase; PBD, p21-binding domain; PI3K, phosphoinositide 3-kinase; PI4K, phosphoinositide 4-kinase; PKC, protein kinase C; ROS, reactive oxygen species; SAPC, 1-stearoyl-2-14C-arachidonyl phosphatidyl choline

INTRODUCTION

Phospholipase A2 catalyses the hydrolysis of the central (sn-2) ester bond of glycerophospholipids, producing a non-esterified fatty acid and lysophospholipid [1]. These products have a variety of cellular functions: they contribute to cell signalling, phospholipid remodelling, membrane perturbation and inflammation [1,2]. Mammalian cells possess structurally diverse forms of phospholipase A2, notably cPLA2 (cytosolic PLA2), sPLA2 (secretory sPLA2) and iPLA2 (Ca2+-independent) forms [2]. Among them, cPLA2 is an 85 kDa cytosolic enzyme that requires micromolar free-Ca2+ and is believed to be a major producer of AA (arachidonic acid) under physiological conditions. AA, a C20 fatty acid released by cPLA2, is further metabolized into eicosanoids such as prostaglandins and leukotrienes by cyclo-oxygenase and lipoxygenase respectively [2,3]. These eicosanoids are potent lipid mediators involved in various homoeostatic functions and inflammation. Although the physiological characteristics of cPLA2 make it an important target for treating eicosanoid-mediated inflammation, the upstream signalling pathway to cPLA2 is still not clear.

cPLA2 is mainly regulated by Ca2+ influx and phosphorylation. It is translocated to the perinuclear membrane region by its C2 domain in a Ca2+-dependent manner [4,5] and it is phosphorylated by MAPK (mitogen-activated protein kinases) such as ERK (extracellular signal-regulated kinase), c-Jun N-terminal kinase or p38 kinase [610]. However, the detailed mechanisms of its regulation remain to be elucidated. Previously, it was demonstrated that cPLA2 is regulated by Rac1, a member of the Rho family of G-proteins, which acts as a molecular switch by cycling between active GTP-bound and inactive GDP-bound forms. Switching is regulated by guanine nucleotide exchange factors, GTPase-activating factors and GDP-dissociation-inhibitory factors [11]. Rac1 binds to effector molecules in its GTP bound form, and influences cellular processes such as cell motility, invasion, degranulation, cell–cell interaction, cell proliferation, cytoskeletal rearrangements and differentiation [1214]. Consistent with the idea of cPLA2 as a downstream mediator of Rac signalling, cPLA2 has been shown to be necessary for Rac in mediating actin remodelling, c-fos serum response element activation, transformation, or c-Jun N-terminal kinase stimulation [1316], and we have obtained evidence of a role for Rac in cPLA2 activation and the subsequent release of AA [1518].

In an approach to understand the signalling mechanism that accounts for Rac-mediated cPLA2 activation, in the present study, we have tested whether PMA stimulates cPLA2 by a Rac-dependent pathway and which signalling mechanism would be involved. PMA is a well-known activator of classical and novel isotypes of PKC (protein kinase C) [19]. We and others have reported that it stimulates AA release in macrophages and fibroblasts probably without the involvement of Ca2+ influx [2022]. In the present study, we report that activation of cPLA2 by Rac is mainly in a p38 kinase-dependent manner. Furthermore, we observed that PMA-induced cPLA2 translocation to the perinuclear membrane region is completely inhibited by the expression of Rac1N17 or treatment with SB203580 (a p38 kinase inhibitor), revealing a novel regulatory mechanism of cPLA2 through the Rac-p38 kinase cascade. Similarly, anisomycin, a very effective activator of p38 kinase, induced cPLA2 activation and translocation to the perinuclear region, further supporting an involved role of p38 kinase in the translocation process of cPLA2.

EXPERIMENTAL

Materials

PMA and EGTA were from Sigma–Aldrich (St. Louis, MO, U.S.A.); GF109203X (PKC inhibitor), SB203580, PD98059 (MAPK/ERK kinase inhibitor) and anisomycin were from Calbiochem (Darmstadt, Germany); wortmannin [23], FBS (fetal bovine serum), DMEM (Dulbecco's modified Eagle's medium), and non-essential amino acids were from GIBCO™ Invitrogen (Carlsbad, CA, U.S.A.); and SAPC (1-stearoyl-2-14C-arachidonyl phosphatidyl choline) was from Amersham Biosciences (Piscataway, NJ, U.S.A.). Silica gel 60 F254 plates were obtained from Merck (Darmstadt, Germany). All other chemicals were from standard sources and were of molecular biology grade or higher. Mouse monoclonal anti-cPLA2 or anti-α-tubulin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Rabbit polyclonal anti-phospho-cPLA2, anti-(phospho-p38 kinase) and anti-(p38 kinase) antibodies were from Cell Signaling Technology (Beverly, MA, U.S.A.). The myc-tagged expression vector pEXV, pEXV-Rac1V12 that encodes Rac1V12 (a constitutively active form of Rac1) and pEXV-Rac1N17 that encodes Rac1N17 (a dominant-negative form of Rac1) were gifts from Dr A. Hall (University College, London, U.K.). cDNAs encoding Rac1V12 and Rac1N17 were cloned between the EcoRI and XhoI sites of HA-tagged pcDNA3 plasmids and were used for the transfection of Rat-2 fibroblasts. pGFP-cPLA2 was a gift from Dr T. Shimizu (University of Tokyo, Tokyo, Japan) [24].

