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• W1992611170 abstract "Cytosolic phospholipase A2 (cPLA2) is activated by phosphorylation at serine-505 (S505) by extracellular regulated kinase 1/2 (ERK1/2). However, rat brain calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates recombinant cPLA2 at serine-515 (S515) and increases its activity in vitro. We have studied the sites of cPLA2 phosphorylation and their significance in arachidonic acid (AA) release in response to norepinephrine (NE) in vivo in rabbit vascular smooth muscle cells (VSMCs) using specific anti-phospho-S515- and -S505 cPLA2 antibodies and by mutagenesis of S515 and S505 to alanine. NE increased the phosphorylation of cPLA2 at S515, followed by phosphorylation of ERK1/2 and consequently phosphorylation of cPLA2 at S505. The CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzene-sulfonyl)]amino-N-(4-chlorocinnamyl)-methylbenzylamine attenuated cPLA2 at S515 and S505, whereas the ERK1/2 inhibitor U0126 reduced phosphorylation at S505 but not at S515. NE in cells transduced with adenovirus carrying enhanced cyan fluorescent protein cPLA2 wild type caused phosphorylation at S515 and S505 and increased AA release. Expression of the S515A mutant in VSMCs reduced the phosphorylation of S505, ERK1/2, and AA release in response to NE. Transduction with a double mutant (S515A/S505A) blocked the phosphorylation of cPLA2 and AA release. These data suggest that the NE-stimulated phosphorylation of cPLA2 at S515 is required for the phosphorylation of S505 by ERK1/2 and that both sites of phosphorylation are important for AA release in VSMCs. Cytosolic phospholipase A2 (cPLA2) is activated by phosphorylation at serine-505 (S505) by extracellular regulated kinase 1/2 (ERK1/2). However, rat brain calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates recombinant cPLA2 at serine-515 (S515) and increases its activity in vitro. We have studied the sites of cPLA2 phosphorylation and their significance in arachidonic acid (AA) release in response to norepinephrine (NE) in vivo in rabbit vascular smooth muscle cells (VSMCs) using specific anti-phospho-S515- and -S505 cPLA2 antibodies and by mutagenesis of S515 and S505 to alanine. NE increased the phosphorylation of cPLA2 at S515, followed by phosphorylation of ERK1/2 and consequently phosphorylation of cPLA2 at S505. The CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzene-sulfonyl)]amino-N-(4-chlorocinnamyl)-methylbenzylamine attenuated cPLA2 at S515 and S505, whereas the ERK1/2 inhibitor U0126 reduced phosphorylation at S505 but not at S515. NE in cells transduced with adenovirus carrying enhanced cyan fluorescent protein cPLA2 wild type caused phosphorylation at S515 and S505 and increased AA release. Expression of the S515A mutant in VSMCs reduced the phosphorylation of S505, ERK1/2, and AA release in response to NE. Transduction with a double mutant (S515A/S505A) blocked the phosphorylation of cPLA2 and AA release. These data suggest that the NE-stimulated phosphorylation of cPLA2 at S515 is required for the phosphorylation of S505 by ERK1/2 and that both sites of phosphorylation are important for AA release in VSMCs. arachidonic acid adenoviral enhanced cyan fluorescent protein cytosolic phospholipase A2 wild type calcium/calmodulin kinase II α cytosolic phospholipase A2 enhanced cyan fluorescent protein extracellular regulated kinase 1/2 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzene-sulfonyl)]amino-N-(4-chlorocinnamyl)-methylbenzylamine multiplicity of infection norepinephrine tissue culture infective dose 50 vascular smooth muscle cell Norepinephrine (NE) released from postganglionic sympathetic fibers stimulates arachidonic acid (AA) release for the synthesis of prostaglandins, which, in turn, act as a physiological modulator of neurotransmitter release in various tissues, including blood vessels (1.Malik K.U. Interaction of arachidonic acid metabolites and adrenergic nervous system.Am. J. Med. Sci. 1988; 31: 280-286Crossref Scopus (32) Google Scholar). In vascular smooth muscle cells (VSMCs), NE stimulates the release of AA via the