Protein Kinases C Translocation Responses to Low Concentrations of Arachidonic Acid*

Joseph T. O'FlahertyDagger, Brad A. Chadwell, Mary W. Kearns§, Susan Sergeant§, and Larry W. Daniel

From the Departments of Internal Medicine and § Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Received for publication, February 5, 2001, and in revised form, April 17, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a >= 20 µM concentration of the fatty acid. We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on PKC, [3H]phorbol dibutyrate (PDB), increased binding of [3H]PDB within 15 s of exposure to >= 10-30 nM AA. Other unsaturated fatty acids, but not a saturated fatty acid, likewise stimulated PDB binding. These responses, similar to those caused by chemotactic factors, resulted from a rise in the number of diacylglycerol receptors that were plasma membrane-associated and therefore accessible to PDB. Unlike chemotactic factors, however, AA was fully active on cells overloaded with Ca2+ chelators. The major metabolites of AA made by PMN, leukotriene B4 and 5-hydroxyicosatetraenoate, did not mimic AA, and an AA antimetabolite did not block responses to AA. AA also induced PMN to translocate cytosolic PKCalpha , beta II, and delta  to membranes. This response paralleled PDB binding with respect to dose requirements, time, Ca2+-independence, resistance to an AA antimetabolite, and induction by another unsaturated fatty acid but not by a saturated fatty acid. Finally, HEK 293 cells transfected with vectors encoding PKCbeta I or PKCdelta fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKCbeta translocation from cytosol to plasma membrane at >= 0.5 µM, and EGFP-PKCdelta translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nM. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca2+ or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Members of the AGC superfamily of kinases move about cells phosphorylating key proteins on serine and threonine. The execution of this movement, or translocation, from one cell compartment to another is crucial to the function of these kinases. Typically, signal pathways entrained by cell stimulation convert resident lipids to products that help guide these kinases to the substrates and milieus appropriate for their phosphorylating activity. One family of AGC kinases, the protein kinases C (PKC),1 affects virtually all aspects of cell physiology. The family was first studied as a phosphorylating activity that developed during the rise in cytosolic Ca2+ ([Ca2+]i) and formation of diacylglycerol (DG) attending cell stimulation. High [Ca2+]i, it was found, caused PKC to move from a latent state in cytosol to plasma membranes. Upon attaching to plasmalemmal phospholipids, particularly phosphatidylserine, the translocated PKC engaged membranous DG and thereby became active. It is now known that human PKC exist as four subfamilies of 13 isoforms (1-4). Conventional PKCalpha , beta I, beta II, and gamma  are sensitive to Ca2+ and DG; novel PKCdelta , epsilon , eta , and theta  are sensitive only to DG; and atypical PKCzeta and iota  are sensitive to neither signal. A more distantly related group of µ, nu , and PKD-2 isoforms binds DG but is Ca2+-insensitive and may or may not translocate depending on cell type (5-7).