Cell culture, transfection and luciferase assay

Rat-2 fibroblasts were obtained from the A.T.C.C. (CRL 1764). The cells were grown in DMEM, supplemented with 0.1 mM non-essential amino acids, 10% (v/v) FBS, penicillin (50 units/ml) and streptomycin (50 μg/ml), at 37 °C in a humidified mixture of air and CO2 (95:5). The stable Rat-2RacN17 clones expressing RacN17, a dominant-negative Rac1 mutant, were described previously [17,25]. Transient transfection was performed by plating approx. 3×105 cells in 60 mm dishes for 24 h and adding calcium phosphate/DNA precipitates prepared with 20 μg of DNA/ml of transfection medium. The quantity of DNA used in each transfection was kept constant (20 μg/ml transfection medium) by adding sonicated calf thymus DNA (Sigma). To control the variation in cell number and transfection efficiency, all clones were co-transfected with 0.3 μg of pCMV-β GAL, a eukaryotic expression vector containing the Escherichia coli β-galactosidase (lacZ) structural gene under the control of the cytomegalovirus promoter. After incubating for 6 h with the calcium phosphate/DNA precipitates, the cells were rinsed twice in PBS before incubating them in fresh DMEM containing 0.5% FBS for an additional 24 h. Each dish of cells was then rinsed twice in PBS and lysed in 0.1 ml of lysis solution (0.2 M Tris (pH 7.6, and 0.1%, v/v, Triton X-100), after which the lysed cells were scraped off and centrifuged for 1 min. The supernatants were assayed for protein and β-galactosidase activity and subjected to Western blotting or subcellular fractionation.

To measure p38 kinase activity with the PathDetect trans-reporting system (Stratagene, La Jolla, CA, U.S.A.; cat. no. 219005), CHOP (C/EBP homologous protein-10, also known as GADD153) fused to a trans-activator plasmid (pFA2-CHOP) was co-transfected with the pFR-Luc reporter plasmid according to the manufacturer's instructions. To examine the role of Rac1 in p38 kinase activation, pcDNA-HA-Rac1V12 was co-transfected with pFR-Luc or pFA2-CHOP using calcium phosphate methods. After 36 h, each dish of cells was rinsed twice in PBS and lysed in 0.1 ml of a lysis solution (0.2 M Tris, pH 7.6, and 0.1% Triton X-100). Next, the supernatants were assayed for luciferase activity as well as protein concentration and β-galactosidase activity.

Luciferase activity was assayed in 10 μl samples of the extract using the Luciferase Assay System (Promega, Madison, WI, U.S.A.) according to the manufacturer's instructions; luciferase luminescence was counted in a luminometer (Turner Designs, Sunnyvale, CA, U.S.A.; TD-20/20) and normalized to co-transfected β-galactosidase activity. Transfection experiments were performed in triplicate with two independently isolated sets of cells and results are expressed as means±S.D. relative to the control.

Assay of cPLA2 activity

The Rat-2 cells Rat-2Rac1N17 cells were starved for 24 h in DMEM containing 0.5% FBS, then rinsed twice in PBS and lysed in a lysis solution (20 mM Tris/Cl, pH 7.5, 0.25 M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM dithiothreitol and protease inhibitors). The cells were lysed by passaging with a 26-guage needle and fractionated by ultracentrifugation at 100000 g for 1 h. The supernatants were assayed for cPLA2 activity using sonicated liposomes prepared as described previously [26,27]. Briefly, a stock solution of the [14C]SAPC radioactive substrate was prepared as follows: the [14C]SAPC substrate (10 nmol) was dried under nitrogen and resuspended in 0.5 ml of distilled water by sonication (3×10 s) in a bath-type sonicator (Ultrasonik 300, J. M. Ney, Broomfield, CT, U.S.A.). The reaction was started by adding each cell lysate (containing ~10 μg of protein/50 μl) to make 200 μl of total reaction mixture that included 50 μl of [14C]SAPC (final concentration 5 μM), 1 mM CaCl2 and 20 mM Tris/Cl at pH 8.5. The assay mixtures were incubated at 37 °C for 30 min and then quenched by adding 500 μl of Dole's reagent (32%, v/v propan-2-ol, 67%, v/v, n-heptane and 1%, v/v, 0.5 M H2SO4) and vortex-mixing. Non-esterified fatty acid was extracted as follows: in the reaction mixture, 300 μl of n-heptane and 200 μl of distilled water were added, the samples were centrifuged and the upper phase (~400 μl) was transferred to a prepared silicic gel column [28]. The released [14C]AA was extracted by adding equal volumes of diethyl ether to silica gel and the eluate was counted for radioactivity in a liquid-scintillation counter. In all experiments, samples were tested in duplicate and adjusted for non-specific release by subtracting the value of the control without cell lysate.

