Inhibition of the MEK1/ERK pathway reduces arachidonic acid release independently of cPLA2 phosphorylation and translocation
© Evans et al; licensee BioMed Central Ltd. 2002
Received: 7 June 2002
Accepted: 8 October 2002
Published: 8 October 2002
The 85-kDa cytosolic phospholipase A2 (cPLA2) mediates arachidonic acid (AA) release in MDCK cells. Although calcium and mitogen-activated protein kinases regulate cPLA2, the correlation of cPLA2 translocation and phosphorylation with MAPK activation and AA release is unclear.
MEK1 inhibition by U0126 inhibited AA release in response to ATP and ionomycin. This directly correlated with inhibition of ERK activation but not with phosphorylation of cPLA2 on Ser505, which was only partially inhibited by ERK inhibition. Inhibition of AA release by U0126 was still observed when stoichiometric phosphorylation of cPLA2 on Ser505 was maintained by activating p38 with anisomycin. Translocation kinetics of wild-type cPLA2 and cPLA2 containing S505A or S727A mutations to Golgi were similar in response to ATP and ionomycin and were not affected by U0126.
These results suggest that the ability of cPLA2 to hydrolyze membrane phospholipid is reduced by inhibition of the MEK1/ERK pathway and that the reduction in activity is independent of cPLA2 phosphorylation and translocation to membrane. The results also demonstrate that cPLA2 mutated at the phosphorylation sites Ser505 and Ser727 translocated with similar kinetic as wild-type cPLA2.
Cytosolic PLA2 specifically hydrolyzes sn-2 arachidonate from phospholipid providing the precursors for many different lipid mediators including prostaglandins and leukotrienes [1, 2]. These lipid metabolites play a role in acute inflammatory responses and also regulate normal physiological processes. Certain prostaglandins are required for female reproduction and kidney function [3–5]. Because of its important role in controlling levels of arachidonic acid (AA), much attention has been focused on the regulation of cPLA2 activation, with particular emphasis on the role of its phosphorylation and Ca2+-mediated translocation [6–8].
cPLA2 is regulated by controlling its cellular localization and access to membrane-phospholipid substrate. An amino terminal, calcium-dependent lipid binding (CaLB or C2) domain regulates Ca2+-mediated cPLA2 translocation to intracellular membranes . In vitro, membrane docking via the C2 domain is necessary and sufficient for catalysis and release of AA . Binding of calcium ions by the cPLA2 C2 domain is essential for the lipid association in vitro [11, 12] and translocation in vivo [13, 14]. In response to an increase in [Ca2+]i, cPLA2 translocates to the Golgi and ER, however translocation to Golgi occurs at a lower [Ca2+]i.
Protein kinase pathways play major roles in cPLA2 activation, and regulation by the mitogen-activated protein kinase kinase (MEK) /extracellular-signal regulated kinase (ERK) signaling pathway has received particular attention. cPLA2 is phosphorylated by mitogen activated protein (MAP) kinases, including p42/p44 ERKs and p38, on Ser505 in vitro [16, 17] and in response to receptor stimulation [16, 18–21]. In addition to phosphorylation by MAP kinase, it has been shown that cPLA2 is also phosphorylated on Ser727 by MAPK-interacting kinase I (MNKI)  and on Ser515 by calcium/calmodulin-dependent protein kinase II . Phosphorylation of these sites may also play a role in regulating cPLA2 function in certain cell models.
Phosphorylation of Ser505 has been extensively studied because it is readily detected due to a characteristic electrophoretic mobility shift when analyzed by SDS-PAGE [13, 16]. The importance of Ser505 phosphorylation in regulating cPLA2 has been demonstrated in different cells and in vitro models by using cPLA2 containing a S505A mutation [16, 22]. However, the mechanism whereby Ser505 phosphorylation regulates cPLA2 function has been elusive. In vitro studies have demonstrated that dephosphorylated cPLA2 is catalytically active and that Ser505 phosphorylation increases activity by only ~30 percent . In contrast, cells expressing the cPLA2 S505A mutation fail to release AA in response to a low dose of calcium ionophore, but release similar amounts of AA as cells expressing wild-type cPLA2 in response to high dose ionophore . From these studies, it has been suggested that cPLA2 Ser505 phosphorylation may have a role in regulating translocation . A previous study demonstrated translocation of cPLA2 S505A in response to Ca2+ ionophore, but did not address the kinetics of translocation, translocation in response to a physiological agonist, or differences in targeting .