α1-adrenergic receptor by the activation of cytosolic phospholipase A2 (cPLA2) (2.Nebigil C. Malik K.U. Comparison of signal transduction mechanisms of alpha-2C and alpha-1A adrenergic receptor-stimulated prostaglandin synthesis.J. Pharmacol. Exp. Ther. 1992; 263: 987-996PubMed Google Scholar, 3.Muthalif M.M. Benter I.F. Uddin M.R. Malik K.U. Calcium/calmodulin-dependent protein kinase II alpha mediates activation of mitogen-activated protein kinase and cytosolic phospholipase A2 in norepinephrine-induced arachidonic acid release in rabbit aortic smooth muscle cells.J. Biol. Chem. 1996; 271: 30149-30157Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), a group IV phospholipase A2 member (4.Schaloske R.H. Dennis E.A. The phospholipase A2 superfamily and its group numbering system.Biochim. Biophys. Acta. 2006; 176: 1246-1259Crossref Scopus (727) Google Scholar). NE-induced cPLA2 activation and AA release require calcium (Ca2+), calmodulin (CaM), and calmodulin-dependent kinase II (CaMKII) in VSMCs (2.Nebigil C. Malik K.U. Comparison of signal transduction mechanisms of alpha-2C and alpha-1A adrenergic receptor-stimulated prostaglandin synthesis.J. Pharmacol. Exp. Ther. 1992; 263: 987-996PubMed Google Scholar, 3.Muthalif M.M. Benter I.F. Uddin M.R. Malik K.U. Calcium/calmodulin-dependent protein kinase II alpha mediates activation of mitogen-activated protein kinase and cytosolic phospholipase A2 in norepinephrine-induced arachidonic acid release in rabbit aortic smooth muscle cells.J. Biol. Chem. 1996; 271: 30149-30157Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). cPLA2 activity has been reported to be regulated by phosphorylation by extracellular regulated kinase 1/2 (ERK1/2), p42/p44 mitogen-activated protein kinase, and Ca2+-dependent translocation to the nuclear envelope, allowing its access to arachidonyl-containing phospholipid substrate at the sn-2 position (6.Leslie C.C. Properties and regulation of cytosolic phospholipase A2.J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar). ERK1/2 phosphorylates cPLA2 at serine-505 (S505) in several cell types (5.Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. cPLA2 is phosphorylated and activated by MAP kinase.Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1656) Google Scholar, 6.Leslie C.C. Properties and regulation of cytosolic phospholipase A2.J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar, 7.Evans J.H. Fergus D.J. Leslie C.C. Inhibition of the MEK1/ERK pathway reduces arachidonic acid release independently of cPLA2 phosphorylation and translocation.BMC Biochem. 2002; 3: 30Crossref PubMed Scopus (42) Google Scholar). However, the phosphorylation and activation of cPLA2 by a mechanism independent of ERK1/2 has also been reported (8.Qiu Z.H. Leslie C.C. 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: 19480-19487Abstract Full Text PDF PubMed Google Scholar) and may be mediated by other members of the mitogen-activated protein kinase family, such as p38 stress-activated protein kinase (9.Murakami M. Hot topics in phospholipase A2 field.Biol. Pharm. Bull. 2004; 27: 1179-1182Crossref PubMed Scopus (19) Google Scholar, 10.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: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). The existence of multiple phosphorylation sites on cPLA2 (S431, S454, S505, and S727) suggested cPLA2 as a substrate for other kinases (11.de Carvalho M.G. McCormack A.L. Olson E. Ghomashchi F. Gelb M.H. Yates J.R. Leslie C.C. Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A2 expressed in insect cells and present in human monocytes.J. Biol. Chem. 1996; 271: 6987-6997Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Studies in platelets, HeLa cells, and CHO cells have shown that cPLA2 is phosphorylated on S505 and S727 (12.Hefner Y. Börsch-Haubold A.G. Murakami M. Wilde J.I. Pasquet S. Schieltz D. Ghomaschchi F. Yates 3rd, J.R. Armstrong C.J. Paterson A. et al.Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases.J. Biol. Chem. 2000; 275: 37542-37551Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). S727 is phosphorylated by MNK1 or a closely related isoform, a protein kinase that is activated by members of the mitogen-activated protein kinase family, and phosphorylation of both S505 and S727 is required for the full activation of cPLA2 (13.Borsch-Haubold A.G. Bartoli F. Asselin J. Dudler T. Kramer R.M. Apitz-Castro R. Watson S.P. Gelb M.H. Identification of the phosphorylation sites of cytosolic phospholipase A2 in agonist-stimulated human platelets and HeLa cells.J. Biol. Chem. 1998; 273: 4449-4458Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). However, it has been shown that cPLA2 phosphorylation is insufficient for its activation; an increase in intracellular Ca2+ is also required, and Ca2+-independent cPLA2 activation by an unknown mechanism has also been reported (14.Gosh M. Tucker D.E. Burchett S.A. Leslie C.C. Properties of the group IV phospholipase A2 family.Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (308) Google Scholar). Studies conducted in rabbit and rat VSMCs have shown that the activity of ERK1/2 in response to agonists such as NE, angiotensin II, or ionomycin is regulated in part by Ca2+/CaMKII and that both CaMKII and ERK1/2 are involved in cPLA2 activation (3.Muthalif M.M. Benter I.F. Uddin M.R. Malik K.U. Calcium/calmodulin-dependent protein kinase II alpha mediates activation of mitogen-activated protein kinase and cytosolic phospholipase A2 in norepinephrine-induced arachidonic acid release in rabbit aortic smooth muscle cells.J. Biol. Chem. 1996; 271: 30149-30157Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 15.Muthalif M.M. Benter I.F. Karzoun N. Fatima S. Harper J. Uddin M.R. Malik K.U. 20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells.Proc. Natl. Acad. Sci. USA. 1998; 95: 12701-12706Crossref PubMed Scopus (185) Google Scholar, 16.Abraham S.T. Benscoter H.A. Schworer C.M. Singer H.A. A role for Ca2+ calmodulin-dependent protein kinase II in the mitogen-activated protein kinase signaling cascade of cultured rat aortic vascular smooth muscle cells.Circ. Res. 1997; 81: 575-584Crossref PubMed Scopus (100) Google Scholar, 17.Muthalif M.M. Benter I.F. Uddin M.R. Harper J.L. Malik K.U. Signal transduction mechanisms involved in angiotensin-(1-7)-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells.J. Pharmacol. Exp. Ther. 1998; 284: 388-398PubMed Google Scholar). Further studies revealed that NE-stimulated CaMKII activates cPLA2 and releases AA and that oxygenated metabolites of AA generated via lipoxygenase and cytochrome P450 stimulate ERK1/2 and amplify cPLA2 activity and release additional AA (15.Muthalif M.M. Benter I.F. Karzoun N. Fatima S. Harper J. Uddin M.R. Malik K.U. 20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells.Proc. Natl. Acad. Sci. USA. 1998; 95: 12701-12706Crossref PubMed Scopus (185) Google Scholar). In vitro surface plasmon resonance, mass spectrometric, and kinetic studies showed that CaMKII binds directly to cPLA2 and phosphorylates it on S515 and increases its enzymatic activity (18.Muthalif M.M. Hefner Y. Canaan S. Harper J. Zhou H. Parmentier J.H. Aebersold R. Gelb M. Malik K.U. Functional interaction of calcium-/calmodulin-dependent protein kinase II and cytosolic phospholipase A(2).J. Biol. Chem. 2001; 276: 39653-39660Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Whether NE promotes cPLA2 phosphorylation on S515 in VSMCs, and whether phosphorylation on S515 mediates ERK1/2 activation and phosphorylation on S505 for AA release in vivo, are not known. Therefore, to assess the significance of cPLA2 phosphorylation by CaMKII on S515 in its activation and its effect on S505 phosphorylation by ERK1/2 and AA release, we examined the expression, distribution, and activation of adenoviral constructs of wild-type cPLA2 and those mutated on S505, S515, and S505/515 to alanine and fused with the enhanced cyan fluorescent protein (ECFP) at the N-terminal end of cPLA2 and AA release in rabbit VSMCs. Our results indicate that NE stimulates phosphorylation on S515 and the activation of endogenous cPLA2 and exogenously expressed enhanced cyan fluorescent protein cytosolic phospholipase A2 wild type (ECFPcPLA2 wt). Moreover, phosphorylation