Rac activity assay

The PAK (p21-activated protein kinase)-PBD (p21-binding domain) binding assay was performed essentially as described in [29]. In brief, the PBD of human PAK1, comprising amino acids 68–166, was subcloned into the bacterial expression vector pGEX-2TK (Amersham Biosciences) and expressed in E. coli as the GST–PAK-PBD fusion protein (where GST stands for glutathione S-transferase), according to the manufacturer's instructions. A 50% (v/v) slurry (15 μl) of GST–PAK-PBD glutathione–Sepharose 4B was added to lysates of Rat-2 fibroblasts and continuously rotated at 4 °C for 60 min. The bound proteins were collected by centrifugation and the pellets were washed three times with cell lysis buffer (25 mM Hepes, pH 7.5, 150 mM NaCl, 25 mM NaF, 10%, v/v, glycerol, 0.25%, w/v, sodium deoxycholate, 10 mM MgCl2, 1 mM EDTA and 1% Triton X-100) and finally suspended in SDS sample buffer. Proteins were size-fractionated by SDS/PAGE, and Rho family GTPases were identified by Western blotting with Rac1 and Cdc42 antibodies.

Subcellular fractionation of lysates

Rat-2 cells were serum-starved for 18 h in serum-free DMEM and incubated with appropriate agonists for the indicated times. The medium was removed and the cells were washed twice with ice-cold PBS, scraped, harvested by microcentrifugation and resuspended in 0.2 ml of buffer A (137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM PMSF and 10 μg/ml leupeptin, pH 7.5). The resuspended cells were then lysed on ice by 20 passes through a 21.1-gauge needle. Lysates were centrifuged at 100000 g for 1 h to prepare cytosolic and particulate membrane fractions and the particulate membrane fractions were washed and suspended in 50 μl of buffer A. The protein concentration was determined routinely with the Bradford reagent and BSA as standard.

Western blotting

Protein samples were heated at 95 °C for 5 min and subjected to SDS/PAGE (8–10% polyacrylamide), followed by transfer to PVDF membranes at 100 V with a Novex wet transfer unit (Novex, San Diego, CA, U.S.A.). The membranes were blocked overnight in Tris-buffered saline with 0.01% (v/v) Tween 20 and 5% (w/v) non-fat dried milk, after which they were incubated for 2 h with primary antibody in Tris-buffered saline, and then for 1 h with horseradish peroxidase-conjugated secondary antibody. The blots were developed using an enhanced chemiluminescence kit (ECL®, Amersham Biosciences).

Confocal microscopy

To localize cPLA2, cells were plated on coverslips and grown for 24 h in DMEM containing 10% FBS. After transfection with calcium phosphate/DNA precipitates (10 μg of DNA/ml transfection medium), the cells were starved in DMEM containing 0.5% FBS for 36 h, and exposed to 20 nM PMA for the indicated times. The reaction was stopped by washing twice with ice-cold PBS and fixing with 4% (w/v) paraformaldehyde. Thereafter, they were mounted on a glass slip and sealed. Images were obtained with a Bio-Rad MRC-1024 laser scanning confocal system (Bio-Rad Laboratories, Hercules, CA, U.S.A.); multiple image sections of thickness 0.2 μm were recorded over the depth of the cells and analysed.

Data analysis and statistics

Data are expressed as means±S.D. or as percentage of control ±S.D. Statistical comparisons between groups were made with Student's t test. Values of P<0.01 were considered significant.

RESULTS

Rac1V12 activates cPLA2

Previously, we have suggested a potential role of Rac as an upstream mediator of cPLA2 stimulation [15]. Consistent with this idea, we observed that overexpression of Rac1V12 induces a highly elevated cPLA2 activity as assayed by the release of 14C-AA. For this assay, subconfluent Rat-2 fibroblasts were transiently transfected with pcDNA-HA-Rac1V12 or pcDNA-HA-Rac1N17 and incubated for 24 h in a serum-free DMEM. As shown in Figure 1, cPLA2 activity was stimulated by Rac1V12 overexpression (Figure 1A) and there was only a small increase of cPLA2 activity in Rac1N17-overexressing cells when compared with pcDNA3-transfected cells (Figure 1B), suggesting that activation of Rac1 is sufficient for cPLA2 stimulation. Next, we investigated the signalling mechanism by which Rac mediates cPLA2 activation. Subconfluent cells were transiently transfected with the plasmid (5 μg) pcDNA-HA-Rac1V12 or pcDNA3 and incubated for an additional 24 h in serum-free DMEM. Various inhibitors (SB203580, PD98059 or EGTA) or DMSO (vehicle) were then added 6 h before harvest. As shown in Figure 1(C), SB203580 (10 μM) significantly attenuated the activation of cPLA2 by Rac1V12 overexpression, whereas an inhibitor of ERKs (PD98059; 10 μM) or a calcium chelator (EGTA; 5 mM) had little effect.