To better understand the regulation of cPLA2 by the MEK1/ERK pathway and Ca2+, we investigated the effect of MEK inhibitors on AA release, cPLA2 phosphorylation of Ser505, cPLA2 translocation kinetics, and [Ca2+]i increase in Madin-Darby canine kidney (MDCK) cells. We found that inhibition of MEK1 by U0126 significantly inhibited AA release and this was correlated with inhibition of ERK activation. However, MEK inhibition only partially affected cPLA2 phosphorylation and had no effect on the kinetics of Ca2+-mediated cPLA2 translocation to membrane. In addition, using cells expressing wild-type cPLA2 and cPLA2 with S505A or S727A mutations, it was found that translocation kinetics and membrane targeting in response to ATP or ionomycin was similar to wild-type cPLA2. These data suggest that MEK1 inhibition reduces cPLA2 catalytic activity and AA release independently of phosphorylation and translocation.
Effect of MEK inhibition on AA release, ERK activation, and cPLA2 Ser505 phosphorylation
Inhibition of AA release by 10 μM U0126 See "Experimental Procedures" for experimental conditions and methods to determine AA release. Percent inhibition of AA release after 3 min of agonist treatment are average ± S.D. for N experiments preformed in duplicate.
100 μM ATP
76.1 ± 6.8
1 μM IONO
80.2 ± 5.9
10 μM IONO
72.2 ± 7.3
The effect of MEK1 inhibition on activation of p42/p44 ERK measured by immunoblot analysis using phospho-specific antibodies in cells treated with U0126 and stimulated as above was determined (Fig. 1B). Work in our laboratory has shown that recognition of ERK by anti-phospho-ERK antibodies correlates with an increase in ERK activity [21, 30, 31]. Interestingly, the anti-phospho-ERK immunoblots revealed that ERKs were constitutively activated in untreated, quiesced MDCK cells and activation was not enhanced further by ATP or IONO (Fig. 1B, left panel). ERK activation was diminished by increasing concentrations of U0126 and was quantitatively inhibited after 15 min incubation in 10 μM U0126. U0126 decreased ERK activation following ATP or IONO stimulation in the same fashion as in unstimulated cells. Consequently, there was a direct correlation between the reduction of AA release (Fig. 1A) and inhibition of ERK activation (Fig. 1B) in MDCK cells treated with U0126.
Because cPLA2 is a target of the MEK1/ERK signaling cascade, we assayed the effect of MEK1 inhibition by U0126 on cPLA2 phosphorylation by analyzing gel shift of cPLA2. Phosphorylation of Ser505 results in a retardation of its electrophoretic mobility (gel shift) [13, 16]. In unstimulated cells, EGFP-tagged and endogenous cPLA2 were nearly completely gel shifted, indicating that most cPLA2 was phosphorylated on Ser505 (Fig. 1C), which is consistent with the observation that ERKs are constitutively activated. Incubation with U0126 resulted in a partial reversal of the gel shift although, at 10 μM U0126, approximately half of cPLA2 remained phosphorylated on Ser505. Thus, unlike the quantitative effect of U0126 on ERK activation, inhibition of MEK1 with U0126 only partially reversed the gel shift of cPLA2. The reversal of the gel shift was similar in cells treated with ATP and 1 and 10 μM IONO. Due to the increased molecular weight of the EGFP-tagged cPLA2, the two forms of cPLA2 did not separate as well, making the gel shift more difficult to visualize, but generally mirrored the gel shift characteristics of the endogenous cPLA2. These results suggest that, in response to MEK1 inhibition, there is a quantitative, dose-dependent decrease in AA release that correlates well with the loss of ERK activation, but not with the extent of cPLA2 Ser505 phosphorylation.
[Ca2+]i increase is independent of MEK1/ERK pathway
cPLA2 translocation is independent of MEK1/ERK pathway
Translocation of phosphorylation site mutants S505A or S727A is similar as wild-type cPLA2
The MEK1/ERK pathway regulates cPLA2 and ERKs phosphorylate cPLA2 on Ser505. The results of this study demonstrate that this pathway is required for cPLA2-mediated AA release independent of Ser505 phosphorylation and extend our previous work in macrophages  by demonstrating that this alternative role of the MEK1/ERK pathway is not involved in regulating [Ca2+]i change or cPLA2 translocation kinetics or targeting, but is required for optimal hydrolytic activity and AA release.