Many factors besides Ca2+, DG, and phospholipids influence the disposition of PKC. Various organelle-associated modulator, scaffold, or substrate proteins bind and thereby recompartmentalize and assist in activating PKC, often in an isoform-specific manner (1-4, 8-18). The phosphorylation of regulatory residues is prerequisite for PKC activity but also may alter their location (1-4, 6, 15, 19, 20). Finally, unsaturated fatty acids (UFAs) share with DG the ability to activate PKC (21). Unlike DG, they target most PKC isoforms and can operate in the absence of phospholipid or Ca2+ cofactors (reviewed in Ref. 22). Because UFAs likewise activate PKC in whole cells (22-32), arachidonic acid (AA) has been proposed to participate in signaling for activation of the enzymes. AA is the major UFA released during cell stimulation and among UFAs has high efficacy in activating PKC. It nonetheless achieves this activation only at >= 20-100 µM, levels unlikely to occur physiologically. On the other hand, UFAs also cause cells to translocate PKC. Observed mainly at UFA concentrations that activate PKC, this effect has been either ignored or viewed as consequential and secondary in importance to PKC activation (21-33). We show here that AA induces PKC translocation in polymorphonuclear neutrophils (PMN) and HEK 293 cells at concentrations 2-4 orders of magnitude below those reported to activate the enzymes. Based on the criterion of potency, AA is better suited as a translocating than activating signal for PKC.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Buffers-- The following were purchased: [3H]phorbol dibutyrate ([3H]PDB) (19.1 Ci/mmol) and polyvinylidene difluoride membranes (PerkinElmer Life Sciences); PDB, phorbol 12-myristate 13-acetate, dioctanoylglycerol, N-formyl-methionyl-leucyl-phenylalanine (FMLP), nordihydroguaiaretic acid, sphinganine, 0.01% poly-L-lysine plates, and fatty acid-free BSA (Sigma); intracellular Ca2+ chelators (Molecular Probes); fatty acids (NuChek Prep, Elysean, MN); PKC isoform standards and rabbit antibodies to these isoforms (PanVera, Madison WI); horseradish peroxidase-linked rabbit anti-IgG (Transduction Laboratories); pPKCbeta -EGFP (CLONTECH); rabbit antibody to enhanced green fluorescent protein (EGFP) (Santa Cruz Biotechnology); Supersignal chemiluminescence kits (Pierce); silicone oil (Versilube F50; General Electric Corp., Westview NY); DH5alpha -competent cells, LipofectAMINE kits, Maxi-prep DNA isolation kits (Promega), and LB medium (Life Technologies, Inc.); and HEK 293 cells (ATCC). Leukotriene (LT)B4 and 5-(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoate (5-HETE) were prepared, and protease and phosphatase inhibitors were purchased (33-35). pPKCdelta -EGFP was a gift from Prof. N. Saito (Biosignal Research Center, Kobe University, Kobe, Japan). Human PMN were suspended in a modified Hanks' balanced solution (33); HEK 293 cells were grown in Dulbecco's modified essential medium (DMEM) with streptomycin (100 µg/ml) and penicillin (100 units/ml), ± 10% fetal calf serum. Where indicated, Hanks' buffer and DMEM were made 100 µM in ascorbic acid plus sufficient NaOH to maintain a constant pH.

[3H]PDB Binding-- PMN (5 × 106) isolated from human donor blood (33) were incubated in 1 ml of Hanks' buffer at 37 °C for 15 min, equilibrated with 125 pM [3H]PDB for 5 min, challenged with 1 µl of ethanol or a UFA dissolved in ethanol, and centrifuged through 400 µl of silicone oil. Cell pellets and supernatant fluids were counted for radioactivity. Results are expressed as fmol of total recovered radiolabel that are cell-associated. [3H]PDB binding attains equilibrium within 1 min regardless of PDB concentration (125 pM-10 µM) or PMN calcium status (34, 35).

PKC Immunodetection-- PMN (2.5 × 108) in 5 ml of Hanks' buffer were incubated at 37 °C for 20 min; challenged with 5 µl of ethanol or UFAs in ethanol; diluted in 40 ml of 4 °C Hanks' buffer; and centrifuged (400 g; 5 min; 4 °C). Cells were suspended in 5 ml of isotonic saline, incubated for 5 min at 4 °C with 2 mM diisopropylfluorophosphate, and washed twice in Hanks' buffer. Cells (2 × 108/ml) were suspended in Hanks' buffer containing 2 mM EGTA, 1 mM PMSF, 1 µg/ml leupeptin, 10 µM benzamidine, 10 µM pepstatin, and 0.2 µg/ml aprotinin. Suspensions were sonicated (4 °C; 5 strokes (3 s) of a Heat Systems sonicator, setting of 2) and centrifuged (800 × g for 10 min at 4 °C) to remove unbroken cells, nuclei, and other debris. Supernatant fluid from the centrifugation was layered onto sucrose gradients and centrifuged (150,000 × g for 30 min at 4 °C) to obtain fractions enriched in cytosol, plasma membrane, and granule markers (36, 37). Fractions were assayed for protein (Bio-Rad protein assay; BSA as standard) and stored in 10% glycerol (-70 °C). Samples of defined protein mass, PKC standards, and prestained Mr markers were resolved on 7% SDS-polyacrylamide gels and electrotransferred to polyvinylidene difluoride membranes. The membranes were washed three times in TBS-T (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween, pH 7.5), blocked for 1 h with 5% Carnation nonfat dry milk in TBS-T, and washed three times in TBS-T. After a 2-h incubation with primary antibody (1/200 in 3% BSA/TBS-T, 0.02% sodium azide), membranes were washed six times in 300 ml of TBS-T, incubated for 1 h with secondary antibody (1/2000 in 5% milk/TBS-T), and washed three times in TBS-T. Blots were developed with enhanced chemiluminescence kits as instructed by the manufacturer. Individual bands were quantified with ImageQuant (Molecular Dynamics, Sunnyvale CA).