Figure 1
Constitutive activation of Rac stimulates cPLA2

p38 kinase as a downstream mediator of Rac1 in the signalling to cPLA2 activation

The above results suggest that Rac stimulates cPLA2 by a p38 kinase-dependent pathway, pointing to p38 kinase as a downstream mediator of Rac1 in signalling to cPLA2 stimulation. To determine further the hierarchical relationship of Rac1 and p38 kinase, we performed Western-blot analysis and phosphorylation-dependent reporter gene assays using the PathDetect transporter system (Stratagene) as described in the Experimental section. To investigate whether activation of Rac1 itself can induce the phosphorylation of p38 kinase, p38 kinase phosphorylation was examined in Rat-2 cells transfected with pcDNA-HA-Rac1V12 by Western blotting. p38 kinase was activated by overexpression of Rac1V12 in a dose-dependent manner (Figure 2A) and p38 kinase luciferase reporter. Gene assays also showed a dose-dependent activation of p38 kinase by overexpressing Rac1V12 (Figure 2B).

Figure 2
p38 kinase as a downstream mediator of Rac in the signalling to cPLA2

Essential role of Rac in the PMA-induced signalling to cPLA2 activation

Next, we examined whether Rac plays a similar mediatory role in the cPLA2 activation in response to PMA in Rat-2 fibroblasts. The cells were exposed to 20 nM PMA and lysates were assayed for cPLA2 activity as described in the Experimental section. As expected, PMA stimulated cPLA2 activity (Figure 3A), and activation was inhibited in Rat-2 cells overexpressing the dominant-negative mutant of Rac1, namely Rat-2Rac1N17 (Figure 3B), indicating that Rac1 plays an essential role in the PMA-induced cPLA2 activation.

Figure 3
PMA activates cPLA2 through Rac in Rat-2 fibroblasts

To study further the role of Rac1 in PMA-induced signalling, we tested if PMA activates Rac or not. For this assay, we performed a Rac-PAK binding assay [29]; Rat-2 cells (1×106 cells) were plated on to 100 mm dishes and incubated in DMEM containing 10% FBS for 24 h. Subconfluent cells were starved in DMEM containing 0.5% FBS for 24 h, and exposed to 20 nM PMA for the indicated times, after which the cells were lysed. Supernatants of lysates were assayed for active Rac1 protein as described in the Experimental section. Rac1 was indeed activated by PMA (20 nM) treatment as shown in Figure 3(C), reaching a maximum at 10 min. At the same time, it appeared to translocate from the cytosolic to particulate membrane regions (Figure 3D), reflecting an activation of Rac1.

PMA activates cPLA2 in a Rac-p38 kinase-dependent pathway

Next, we examined the signalling mechanism by which PMA mediates cPLA2 activation. For this assay, serum-starved Rat-2 fibroblasts were exposed to various inhibitors 30 min before treatment with PMA (20 nM, 30 min), and then cPLA2 was assayed. In agreement with the above results, SB203580 inhibited the activation of cPLA2 in the PMA-stimulated Rat-2 cells, whereas PD98059 and EGTA had little effect (Figure 4A). GF109203X (5 μM), a general inhibitor of PKC isotypes, also inhibited the activation of cPLA2 by PMA, thus supporting a role for PKC in PMA-induced cPLA2 activation; this is consistent with the fact that PMA is a potent PKC activator [19]. Wortmannin (100 nM), a PI3K (phosphoinositide 3-kinase)-inhibitor, had no effect. To demonstrate the role of p38 kinase in the PMA signalling to cPLA2 activation, we first examined the time course of p38 kinase phosphorylation in response to PMA treatment. As shown in Figure 4(B), p38 kinase was phosphorylated by PMA (50 nM) treatment in a time-dependent manner, reaching a maximum at 45 min. In addition, PMA treatment led to the phosphorylation of p38 kinase in wild-type Rat-2 cells but not in Rat-2Rac1N17 cells (Figure 4C), indicating that PMA activates p38 kinase through Rac. We have also examined whether PMA activates the p38 kinase/CHOP-luciferase reporter gene activity. As shown in Figure 4(D), a significant stimulation of p38 kinase activity (~2.3-fold increase compared with the control) was observed in response to PMA (50 nM), and this activation was significantly attenuated in Rat-2Rac1N17 cells. Together, p38 kinase is suspected to act as a downstream mediator of cPLA2 stimulation in response to PMA or activated Rac1.

Figure 4
PMA activates cPLA2 in a Rac-p38 kinase-cascade-dependent manner

To assess further the involvement of p38 kinase in the activation of cPLA2, Rat-2 cells were exposed to anisomycin, a very effective p38 kinase activator [30]. As expected, anisomycin induced the activation of p38 kinase in Rat-2 fibroblasts, leading to the activation of cPLA2 activity (Figures 5A and and5C).5C). Interestingly, activation of p38 kinase in response to anisomycin was also observed in Rat-2Rac1N17 cells (Figure 5A), suggesting that anisomycin activates p38 kinase in a Rac-independent manner.