The results shown here demonstrate that inhibition of MEK with U0126 quantitatively inhibits both ERK phosphorylation and AA release in MDCK cells in response to [Ca2+]i mobilization. The MEK inhibitor PD098059, a less potent inhibitor [28, 29], also inhibited ATP- and IONO-induced AA release (data not shown). ERK was found to be constitutively phosphorylated in our study using MDCK cells from ATCC, in contrast to what has been reported previously for MDCK-D1, a subclone of MDCK selected for adrenergic receptor expression [39, 40]. However, a side-by-side comparison of MDCK cells from ATCC and the MDCK-D1 subclone (kindly provided by Dr. Paul Insel, UCSD) demonstrated constitutive activation of ERKs when both are grown at low density (not shown). However, at high density ERKs are less active and can be further activated by phorbol ester (not shown).
In MDCK-D1 cells, without constitutively active ERKs, AA release is delayed after [Ca2+]i mobilization, and is temporally correlated with ERK activation , whereas in MDCK cells with constitutively active ERKs, AA release is rapid, with significant AA release measured 30 s after [Ca2+]i increase . This temporal correlation between AA release and ERK activation has also been reported in CHO cells in response to PAF stimulation [35, 41]. These results support the observations made here that ERK activity is required for phospholipid hydrolysis independently of cPLA2 translocation.
cPLA2-mediated AA release must be preceded by translocation of the enzyme to its membrane substrate which is a Ca2+-dependent process and is a function of the calcium-dependent lipid-binding (C2) domain. cPLA2 translocates primarily to Golgi in response to a transient [Ca2+]i changes and to Golgi and ER in response to a sustained [Ca2+]i increase . The reduction in AA release by MEK inhibition did not involve a failure in [Ca2+]i release or translocation. These results show that translocation is necessary but not sufficient for optimum hydrolytic activity.
Measuring cPLA2 translocation is not a trivial matter when investigating mechanisms of AA release. For example, cPLA2 constructs with a S505A mutation have long been recognized as unable to support AA release in response to physiological agonists or low-dose ionophore when transfected in cells. In contrast, cPLA2 S505A is active in vitro and phosphorylation only modestly increases the activity of the enzyme [16, 17, 24]. Interestingly, the inhibitory effect of the S505A mutation on AA release is obviated by a high [Ca2+]i increase. In light of these observations, it is possible that Ser505 phosphorylation may alter the [Ca2+]i sensitivity of the enzyme, its rate of translocation, its intracellular targeting or, as has been previously suggested , the ability of cPLA2 to release from a non-membrane sequestering agent. Although one report has shown that cPLA2 S505A translocates in CHO cells in response to ionophore, we were able to directly compare rates of translocation between cPLA2 and cPLA2 S505A to a physiological agonist and found no difference between translocation rates or intracellular targeting. We also demonstrated that there was no difference in translocation rates or intracellular targeting between wild-type cPLA2 and cPLA2 S727A, which has the same phenotype as S505A with regard to AA release when transfected into cells. Thus, the role of cPLA2 phosphorylation in mediating AA release remains unclear.
Although the alternative mechanism whereby the MEK1/ERK pathway regulates cPLA2 is not known, it is possible that it affects membrane properties and/or cPLA2 conformation that promotes optimal hydrolytic activity. It is also possible that the alternative mechanism is due to phosphorylation of cPLA2 on a novel site by a kinase that is downstream of the MEK1/ERK pathway or phosphorylation of a regulatory protein.
Translocation to membrane is a critical regulatory step for the action of cPLA2 because it is necessary for access to substrate. In this study we demonstrate, however, that association of cPLA2 with membrane when phosphorylated on Ser505 is not sufficient for its full activity in vivo. This is demonstrated by the results showing that inhibition of the MEK1/ERK pathway significantly blocks AA release but has no effect on [Ca2+]i mobilization or cPLA2 translocation and targeting. Diminution of AA release by MEK1/ERK is also independent of cPLA2 phosphorylation on Ser505. Consequently, our results demonstrate in living cells that the translocation process and subsequent catalytic activity on the membrane are two independently regulated steps.