Ca2+ Studies-- PMN (1 × 107/ml) were incubated (37 °C) for 1 h with fura-2 AM or Quin2 AM in Hanks' buffer (1 µM EGTA, no Ca2+), washed twice in this buffer, and incubated (37 °C) for 20 min in Hanks' buffer ± CaCl2. Cells loaded with 1 µM fura-2 or Quin2 and incubated with 0 or 1.4 mM Ca2+ are termed Ca2+-depleted and Ca2+-repleted, respectively; cells incubated with a 30 µM concentration of a probe and no Ca2+ are termed Ca2+-chelated. To assay [Ca2+]i, PMN loaded with fura-2 were incubated at 1 × 107/ml in Hanks' buffer ± 1.4 mM CaCl2 at 37 °C, excited alternately at 340 and 380 nm, and monitored at 510 nm with an Aminco-Bowman spectrofluorometer. To obtain [Ca2+]i, the ratio of emission intensities at the exciting wavelengths were compared with those of a buffer containing 1 µM fura-2 pentapotassium salt and CaCl2 clamped at 0-800 nM free Ca2+ with EDTA (34). Results are given in nM [Ca2+]i.

In Situ PKC Translocation-- Our two vectors encoded for human PKCbeta I and PKCdelta fused to EGFP. pPKCbeta -EGFP was transformed in DH5alpha -competent cells and grown on LB agar (kanamycin, 30 µg/ml). Colonies were picked and expanded in LB medium (kanamycin, 30 µg/ml). After isolation with Maxi-prep DNA isolation kits, DNA was digested with KpnI and BamI to yield a fragment running with the appropriate size (2027 base pairs) on 1% agarose gels. pPKCdelta -EGFP was transformed in DH5alpha -competent cells and plated on LB agar (ampicillin, 50 µg/ml). Colonies were selected and expanded in LB medium (ampicillin, 50 µg/ml). On reaching an absorbance of 0.4 AU (400 nm), cultures were treated with 170 µg/ml chloramphenicol and grown overnight. Digestion of isolated DNA with HindIII gave appropriately sized fragments of 1400 and 5600 base pairs on agarose gels. HEK-293 cells (2 × 105 cells on 35-mm poly-L-lysine-coated dishes) were transfected with 2 µg of a vector using the Life Technologies, Inc. LipofectAMINE kit and protocol. Cells were grown for 10-12 days in DMEM, 10% fetal calf serum with 700 µg/ml gentamycin. Viable cells were diluted to single cells and expanded in DMEM, 10% fetal calf serum, 400 µg/ml gentamycin. Final colonies, which expressed appropriate molecular weight proteins that reacted with antibody to EGFP and either PKCbeta I or PKCdelta on Western blots, were maintained in this medium and transferred to serum-free DMEM supplemented with insulin, transferrin, and selenium for 18 h. Cells were challenged while being monitored by fluorescent (excitation at 488 nm, emission at 510 nm) and Nomarsky optics with a Zeiss LSM-510 confocal laser scanning system.