Figure 5
Anisomycin induces cPLA2 activation of Rat-2 fibroblasts

p38 kinase-linked cascade mediates the translocation of cPLA2

A previous study has reported that, in primary murine astrocytes, PMA stimulated the phosphorylation of ERKs and cPLA2 as well as evoked AA release and that, however, complete inhibition of phospho-ERK by U0126, an inhibitor of MAPK/ERK kinase, did not completely inhibit PMA-stimulated cPLA2 and AA release, suggesting that phosphorylation of cPLA2 due to phospho-ERK is not sufficient to evoke AA release [31]. Therefore, similar to that report, we undertook to test whether Rac-p38 kinase-mediated activation of cPLA2 is not enhanced by phosphorylation of cPLA2 through ERKs in the PMA signalling to cPLA2 activation. We therefore examined the phosphorylation of cPLA2 on Ser505 in subconfluent Rat-2 and Rat-2Rac1N17 fibroblasts using an antibody to Ser505-phosphorylated cPLA2 [3234]. As shown in Figure 6(A), although PMA caused a significant phosphorylation of cPLA2 on Ser505, this phosphorylation on Ser505 by PMA was not inhibited by Rac1N17, suggesting that the Rac-linked pathway is independent of the phosphorylation mediated by ERKs. Similarly, phosphorylation on Ser505 was not blocked by pretreatment with SB203580, although PD98059 completely decreased the cPLA2 phosphorylation by PMA (Figure 6A). Consistent with these results, transient expression of Rac1V12 did not show any enhanced level of Ser505 phosphorylation (results not shown). Together, these and earlier results (Figures 1C and and4A)4A) suggest that PMA-Rac-p38 kinase-mediated cPLA2 activation does not involve ERK-mediated phosphorylation of cPLA2 on Ser505, at least in Rat-2 cells.

Figure 6
Rac-p38 kinase mediates the translocation of cPLA2

In a further approach to determine the signalling mechanism by which the Rac-p38 kinase cascade mediates cPLA2 activation in response to PMA, we next examined whether cPLA2 translocation is affected by this cascade or not. By subcellular fractionation of lysates, Rac-induced cPLA2 translocation was significantly inhibited by SB203580 (Figure 6B) and, similarly, PMA was shown to induce the translocation of cPLA2 from the cytosol to the membrane fraction in a Rac-dependent manner, and this was further confirmed by confocal microscopy of GFP–cPLA2 in Rat-2 fibroblasts (Figures 6C and and6D).6D). Clearly, translocation of cPLA2 was inhibited in Rat-2Rac1N17 cells, suggesting a mediatory role for the Rac-p38 kinase cascade in the translocation of cPLA2. By pretreatment with SB203580 (Figure 6E), PMA-induced cPLA2 translocation was completely blocked, suggesting the involvement of p38 kinase in the translocation of cPLA2 in response to PMA.

In addition, anisomycin caused cPLA2 to be translocated to the membrane fraction from the cytosolic fraction (Figure 6F). Therefore we propose a hypothetic model, and, in this model, the Rac-p38 kinase cascade mediates the translocation of cPLA2 and this pathway does not involve ERK-dependent phosphorylation of cPLA2 on Ser505 in Rat-2 fibroblasts (Scheme 1).

DISCUSSION

The present study showed that Rac1 stimulates cPLA2 by a signalling pathway in a p38 kinase-dependent manner. The activation of cPLA2 after PMA stimulation or Rac1V12 overexpression was completely inhibited by SB203580, but not by PD98059 (Figures 1C and and4A),4A), suggesting that Rac1 stimulates cPLA2 by an ERK-independent pathway. An essential role of Rac1 was demonstrated by comparing the effects of Rac1V12 overexpression in Rat-2 fibroblasts, and the effect of PMA on cPLA2 activation in Rat-2 and Rat-2Rac1N17 cells (Figures 1 and and3).3). These findings support our previous results showing that Rac liberates AA by a cPLA2-dependent pathway [1518]. Activation of Rac1 by PMA was further confirmed by the fact that it is rapidly translocated from the cytosol to the membrane fraction when cells are exposed to PMA (Figure 3D).