Materials and Methods
Fluorescent protein-cPLA2 fusion constructs
DNA encoding the full-length human cPLA2 was cloned into the vector pEGFP-C3 (Clontech) to create pEGFP-cPLA2, as previously described . The XbaI/PstI fragment from a cPLA2α clone containing S505A or S727A mutations  was inserted into an XbaI/PstI site in pEGFP-cPLA2 to generate pEGFP-cPLA2S505A and pECFP-cPLA2S727A. Different fluorescent-protein tagged constructs were produced by exchanging the NheI/BsrGI fragment containing the fluorescent protein coding sequence between EGFP, EYFP, and ECFP. All constructs were confirmed by sequencing.
MDCK cells obtained from ATCC were cultured in DMEM containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.292 mg/ml glutamine (growth medium) in 5% CO2 at 37°C. Subconfluent cells (5 × 103 cells/cm2) were transfected with 2 μg of the relevant plasmid using Fugene-6 (Boehringer Mannheim) in DMEM containing 0.2% BSA, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.292 mg/ml glutamine (serum-free medium) following the manufacturer's protocol. Stable lines expressing EGFP-cPLA2 were generated by growing transfected cells in growth medium for 3 d, supplementing the growth medium with 5 mg/ml Geneticin (antibiotic G418-sulfate), and culturing for an additional 2 wk in Geneticin. Cells expressing EGFP fluorescence were selected using a fluorescence-activated cell sorter. The EGFP-positive cells were maintained in growth medium supplemented with 5 mg/ml Geneticin. For imaging studies, MDCK cells were plated on glass-bottomed 35 mm culture dishes (MatTek) at 5 × 103 cells/cm2 in growth medium and incubated overnight, transfected with the relevant plasmid(s), changed into serum-free medium to quiesce the cells, incubated overnight, and used the next day.
Stable EGFP-cPLA2 transfectants were grown on 100 mm dishes at 5 × 103 cells/cm2 in growth medium for one day, then quiesced in serum-free medium overnight. Cells were scraped into ice-cold lysis buffer: 50 mM HEPES, pH 7.4, 150 mM sodium chloride, 1.5 mM magnesium chloride, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 200 μM sodium vanadate, 10 mM tetrasodium pyrophosphate, 100 mM sodium fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Lysates were centrifuged at 15,000 × g for 15 min, and protein concentration of the supernatant was determined by the bicinchoninic acid method. Laemmli electrophoresis sample buffer (5×) was added to the lysates, and SDS-polyacrylamide gel electrophoresis and immunoblotting were performed using 35 μg lysate protein, phospho-specific antibodies for ERK and p38, and rabbit polyclonal antibody for cPLA2.
Dual imaging microscopy of fluorescent protein translocation
In order to compare the characteristics of full-length cPLA2 and cPLA2S505A or cPLA2S727A translocation, while controlling for cell-to-cell heterogeneity, we used a dual CFP/YFP imaging approach. EYFP-cPLA2/ECFP-cPLA2S505A- or pECFP-cPLA2S727A-transfected MDCK cells grown on MatTek plates were quiesced overnight in serum-free medium, washed with and incubated in Hank's balanced salt solution (HBSS) additionally buffered with 25 mM HEPES pH 7.4 (HHBSS). Cells were imaged using an Olympus inverted microscope equipped with a 60×, 1.25 NA oil immersion objective, CFP and YFP emission filters (Chroma) in a Sutter filter wheel, a dual CFP/YFP dichroic mirror, and a TILL Imago CCD camera (TILL Photonics). Excitation light of 430 and 510 nm for CFP and YFP, respectively, was provided using a Polychrome IV monochromator (TILL Photonics). TILLvisION software was used for acquisition and analysis. Bleach values for ECFP and EYFP were calculated by determining the background-corrected fluorescence for the entire cell with respect to time and normalizing each value to the initial value. ECFP/EYFP fluorescence changes with respect to time for regions of interest corresponding to an area of Golgi membrane were determined by calculating the Ft/F0, where Ft is the background- and bleach-corrected ECFP or EYFP fluorescence at time = t and F0 is the background-corrected ECFP or EYFP fluorescence at time = 0 s. Fluorescence was normalized to the F0 value, which resulted in Ft/F0 representing the fraction of total cell fluorescence at Golgi. Final images were produced using Adobe Photoshop.