of cPLA2 on S505 is dependent on its phosphorylation at S515 elicited by CaMKII, and both sites of phosphorylation are required for AA release in response to NE in VSMCs. Arterenol NE and ampicillin were from Sigma (St. Louis, MO); the CaMKII inhibitor 2-[N-(2-hydroxyethyl)]N-(4-methoxybenzene-sulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-93) and its inactive analog 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-92) were from Calbiochem (San Diego, CA). [3H]AA (0.1 μCi/ml) was from ARC, Inc. (St. Louis, MO); agarose was from Invitrogen-Life Technologies (Gaithersburg, MD); FuGENE 6 transfection reagent was from Roche (Indianapolis, IN); and HEK293 cells were obtained from the American Type Culture Collection (Manassas, VA). Male New Zealand White rabbits (1–2 kg) were anesthetized with 30 mg/kg pentobarbital (Abbott Laboratories, North Chicago, IL), and the thorax and abdomen were opened by a midline incision. The aorta was rapidly removed, and VSMCs were isolated as described previously (2.Nebigil C. Malik K.U. Comparison of signal transduction mechanisms of alpha-2C and alpha-1A adrenergic receptor-stimulated prostaglandin synthesis.J. Pharmacol. Exp. Ther. 1992; 263: 987-996PubMed Google Scholar). Cells between the fourth and eighth passages were cultured in 6- or 24-well, or 60 or 100 mm, plates for experiments. Cells were maintained under 5% CO2 in M-199 medium (Sigma Aldrich, St. Louis, MO) containing 10% FBS, 1% penicillin/streptomycin, and 0.1% amphotericin B. Cells were cultured in Dulbecco's modified Eagle's medium that was supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 μg/ml streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was changed three times per week, and the cells were passaged two times per week. Cells were detached from the culture flask by adding 0.25% trypsin and 2.21 mM EDTA in HBSS. Transient transfection by liposomic reagent usually leads to a low level of transfection and variable levels of expression from cell to cell as well as to nonphysiological levels of expression in some cells (19.Han W.K. Sapirstein A. Hung C.C. Alessandrini A. Bonventre J.V. Cross-talk between cytosolic phospholipase A2 alpha (cPLA2 alpha) and secretory phospholipase A2 (sPLA2) in hydrogen peroxide-induced arachidonic acid release in murine mesangial cells: sPLA2 regulates cPLA2 alpha activity that is responsible for arachidonic acid release.J. Biol. Chem. 2003; 278: 24153-24163Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Therefore, we used adenovirus carrying ECFPcPLA2 wt and its mutants. Plasmids carrying ECFPcPLA2 wt and its mutants S515A, S505A, and S515A/S505A were prepared as described (20.Anderson R.D. Haskell R.E. Xia H. Roessler B.J. Davidson B.L. A simple method for the rapid generation of recombinant adenovirus vectors.Gene Ther. 2000; 7: 1034-1038Crossref PubMed Scopus (225) Google Scholar, 21.Mittereder N. March K.L. Trapnell B.C. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.J. Virol. 1996; 70: 7498-7509Crossref PubMed Google Scholar). These plasmids contained coding sequence (methionine-1 to alanine-749) of the human cPLA2 gene (GenBank accession number M72393). This gene was inserted into multiple cloning sites HindIII/SmaI of pECFP-C3 plasmid (Clontech, Mountain View, CA) so that fluorescent tag ECFP was at the N terminus of cPLA2. Two restriction sites were chosen for subcloning into adenoviral shuttle vector pacAd5CMV (obtained from the University of Iowa, Iowa Viral Vector Facility, Iowa City): 5′ end Eco47III isoschizomer AfeI, and SmaI at the 3′ end; this restriction digest contained the sequence of ECFPcPLA2 wt. Adenoviral pacAd5CMV vector was linearized using the EcoRV site in multiple cloning sites of the vector. Ligation reaction of the ECFPcPLA2 wt fragment and adenoviral pacAd5CMV vector was made using the Ready-To-Go T4 DNA Ligase kit (Amersham Pharmacia Biotech, Piscataway, NJ). The ligation product was transformed into chemically competent DH5α cells according to the manufacturer's instructions (Invitrogen, Carlsbad, CA) and grown on agar plates containing ampicillin. Single colonies were picked, and