Although PMA caused the phosphorylation of cPLA2 on Ser505 [3234], the phosphorylation on Ser505 by PMA was not inhibited by Rac1N17 (Figure 6A), suggesting that the Rac-linked pathway is independent of ERK-mediated phosphorylation of cPLA2. Similarly, phosphorylation on Ser505 was not blocked by pretreatment with SB203580, although PD98059 completely decreased cPLA2 phosphorylation by PMA (Figure 6A). While p38 kinase is involved in the phosphorylation of cPLA2 on Ser505 in thrombin-stimulated platelets [7], cPLA2 is also phosphorylated by p38 kinase on four residues, namely Ser437, Ser454, Ser505 and Ser727 [10,3134], and thus it remains to be determined whether p38 kinase causes phosphorylation on any of the other serine residues under our experimental conditions. For instance, p38 kinase has been reported to lead to phosphorylation of cPLA2 on Ser727 through MAPK-interacting kinase 1 in Chinese-hamster ovary and HEK-293 cells (human embryonic kidney 293 cells) [32]. Interestingly, a significant fraction of cPLA2 is constitutively phosphorylated on Ser505 in Sf9 cells, yet AA is not released unless the cells are treated with okadaic acid or agonists that increase intracellular Ca2+ [32]. Previously, we reported that Rac1-ERK signalling to cPLA2 is implicated in LTB4 (leukotriene B4)-induced ROS (reactive oxygen species) generation for chemotaxis [35]. Thus, depending on the agonists, ERKs or p38 kinase may be involved in cPLA2 regulation and in fact, for an LTB4-induced cPLA2 stimulation, cPLA2 was assumed to act in a positive feedback loop (Rac-ERK-cPLA2-AA-NADPH oxidase cascade) to modulate NADPH oxidase to mediate ROS generation. Therefore an ERK-cPLA2-linked cascade does not constitute the main signalling route to ROS generation in LTB4 signalling. In contrast, for PMA, the Rac-p38 kinase-cascade is the main signalling mediator of the PMA-induced signalling leading to cPLA2 stimulation, regulating the formation of eicosanoids. Thus we suspect that p38 kinase acts as a general downstream mediator of Rac in cPLA2 stimulation in response to a variety of agonists acting through Rac. In any event, from these reports and our present results, we conclude that the role of Ser505 phosphorylation in the regulation of cPLA2 is cell-type- and agonist-dependent and that it is not essential for the Rac-p38 kinase-stimulated cPLA2 activation in Rat-2 fibroblasts.

We were then interested in the translocation of cPLA2, the other major mechanism of cPLA2 activation, and found that PMA caused the translocation of cPLA2, especially to the perinuclear membrane region, through the p38 kinase-linked cascade. This was further supported by the effect of anisomycin on cPLA2 translocation in Rat-2 fibroblasts. Recently, Evans et al. [36,37] reported that the [Ca2+]i level controls the targeting of cPLA2 to intracellular membranes such as the Golgi, endoplasmic reticulum and perinuclear membrane. According to Evans et al., cPLA2 translocation to the perinuclear membrane region requires high [Ca2+]i. However, it has been shown that PMA provides cPLA2-activating signals without inducing Ca2+ influx [2022]. This has led to the suggestion that, under certain conditions, cPLA2 activity may be modulated independent of Ca2+ mobilization in some cell types [19]; it remains to be elucidated whether this is the case for Rac-p38 kinase-dependent cPLA2 translocation in Rat-2 fibroblasts. Although removal of extracellular free-Ca2+ with EGTA did not inhibit PMA- or Rac1-induced activation of cPLA2 (Figures 1C and and4A),4A), the effect of Ca2+ in the translocation of cPLA2 by the Rac-p38 kinase cascade is still not clear. The detailed mechanism by which the Rac-p38 kinase cascade mediates cPLA2 stimulation in response to PMA remains to be characterized, especially regarding the downstream targets of p38 kinase.

Another potential mechanism by which Rac mediates cPLA2 activation has been previously reported. For example, Dennis and co-workers [38,39] reported that cPLA2 could be activated by direct binding to phosphatidylinositol 4,5-bisphosphate, a product of PI4K (phosphoinositide 4-kinase), which is activated by Rac1 [38,39]. In addition, polyphosphoinositides decreased the Ca2+ requirement of cPLA2 such that, under certain conditions, its activity was truly Ca2+-independent [24]. However, we have shown that PMA/Rac1-mediated cPLA2 activation in Rat-2 fibroblasts is independent of PI4K, since translocation of cPLA2 was not blocked by 2,3-dihydroxy-benzaldehyde, a PI4K inhibitor (results not shown) [40,41]. The involvement of PI3K, another molecule closely related to PI4K, in the PMA-mediated cPLA2 activation, was also excluded by the fact that wortmannin did not attenuate cPLA2 activation in response to PMA (Figure 4A). The C2 domain of cPLA2 is known to be involved in the preferential translocation of cPLA2 to the nuclear region [5,6,36]. However, we found that PMA induced the translocation of full-length cPLA2 but not the C2 domain alone (results not shown). Our results thus suggest that a region other than C2 is required, depending on the agonists, for the translocation of cPLA2.

Taken together, our findings indicate that Rac1 induces cPLA2 activation in Rat-2 fibroblasts through a novel mechanism involving a p38 kinase. Although the downstream targets of p38 kinase still need to be identified, our present results suggest that the Rac-p38 kinase cascade acts by mediating the translocation of cPLA2. Further characterization of the mechanism of cPLA2 activation by the Rac-p38 kinase cascade would be a pivotal step towards a better understanding of the regulatory mechanism leading to cPLA2 activation.

Scheme 1
A hypothetic model for the role of the Rac-p38 kinase cascade in the activation of cPLA2

Acknowledgments

We thank Dr T. Shimizu (University of Tokyo, Tokyo, Japan) for kindly providing pGFP-cPLA2 expression plasmids. This study was supported by a grant from the Korea Health 21 R&D project, Ministry of Health and Welfare, Republic of Korea (0405-B502-0205-0001).