MDCK cells grown on MatTek plates were quiesced overnight in serum-free medium, washed with HHBSS containing 1 mM probenecid and incubated with 5 μM Fura2-AM (Calbiochem) in HHBSS, 1 mM probenecid, and 1% DMSO for 45 min at 37°C. Cells were then washed with HHBSS containing 1 mM probenecid and imaged after a 30 min incubation for de-esterification of the Fura2-AM. Single-cell imaging was performed on the Olympus system described above, but using a 40×, 1.35 NA oil immersion objective and a Fura2 dichroic mirror and emission filter (Chroma). Fura2 image pairs illuminated at 340 and 380 nm were taken at 1 Hz. The [Ca2+]i increase is expressed as the ratio of the background-corrected Fura2 fluorescence at 340 and 380 nm .
Measurement of AA release
The protocol for determining AA release is essentially as described [15, 21]. MDCK cells stably expressing EGFP-cPLA2 were plated in 12-well plates at 5 × 103 cells/cm2 and incubated in growth medium overnight. Cells were then washed twice with serum-free medium and incubated with 0.25 μCi [3H]-AA/well in serum-free medium overnight. U0126 or vehicle was added to each well and the cells were then incubated for 15 min at 37°C in 5% CO2. Cells were washed to remove unincorporated [3H]-AA and then incubated in HHBSS supplemented with 0.05% BSA with either U0126 or vehicle. Cells were stimulated with the agonist of choice and the medium was collected at appropriate time points. The medium was centrifuged at 500 g for 5 min, and the amount of radioactivity in the supernatant was determined by scintillation counting. Cells were scraped in 0.5 ml 0.1% Triton X-100 for determining the total cellular radioactivity.
This work was supported by National Institutes of Health grants HL61378 and HL34303 (to C.C.L.), an Individual National Research Service Award (HL10507), and an Aronsen Fellowship in Pediatric Pulmonary Medicine from National Jewish Medical and Research Center (to J.H.E.).
- Sapirstein A, Bonventre JV: Specific physiological roles of cytosolic phospholipase A2 as defined by gene knockouts. Biochim Biophys Acta. 2000, 1488: 139-48. 10.1016/S1388-1981(00)00116-5.PubMedView ArticleGoogle Scholar
- Bonventre JV: Phospholipase A2 and signal transduction. J Am Soc Nephrol. 1992, 3: 128-150.PubMedGoogle Scholar
- Majerus PW: Prostaglandins: critical roles in pregnancy and colon cancer. Curr Biol. 1998, 8: R87-9.PubMedView ArticleGoogle Scholar
- Breyer MD, Breyer RM: Prostaglandin E receptors and the kidney. Am J Physiol Renal Physiol. 2000, 279: F12-23.PubMedGoogle Scholar
- Breyer MD, Harris RC: Cyclooxygenase 2 and the kidney. Curr Opin Nephrol Hypertens. 2001, 10: 89-98. 10.1097/00041552-200101000-00014.PubMedView ArticleGoogle Scholar
- Hirabayashi T, Shimizu T: Localization and regulation of cytosolic phospholipase A2. Biochim Biophys Acta. 2000, 1488: 124-38. 10.1016/S1388-1981(00)00115-3.PubMedView ArticleGoogle Scholar
- Leslie CC: Properties and regulation of cytosolic phospholipase A2. J Biol Chem. 1997, 272: 16709-16712. 10.1074/jbc.272.27.16709.PubMedView ArticleGoogle Scholar
- Clark JD, Schievella AR, Nalefski EA, Lin L-L: Cytosolic phospholipase A2. J Lipid Mediat Cell Signal. 1995, 12: 83-117. 10.1016/0929-7855(95)00012-F.PubMedView ArticleGoogle Scholar
- Dessen A, Tang J, Schmidt H, Stahl M, Clark JD, Seehra J, Somers WS: Crystal structure of human cytosolic phospholipase A2 reveals a novel topology and catalytic mechanism. Cell. 1999, 97: 349-360.PubMedView ArticleGoogle Scholar
- Nalefski EA, Sultzman LA, Martin DM, Kriz RW, Towler PS, Knopf JL, Clark JD: 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.PubMedGoogle Scholar