adenoviral pshuttle vector containing ECFPcPLA2 wt was isolated using the Maxi-prep kit (Qiagen, Valencia, CA). The new construct was confirmed by digestion with a unique PacI restriction site that gave a band of 10 kb on 1% agarose gels and by sequencing. Mutants of ECFPcPLA2 (505, 515, and 505/515) were made using ECFPcPLA2 wt inserted in pacAd5CMV vector as the template and appropriate primers [for mutation of S505 to alanine (S505A), forward primer 5′-CATCTTATCCACTGGCTCCTTTGAGTGAC-3′ and reverse primer 5′-GTCACTCAAAGGAGCCAGTGGATAAGATG-3′; for mutation of S515 to alanine (S515A), forward primer 5′-GACTTTGCCACACAGGACGCCTTTGATGATGATGAACTG-3′ and reverse primer 5′-CAGTTCATCATCATCAAAGGCGTCCTGTGTGGCAAAGTC-3′ (IDT, Inc., Caralville, IA)] for each mutant and the Quick-Change XL Site-Directed Mutagenesis Kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). To make the double S505A/S515A mutant, single mutants were used as the templates in reactions of Quick-Change XL site-directed mutagenesis. ECFPcPLA2 wt and its mutants S505A, S515A, and S505A/S515A inserted in pacAd5CMV were cotransfected with adenoviral ΔE1 backbone vector into low-passage HEK293 cells (American Type Culture Collection) using the FuGENE 6 transfection reagent (Roche, Palo Alto, CA). A cytopathic effect signaled virus formation, and fluorescence measurements were used to check the efficiency of cotransfection. Viruses were amplified in HEK293 cells and purified using ultracentrifugation in a CsCl2 gradient and dialysis in PBS. Viral particle concentration was determined by measuring optical density at 260 nm and by tissue culture infection dose 50 (20.Anderson R.D. Haskell R.E. Xia H. Roessler B.J. Davidson B.L. A simple method for the rapid generation of recombinant adenovirus vectors.Gene Ther. 2000; 7: 1034-1038Crossref PubMed Scopus (225) Google Scholar). Adenoviruses carrying cDNA ECFPcPLA2 wt and its mutants S515A, S505A, and S515A/S505A were transduced in rabbit VSMCs at a concentration of 60 or 120 multiplicity of infection (MOI) per cell. The cells were then incubated with viral particles for 48 h, and expression of ECFPcPLA2 protein was confirmed by fluorescence microscopy (Olympus IX 50) using filter set exciter wavelength 440 nm, dichroic wavelength 455 nm, and emitter wavelength 480 nm at 40× magnification and by Western blot analysis of cell lysates using anti-green fluorescent protein antibody (Santa Cruz Biotechnology, Santa Cruz, CA) that cross-reacts with ECFP and anti-cPLA2 antibody (Santa Cruz Biotechnology). The transduction efficiency of VSMCs with adenoviral enhanced cyan fluorescent protein cytosolic phospholipase A2 wild type (AdECFPcPLA2 wt) and its mutants, as determined by fluorescence microscopy, was >95% and was similar for both AdECFPcPLA2 wt and its mutants. Anti-phospho-S515 antibody was produced against human cPLA2 sequence amino acids (510–520) by Quality Controlled Biochemicals (Hopkinton, MA). This antibody was raised in rabbits immunized with Ac-CFATQD(pS)FDDDE-amide peptide and purified from serum of immunized animals by affinity purification. The specificity of this antibody was examined by transfecting HEK293 cells with plasmid pacAd5CMV containing ECFPcPLA2 wt and mutants S515A, S505A, and S505A/S515A ECFPcPLA2. The cell lysates were subjected to SDS-PAGE, and Western blot analysis was performed. The blots were probed with anti-phospho-S515 and anti-phospho-S505 antibodies. To further determine the specificity of the phospho-S515 antibody, competition experiments were performed by adding phospho-S515 peptide Ac-CFATQD(pS)FDDDE-amide to the same blocking solution used for Western blot analysis. Phospho-S515 cPLA2 antibody prepared against human cPLA2 peptide sequence (FATQDpSFDDDE) was able to recognize the rabbit cPLA2 peptide sequence (DFTQEpSFDDDE). To determine the localization of ECFPcPLA2 wt and its mutants S505A, S515A, and S505A/S515A and ECFP (from empty virus) expressing VSMCs, we used fluorescence microscopy (Olympus IX 50) at 40× magnification with filter set exciter wavelength 440 nm, dichroic wavelength 455 nm, and emitter wavelength 480 nm. AdECFPcPLA2 