Cell Toxicity-- As little as a 20 µM concentration of a UFA caused PMN to leak preloaded fura-2 or Quin2, leak cytosolic LDH, and take up trypan blue (assayed as in Ref. 34). At 30 µM, AA induced leakage of LDH and the two chromophores but only several minutes after the peak in PKC translocation responses; at >= 50 µM, it caused leakage by 2 min of PMN challenge. 5,8,11,14,17-Eicosapentaenoic acid had these respective immediate and delayed effects at 20 and 30 µM; 8,11,14-eicosatrienoic acid and 11,14,17-eicosatrienoic acid at 30 and 50 µM; and 8,11-eicosadienoic acid at >= 100 µM and >=  100 µM. Eicosanoic acid lacked these effects at 100 µM. We restricted most PMN analyses to UFA concentrations <= 10-fold below the level in which each fatty acid caused delayed cytolysis. HEK-293 cells did not leak LDH or EGFP-PKC isoforms nor alter uptake of trypan blue in the presence of 100 µM AA. We did note that AA, at >= 1-10 µM, caused HEK 293 cells to round, shrink, and, mostly in EGFP-PKCbeta -expressing transfectants, form blebs. Because resting cells had blebs that were only faintly fluorescent and therefore hard to capture in our reproductions, AA, by causing cytosolic EGFP-PKCbeta to attach to plasmalemma, may have highlighted preexisting blebs more than caused new bleb formation. In any case, the data indicate that AA produces morphology changes that are not due to overt alterations in plasma membrane integrity.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PDB Binding-- [3H]PDB has been used to track PKC in astrocytoma cells (38), macrophage tumor cells (29), platelets (39), and PMN (34, 40). The label binds to DG receptors. In consequence, its association with cells rises as these receptors move from the cell interior to surface membrane (34, 38). The top panels of Fig. 1 give the PDB binding of PMN exposed to a 100 nM concentration of the leukocyte chemotactic factor (CF) FMLP. Cells were loaded with 1 µM Quin2 and incubated with 1 µM EGTA plus 0 (Ca2+-fixed) or 1.4 mM (Ca2+-repleted) CaCl2 or, alternatively, loaded with 30 µM Quin2 and incubated with 1 µM EGTA and 0 µM Ca2+ (Ca2+-chelated). On stimulation, Ca2+-repleted PMN rapidly increased binding of [3H]PDB. Ca2+-fixed PMN had slower changes, and Ca2+-chelated PMN had no such changes. Only Ca2+-repleted PMN mounted Ca2+ transients (Fig. 1, bottom panels). The refractoriness of Ca2+-chelated PMN was not due to the side effects of Ca2+ deprivation or chelators; after 5 min of incubation with 1.4 mM CaCl2, Ca2+-chelated PMN responded fully to FMLP (data not shown). These results, which agree with those found in PMN challenged by other CFs, imply PDB binding responses to CFs consist of an early component associated with [Ca2+]i rises and a late component that is independent of these rises but still requires some minimal level of cell Ca2+ (34, 35).


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Fig. 1.   PDB binding and Ca2+ transient responses to FMLP. For PDB binding (top panels), PMN were Ca2+-repleted, Ca2+-fixed, or Ca2+-chelated with Quin2 (fura-2 gave similar results); equilibrated with 125 pM [3H]PDB ± 5 µM PDB; exposed to 100 nM FMLP; and counted for radioactivity. Results are fmol (mean ± S.E. for 5-7 experiments) of label specifically bound (bound by cells incubated with [3H]PDB minus that bound by cells incubated with [3H]PDB plus PDB) after correction for the specific binding of PMN stimulated by the vehicle for FMLP, 62.5 µg of BSA. For Ca2+ transients (bottom panels), PMN were Ca2+-manipulated with fura-2 and challenged with 100 nM FMLP. Results are mean ± S.E. [Ca2+]i (nM) for 4-6 experiments. By itself, BSA did not stimulate PDB binding or [Ca2+]i rises.

Similar to CFs, >= 10 nM AA stimulated Ca2+-repleted PMN to bind [3H]PDB (Fig. 2). Unlike CFs, however, AA was equally active on Ca2+-fixed and Ca2+-chelated cells (Fig. 3, top panels). AA provoked [Ca2+]i rises only in Ca2+-repleted PMN (Fig. 3, bottom panels), although these rises were slow-paced and, in view of the response to AA executed by Ca2+-fixed and Ca2+-chelated PMN, contributed little to the effect of the fatty acid on PDB binding. Neither LTB4 nor 5-HETE, the major metabolites of AA in PMN, mediated the action of AA. 5-HETE (5 µM) did not stimulate PDB binding (data not shown), and LTB4, although it elicited the response in Ca2+-repleted and Ca2+-fixed PMN, differed from AA in having no effect on Ca2+-chelated PMN (34). Moreover, PMN pretreated with LTB4 had reduced or down-regulated PDB binding responses to a second LTB4 challenge yet responded fully to AA (data not shown) and an inhibitor of AA oxygenases, nordihydroguaiaretic acid, failed to reduce responses to AA (Table I). Finally, UFAs that are not substrates for lipoxygenases or cyclooxygenases stimulated PDB binding. Their potencies were inversely related to saturation level, with the saturated fatty acid, arachidic acid, being devoid of activity (Fig. 4).


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Fig. 2.   PDB binding responses to AA. PMN were Ca2+-repleted with Quin2 (fura-2 gave similar results), exposed to AA, and analyzed for fmol of [3H]PDB-specific binding (mean ± S.E. for 5-7 experiments) as in Fig. 1 except that data were corrected for the action of ethanol (1 µl/ml), which by itself did not stimulate PDB binding or [Ca2+]i rises.