References

1. Dessen A. Structure and mechanism of human cytosolic phospholipase A2. Biochim. Biophys. Acta. 2000;1488:40–47. [PubMed]
2. Six D. A., Dennis E. A. The expanding superfamily of phospholipase A2 enzymes: classification and characterization. Biochim. Biophys. Acta. 2000;1488:1–19. [PubMed]
3. Hirabayashi T., Shimizu T. Localization and regulation of cytosolic phospholipase A2. Biochim. Biophys. Acta. 2000;1488:124–138. [PubMed]
4. Clark J. D., Lin L. L., Kriz R. W., Ramesha C. S., Sultzman L. A., Lin A. Y., Milona N., Knopf J. L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell (Cambridge, Mass.) 1991;65:1043–1051. [PubMed]
5. Nalefski E. A., Sultzman L. A., Martin D. M., Kriz R. W., Towler P. S., Knopf J. L., Clark J. D. Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca2+-dependent lipid-binding domain and a Ca2+-independent catalytic domain. J. Biol. Chem. 1994;269:18239–18249. [PubMed]
6. Nemenoff R. A., Winitz S., Qian N. X., Van Putten V., Johnson G. L., Heasley L. E. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J. Biol. Chem. 1993;268:1960–1964. [PubMed]
7. Kramer R. M., Roberts E. F., Um S. L., Borsch-Haubold A. G., Watson S. P., Fisher M. J., Jakubowski J. A. p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2. J. Biol. Chem. 1996;271:27723–27729. [PubMed]
8. Waterman W. H., Molski T. F., Huang C. K., Adams J. L., Sha'afi R. I. Tumor necrosis factor-alpha-induced phosphorylation and activation of cytosolic phospholipase A2 are abrogated by an inhibitor of the p38 mitogen-activated protein kinase cascade in human neutrophils. Biochem. J. 1996;319:17–20. [PMC free article] [PubMed]
9. Hernandez M., Bayon Y., Sanchez Crespo M., Nieto M. L. Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1. Biochem. J. 1997;328:263–269. [PMC free article] [PubMed]
10. Coulon L., Calzada C., Moulin P., Vericel E., Lagarde M. Activation of p38 mitogen-activated protein kinase/cytosolic phospholipase A2 cascade in hydroperoxide-stressed platelets. Free Radical Biol. Med. 2003;35:616–625. [PubMed]
11. Takai Y., Sasaki T., Matozaki T. Small GTP-binding proteins. Physiol. Rev. 2001;81:153–208. [PubMed]
12. Nobes C. D., Hall A. Rho, Rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell (Cambridge, Mass.) 1995;81:53–62. [PubMed]
13. Peppelenbosch M. P., Qiu R. G., de Vries-Smits A. M., Tertoolen L. G., de Laat S. W., McCormick F., Hall A., Symons M. H., Bos J. L. Rac mediates growth factor-induced arachidonic acid release. Cell (Cambridge, Mass.) 1995;81:849–856. [PubMed]
14. Hong-Geller E., Holowka D., Siraganian R. P., Baird B., Cerione R. A. Activated Cdc42/Rac reconstitutes Fcepsilon RI-mediated Ca2+ mobilization and degranulation in mutant RBL mast cells. Proc. Natl. Acad. Sci. U.S.A. 2001;98:1154–1159. [PMC free article] [PubMed]
15. Kim B. C., Kim J. H. Nuclear signalling by Rac GTPase: essential role of phospholipase A2. Biochem. J. 1997;326:333–337. [PMC free article] [PubMed]
16. Yoo M. H., Woo C. H., You H. J., Cho S. H., Kim B. C., Choi J. E., Chun J. S., Jhun B. H., Kim T. S., Kim J. H. Role of the cytosolic phospholipase A2-linked cascade in signaling by an oncogenic, constitutively active Ha-Ras isoform. J. Biol. Chem. 2001;276:24645–24653. [PubMed]
17. Woo C. H., Eom Y. W., Yoo M. H., You H. J., Han H. J., Song W. K., Yoo Y. J., Chun J. S., Kim J. H. Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. J. Biol. Chem. 2000;275:32357–32362. [PubMed]
18. Woo C. H., Lee Z. W., Kim B. C., Ha K. S., Kim J. H. Involvement of cytosolic phospholipase A2, and the subsequent release of arachidonic acid, in signaling by Rac for the generation of intracellular reactive oxygen species in Rat-2 fibroblasts. Biochem. J. 2000;348:525–530. [PMC free article] [PubMed]
19. Liu W. S., Heckman C. A. The sevenfold way of PKC regulation. Cell. Signal. 1998;10:529–542. [PubMed]
20. Gijon M. A., Spencer D. M., Siddiqi A. R., Bonventre J. V., Leslie C. C. Cytosolic phospholipase A2 is required for macrophage arachidonic acid release by agonists that do and do not mobilize calcium. Novel role of mitogen-activated protein kinase pathways in cytosolic phospholipase A2 regulation. J. Biol. Chem. 2000;275:20146–20156. [PubMed]
21. Oh J., Rhee H. J., Kim S., Kim S. B., You H., Kim J. H., Na D. S. Annexin-1 inhibits PMA-induced c-fos SRE activation by suppressing cytosolic phospholipase A2 signal. FEBS Lett. 2000;477:244–248. [PubMed]