- Ball A, Nielsen R, Gelb MH, Robinson BH: Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance. Proc Natl Acad Sci U S A. 1999, 96: 6637-6642. 10.1073/pnas.96.12.6637.PubMed CentralPubMedView ArticleGoogle Scholar
- Xu G-Y, McDonagh T, Hsiang-Ai Y, Nalefski EA, Clark JD, Cumming DA: Solution structure and membrane interactions of the C2 domain of cytosolic phospholipase A2. J Mol Biol. 1998, 280: 485-500. 10.1006/jmbi.1998.1874.PubMedView ArticleGoogle Scholar
- Gijón MA, Spencer DM, Kaiser AL, Leslie CC: Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2. J Cell Biol. 1999, 145: 1219-1232. 10.1083/jcb.145.6.1219.PubMed CentralPubMedView ArticleGoogle Scholar
- Perisic O, Paterson HF, Mosedale G, Lara-González S, Williams RL: Mapping the phospholipid-binding surface and translocation determinants of the C2 domain from cytosolic phospholipase A2. J Biol Chem. 1999, 274: 14979-14987. 10.1074/jbc.274.21.14979.PubMedView ArticleGoogle Scholar
- Evans JH, Spencer DM, Zweifach A, Leslie CC: Intracellular calcium signals regulating cytosolic phospholipase A2 translocation to internal membranes. J Biol Chem. 2001, 276: 30150-30160. 10.1074/jbc.M100943200.PubMedView ArticleGoogle Scholar
- Lin L-L, Wartmann M, Lin AY, Knopf JL, Seth A, Davis RJ: cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993, 72: 269-278.PubMedView ArticleGoogle Scholar
- Nemenoff RA, Winitz S, Qian N-X, Van Putten V, Johnson GL, Heasley LE: 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.PubMedGoogle Scholar
- Kramer RM, Roberts EF, Strifler BA, Johnstone EM: Thrombin induces activation of p38 MAP kinase in human platelets. J Biol Chem. 1995, 270: 27395-27398. 10.1074/jbc.270.43.25885.PubMedView ArticleGoogle Scholar
- Kramer RM, Roberts EF, Um SL, Börsch-Haubold AG, Watson SP, Fisher MJ, Jakubowski JA: p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. J Biol Chem. 1996, 271: 27723-27729. 10.1074/jbc.271.37.22915.PubMedView ArticleGoogle Scholar
- Lin L-L, Lin AY, Knopf JL: Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc Natl Acad Sci USA. 1992, 89: 6147-6151.PubMed CentralPubMedView ArticleGoogle Scholar
- Gijon MA, Spencer DM, Siddiqi AR, Bonventre JV, Leslie CC: 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. 10.1074/jbc.M908941199.PubMedView ArticleGoogle Scholar
- Hefner Y, Borsch-Haubold AG, Murakami M, Wilde JI, Pasquet S, Schieltz D, Ghomashchi F, Yates JR, Armstrong CG, Paterson A: Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases. J Biol Chem. 2000, 275: 37542-51. 10.1074/jbc.M003395200.PubMedView ArticleGoogle Scholar
- Muthalif MM, Hefner Y, Canaan S, Harper J, Zhou H, Parmentier JH, Aebersold R, Gelb MH, Malik KU: Functional interaction of calcium-/calmodulin-dependent protein kinase II and cytosolic phospholipase A2. J Biol Chem. 2001, 276: 39653-60. 10.1074/jbc.M103136200.PubMedView ArticleGoogle Scholar
- Bayburt T, Gelb MH: Interfacial catalysis by human 85 kDa cytosolic phospholipase A2 on anionic vesicles in the scooting mode. Biochemistry. 1997, 36: 3216-3231. 10.1021/bi961659d.PubMedView ArticleGoogle Scholar
- Schievella AR, Regier MK, Smith WL, Lin L-L: Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum. J Biol Chem. 1995, 270: 30749-30754. 10.1074/jbc.270.51.30749.PubMedView ArticleGoogle Scholar
- Stewart A, Ghosh M, Spencer DM, Leslie CC: Enzymatic properties of human cytosolic phospholipase A2 gamma. J Biol Chem. 2002, 277: 29526-36. 10.1074/jbc.M204856200.PubMedView ArticleGoogle Scholar
- Ghomashchi F, Stewart A, Hefner Y, Ramanadham S, Turk J, Leslie CC, Gelb MH: A pyrrolidine-based specific inhibitor of cytosolic phospholipase A2 alpha blocks arachidonic acid release in a variety of mammalian cells. Biochim Biophys Acta. 2001, 1513: 160-6. 10.1016/S0005-2736(01)00349-2.PubMedView ArticleGoogle Scholar
- Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F: Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem. 1998, 273: 18623-18632. 10.1074/jbc.273.29.18623.PubMedView ArticleGoogle Scholar
- Davies SP, Reddy H, Caivano M, Cohen P: Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000, 351: 95-105. 10.1042/0264-6021:3510095.PubMed CentralPubMedView ArticleGoogle Scholar
- Qiu Z-H, Leslie CC: Protein kinase C-dependent and -independent pathways of mitogen-activated protein kinase activation in macrophages by stimuli that activate phospholipase A2. J Biol Chem. 1994, 269: 19480-19487.PubMedGoogle Scholar
- Qiu Z-H, Gijón MA, de Carvalho MS, Spencer DM, Leslie CC: The role of calcium and phosphorylation of cytosolic phospholipase A2 in regulating arachidonic acid release in macrophages. J Biol Chem. 1998, 273: 8203-8211. 10.1074/jbc.273.14.8203.PubMedView ArticleGoogle Scholar
- Cano E, Hazzalin CA, Mahadevan LC: Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun. Mol Cell Biol. 1994, 14: 7352-7362.PubMed CentralPubMedView ArticleGoogle Scholar
- Magne S, Couchie D, Pecker F, Pavoine C: Beta(2)-adrenergic receptor agonists increase intracellular free Ca2+ concentration cycling in ventricular cardiomyocytes through p38 and p42/44 MAPK-mediated cytosolic phospholipase A2 activation. J Biol Chem. 2001, 276: 39539-48. 10.1074/jbc.M100954200.PubMedView ArticleGoogle Scholar
- Xing M, Firestein BL, Shen GH, Insel PA: Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and -independent pathways. J Clin Invest. 1997, 99: 805-814.PubMed CentralPubMedView ArticleGoogle Scholar
- 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 of cytosolic phospholipase A2. J Biol Chem. 1999, 274: 5163-5169. 10.1074/jbc.274.8.5163.PubMedView ArticleGoogle Scholar
- Glover S, de Carvalho MS, Bayburt T, Jonas M, Chi E, Leslie CC, Gelb MH: Translocation of the 85-kDa phospholipase A2 from cytosol to the nuclear envelope in rat basophilic leukemia cells stimulated with calcium ionophore or IgE/antigen. J Biol Chem. 1995, 270: 15359-15367. 10.1074/jbc.270.25.15359.PubMedView ArticleGoogle Scholar
- Nakatani Y, Tanioka T, Sunaga S, Murakami M, Kudo I: Identification of a cellular protein that functionally interacts with the C2 domain of cytosolic phospholipase A2 alpha. J Biol Chem. 2000, 275: 1161-1168. 10.1074/jbc.275.2.1161.PubMedView ArticleGoogle Scholar
- Das S, Cho W: Roles of catalytic domain residues in interfacial binding and activation of group IV cytosolic phospholipase A2. J Biol Chem. 2002, 277: 23838-46. 10.1074/jbc.M202322200.PubMedView ArticleGoogle Scholar
- Meier KE, Sperling DM, Insel PA: Agonist-mediated regulation of alpha 1- and beta 2-adrenergic receptors in cloned MDCK cells. Am J Physiol. 1985, 249: C69-77.PubMedGoogle Scholar
- Xing M, Insel PA: Protein kinase C-dependent activation of cytosolic phospholipase A2 and mitogen-activated protein kinase by alpha 1-adrenergic receptors in Madin-Darby canine kidney cells. J Clin Invest. 1996, 97: 1302-1310.PubMed CentralPubMedView ArticleGoogle Scholar
- Hirabayashi T, Kume K, Shimizu T: Conditional expression of the dual-specificity phosphatase PYST1/MKP-3 inhibits phosphorylation of cytosolic phospholipase A2 in Chinese hamster ovary cells. Biochem Biophys Res Commun. 1998, 253: 485-8. 10.1006/bbrc.1998.9807.PubMedView ArticleGoogle Scholar
- Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985, 260: 3440-3450.PubMedGoogle Scholar
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