wt and mutants were transduced into VSMCs, grown on tissue culture plates at 60 MOI in M199 medium containing 0.1% FBS, and incubated for 48 h. Cells were imaged using an Olympus IX 50 inverted fluorescence microscope equipped with a 40× objective. To determine the distribution of ECFPcPLA2 wt and its mutants in response to NE, VSMCs were transduced with AdECFPcPLA2 wt and its mutants at 60 MOI as described above and then washed with serum-free medium. After this period, the cells were exposed to NE (10 μM) or its vehicle and viewed with a fluorescence microscope (Nikon Eclipse TE300) using Metamorph software version 6.1. Time-lapse images were taken every 5 s before and after adding NE for 15 min. Two time points were selected, 0 and 20 s after adding NE for data analysis. Experiments were performed in triplicate for each ECFP alone, ECFPcPLA2 wt, and mutant ECFP (S505A, S515A, and S505A/S515A) expressing VSMCs. Images were analyzed using ImageJ software. Lysates of VSMCs with and without transduction with AdECFPcPLA2 wt and its mutants were prepared in RIPA-modified lysis buffer (Upstate Biotechnology, Charlottesville, VA). Samples containing ∼30 μg of proteins were resolved by SDS-PAGE before transferring to nitrocellulose membranes. The membranes were blocked with 3% milk in TBST at room temperature for 1 h and then in a cold room overnight with primary monoclonal antibodies (1:1,000 dilution) against phospho-S505 cPLA2, phospho-ERK1/2, ERK1/2 (Cell Signaling Technology, Danvers, MA), and α-actin (Sigma). Anti-phospho-S515 cPLA2 antibody was used at a dilution of 1:100. The blots were developed using biotinylated secondary antibodies and horseradish peroxidase (Amersham Pharmacia Biotech), and signals were detected using ECL Western blot detection reagent SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). The density of bands was measured using ImageJ software. Western blot experiments were performed at least three times on the cells transfected with mutants. The phosphorylation of endogenously and exogenously expressed ECFPcPLA2 wt and its mutants S505A, S515A, and S505A/S515A in VSMCs in response to NE was studied using specific anti-phospho-S515 and anti-phospho-S505 antibodies. The cells with and without transduction with AdECFPcPLA2 wt and its mutants as described above were treated with NE (10 μM) for 10 min and lysed; total cell proteins were determined using the Bradford assay (22.Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216334) Google Scholar) and separated by SDS-PAGE, and blots were probed with anti-phospho-S515 or anti-phospho-S505 cPLA2 antibody. VSMCs were plated on 24-well plates at a density of 105 cells per well and infected with viral particles carrying AdECFPcPLA2 wt or its mutants (S515A, S505A, and S505A/S515A) at 60 or 120 MOI. Cells were incubated with viral particles for 48 h in serum-free M199 medium, and then they were labeled with 0.1 μCi/ml [3H]AA in serum-free M199 medium for 18 h at 37°C. After that, the medium was removed and the cells were washed twice with HBSS, incubated with M199 containing 0.1% BSA, and treated with NE (10 μM) or its vehicle for 15 min. The reaction medium was then removed, and radioactivity was measured by liquid scintillation spectroscopy (Beckman, Irvine, CA). The cells were digested in 1 N NaOH overnight for measurement of total cell radioactivity. [3H]AA release into the medium was expressed as a percentage of the total radioactivity of cells plus supernatant. To determine any possible cytotoxic effect on the viability of VSMCs transduced with AdECFPcPLA2 wt and its mutants, we counted the number of viable cells at 72 h after transduction with AdECFPcPLA2 wt and its mutants using trypan blue 0.4% solution (Sigma). More than 99% of cells were trypan blue-negative. We did not find any difference in the number of trypan blue-positive cells between transduced and nontransduced cells. The results of [3H]AA release are presented as mean relative increases elicited by NE as a percentage of that obtained in samples treated with NE vehicle (±SEM). The n value in figure legends refers to the number of experiments