22. Qiu Z. H., Gijon M. A., de Carvalho M. S., Spencer D. M., Leslie C. C. The role of calcium and phosphorylation of cytosolic phospholipase A2 in regulating arachidonic acid release in macrophages. J. Biol. Chem. 1998;273:8203–8211. [PubMed]
23. Wymann M. P., Bulgarelli-Leva G., Zvelebil M. J., Pirola L., Vanhaesebroeck B., Waterfield M. D., Panayotou G. Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol. Cell. Biol. 1996;16:1722–1733. [PMC free article] [PubMed]
24. Hirabayashi T., Kume K., Hirose K., Yokomizo T., Iino M., Itoh H., Shimizu T. Critical duration of intracellular Ca2+ response required for continuous translocation and activation pf cytosolic phospholipase A2. J. Biol. Chem. 1999;274:5163–5169. [PubMed]
25. Kim B. C., Yi J. Y., Yi S. J., Shin I. C., Ha K. S., Jhun B. H., Hwang S. B., Kim J. H. Rac GTPase activity is essential for EGF-induced mitogenesis. Mol. Cell. 1998;8:90–95. [PubMed]
26. Kim K. M., Kim D. K., Park Y. M., Kim C. K., Na D. S. Annexin-1 inhibits phospholipase A2 by specific interaction, not by substrate depletion. FEBS Lett. 1994;343:251–255. [PubMed]
27. Favier-Perron B., Lewit-Bentley A., Russo-Marie F. The high-resolution crystal structure of human annexin III shows subtle differences with annexin V. Biochemistry. 1996;35:1740–1744. [PubMed]
28. Huwiler A., Staudt G., Kramer R. M., Pfeilschifter J. Cross-talk between secretory phospholipase A2 and cytosolic phospholipase A2 in rat renal mesangial cells. Biochim. Biophys. Acta. 1997;1348:257–272. [PubMed]
29. Benard V., Bohl B. P., Bokoch G. M. Characterization of Rac and Cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J. Biol. Chem. 1999;274:13198–13204. [PubMed]
30. Hazzalin C. A., Cuenda A., Cano E., Cohen P., Mahadevan L. C. Effects of the inhibition of p38/RK MAP kinase on induction of five fos and jun genes by diverse stimuli. Oncogene. 1997;15:2321–2331. [PubMed]
31. Xu J., Weng Y. I., Simonyi A., Krugh B. W., Liao Z., Weisman G. A., Sun G. Y., Simoni A. Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachidonic acid release in primary murine astrocytes. J. Neurochem. 2002;83:259–270. [PubMed]
32. Hefner Y., Borsch-Haubold A. G., Murakami M., Wilde J. I., Pasquet S., Schieltz D., Ghomashchi F., Yates J. R., III, Armstrong C. G., Paterson A., et al. Ser727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases. J. Biol. Chem. 2000;275:37542–37551. [PubMed]
33. Gijon M. A., Spencer D. M., Kaiser A. L., Leslie C. C. Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2. J. Cell Biol. 1999;145:1219–1232. [PMC free article] [PubMed]
34. Das S., Rafter J. D., Kim K. P., Gygi S. P., Cho W. Mechanism of group IVA cytosolic phospholipase A2 activation by phosphorylation. J. Biol. Chem. 2003;278:41431–41442. [PubMed]
35. Woo C. H., You H. J., Cho S. H., Eom Y. W., Chun J. S., Yoo Y. J., Kim J. H. Leukotriene B(4) stimulates Rac-ERK cascade to generate reactive oxygen species that mediates chemotaxis. J. Biol. Chem. 2002;277:8572–8578. [PubMed]
36. Evans J. H., Spencer D. M., Zweifach A., Leslie C. C. Intracellular calcium signals regulating cytosolic phospholipase A2 translocation to internal membranes. J. Biol. Chem. 2001;276:30150–30160. [PubMed]
37. Evans J. H., Gerber S. H., Murray D., Leslie C. C. The calcium binding loops of the cytosolic phospholipase A2 C2 domain specify targeting to Golgi and ER in live cells. Mol. Biol. Cell. 2004;15:371–383. [PMC free article] [PubMed]
38. Mosior M., Six D. A., Dennis E. A. Group IV cytosolic phospholipase A2 binds with high affinity and specificity to phosphatidylinositol 4, 5-bisphosphate resulting in dramatic increases in activity. J. Biol. Chem. 1998;273:2184–2191. [PubMed]
39. Balsinde J., Balboa M. A., Li W. H., Llopis J., Dennis E. A. Cellular regulation of cytosolic group IV phospholipase A2 by phosphatidylinositol bisphosphate level. J. Immunol. 2000;164:5398–5402. [PubMed]
40. Nishioka H., Imoto M., Sawa T., Hamada M., Naganawa H., Takeuchi T., Umezawa K. Screening of phosphatidylinositol kinase inhibitors from Streptomyces. J. Antibiot. (Tokyo) 1989;42:823–825. [PubMed]
41. Carpenter C. L., Cantley L. C. Phsophatidylinositide kinases. Biochemistry. 1990;29:11147–11156. [PubMed]

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