that were performed in triplicate for each treatment. The data were analyzed by one-way ANOVA; the unpaired Student's t-test was used to determine the difference between two groups, and the Newman-Keuls a posteriori test was used to determine the difference between multiple groups. P < 0.05 was considered significant. The phospho-S515/total cPLA2, phospho-S505/total cPLA2 and phospho-ERK1/2/total ERK1/2 ratios were calculated from the densitometry analysis of the bands; the value at time point 0 min was arbitrarily chosen as 100%. Values are expressed as x-fold change from the value obtained at time 0 for the subsequent time course during treatment with NE. Means ± SEM of each ratio were calculated from three different Western blot analyses. To determine the specificity of anti-phospho-S515 cPLA2 antibody, HEK293 cells were transfected with plasmid pacAd5CMV carrying ECFPcPLA2 wt (pECFPcPLA2 wt) and its mutants S515A and S505A, as described in Experimental Procedures. The expression of pECFPcPLA2 wt and its mutants relative to the levels of endogenous cPLA2 in HEK293 cells as detected by anti-cPLA2 antibody is shown in Fig. 1A . Exogenous ECFPcPLA2 and its mutants expressed in HEK293 cells were detected as a single band (∼130 kDa), but the level of endogenous cPLA2 was very low and was not detected consistently. Analysis of cell lysates by Western blots that were probed with anti-phospho-S515 antibody showed a single phosphorylated band from lysates of HEK293 cells transfected with pECFPcPLA2 wt and pECFPcPLA2S505A mutant but not pECFPcPLA2S515A (Fig. 1B, upper panel). When the blots were probed with anti-phospho-S505 antibody, a single phosphorylated band was observed on Western blots from the samples of HEK293 cells transfected with pECFPcPLA2 wt and pECFPcPLA2S515A mutant but not pECFPcPLA2S505A (Fig. 1B, middle panel). The level of total cPLA2 in HEK293 cells transfected with these mutants was detected by probing the blots with anti-cPLA2 (Fig. 1B, lower panel). These results show that anti-phospho-S515 antibody and anti-phospho-S505 antibody are specific in recognizing phospho-S515 cPLA2 and phospho-S505 cPLA2, respectively. Moreover, in a competition experiment, when anti-phospho-S515 antibody and phospho-S515 peptide were added together in the same blocking solution, no phospho-S515 band was observed on the Western blots prepared from lysates of cells transfected with pECFPcPLA2 wt (Fig. 1C). This further confirms the specificity of antiphospho-S515 cPLA2 antibody. In HEK293 cells expressing pECFPcPLA2 wt, stimulation with ionomycin (5 μM) for 10 min increased its phosphorylation by 5-fold at S515, by 2-fold at S505, and by 7-fold at ERK1/2 above basal (Fig. 1D). These results indicate that pECFPcPLA2 wt overexpressed in HEK293 cells is constitutively phosphorylated on both S515 and S505 and that its phosphorylation is enhanced by ionomycin, an agent known to increase the influx of extracellular Ca2+. To determine whether NE-activated CaMKII phosphorylates cPLA2 at S515 and leads to the phosphorylation and activation of ERK1/2 and, in turn, the phosphorylation of cPLA2 at S505 in vivo in VSMCs, we examined the time course of phosphorylation of endogenous cPLA2 at S515 and S505 using specific anti-phospho-S515 and -S505 antibodies. Stimulation of rabbit VSMCs with NE (10 μM) at different time points increased the phosphorylation of cPLA2 at S515, as detected by anti-phospho-S515 antibody. CaMKII was rapidly activated (<1 min) by NE (unpublished data) and phosphorylated cPLA2 at S515 that was sustained for up to 20 min (Fig. 2A). NE (10 μM) increased ERK1/2 phosphorylation at 5 min, which reached its maximum at 10 min (Fig. 2B). The phosphorylation of ERK1/2" @default.
• W1992611170 created "2016-06-24" @default.
• W1992611170 creator A5002098518 @default.
• W1992611170 creator A5080736496 @default.
• W1992611170 creator A5091457300 @default.
• W1992611170 date "2008-04-01" @default.
• W1992611170 modified "2023-10-16" @default.
• W1992611170 title "cPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells" @default.
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