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Fig. 3.   PDB binding and Ca2+ transient responses to AA. PMN were Ca2+-manipulated with Quin2 (fura-2 gave similar results), exposed to 1 µM AA, and assayed for fmol of [3H]PDB specifically bound (top panels) or nM [Ca2+]i (bottom panels) as in Fig. 1. Data are mean ± S.E. for 5-8 experiments.

                              
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Table I
PDB binding responses in variably treated PMN
PMN were Ca2+-repleted with Quin2, equilibrated with 125 pM [3H]PDB for 4 min, pretreated with an agent for 2 min, filtered, and counted for radioactivity. Alternatively, PMN were treated with nordihydroguaiaretic acid, PDB, PMA, dioctanoyl-glycerol8, or sphinganine for 30 min; equilibrated with 125 pM [3H]PDB for 5 min; and stimulated with AA for 2 min before filtering and assaying PDB binding.


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Fig. 4.   PDB binding responses to fatty acids. PMN were Ca2+-repleted with Quin2, exposed to an indicated fatty acid for 2 min, and assayed for fmol of PDB specifically bound as in Fig. 1. Data are mean ± S.E. for 3-8 experiments. Unsaturated fatty acids had 2-5 cis double bounds at the indicated carbon positions.

PMN pretreated with a DG receptor agonist, either PDB, PMA, or dioctanoylglycerol, bound PDB minimally and did not raise this binding in response to UFAs (Table I). The same agents, when added 2 min after UFAs, reversed the rise in PDB binding by >= 95% within 0.5 min (data not shown). Sphinganine, which blocks PKC attachment to membranes, also inhibited responses to UFAs (Table I). Thus, baseline as well as stimulated PDB binding is reversible and sensitive to a drug disrupting PKC-membrane interactions. We also observed that Ca2+-repleated PMN treated with 100 µM ascorbic acid for 10 min increased the binding of [3H]PDB by 10.5 ± 2 fmol (mean ± S.E. for three experiments) 2 min after challenge with 1 µM AA. This value was 9.9 ± 0.8 fmol for cells not treated with the antioxidant. Finally, UFAs increased the number of sites bound by PDB in whole cells but did not alter the Kd for this binding or the number of sites detected in sonicated PMN (Table II). We conclude that AA and other UFAs induce PMN to raise the number of receptors available to PDB. In achieving the effect, they do not require processing by oxygenases, [Ca2+]i rises, the generation of reactive oxygen species, or, because dioctanoylglycerol blocked but never increased PDB binding (Table I), the endogenous formation of DG.

                              
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Table II
Number of receptors accessed by PDB in variably stimulated intact or sonicated PMN
5 × 106 PMN were Ca2+-repleted, Ca2+-fixed, or Ca2+-chelated with Quin2 and sonicated and assayed for PDB receptors or, alternatively, equilibrated with 125 pM [3H]PDB for 4 min; stimulated for 15 s; treated with 0.25, 1, 4, 8, 16, 256, 1024, or 10,000 nM PDB for 1 min; filtered; and counted for radioactivity.

PKC in PMN-- PMN express alpha , beta I, beta II, delta , and zeta  but not several other PKC isoforms (25, 29, 30, 43-49). We confirmed that PMN contain the former but lack epsilon , eta , theta , or iota  isoforms (36, 37). PKCalpha , beta I, beta II, and delta  localized mostly to cytosol with small amounts in plasmalemma and traces of PKCzeta and delta  in granule fractions of resting PMN. The antibodies used here had high isoform specificity except that PKCbeta I antibody cross-reacted with PKCbeta II. Given these findings and the toxicity of UFAs (see "Cell Toxicity" under "Experimental Procedures"), we focused on PKCalpha , beta II, and delta  in experiments on Ca2+-repleted and Ca2+-chelated PMN exposed to <= 1 µM AA. These restrictions still allow for testing the effect of AA at low concentrations on the translocation of Ca2+-sensitive and Ca2+-insensitive PKC isoforms as a function of cell Ca2+. Ca2+-repleted and Ca2+-chelated PMN were challenged with AA and separated into membrane, granule, and cytosol fractions. Fractions were resolved by SDS-polyacrylamide gel electrophoresis, transferred to membranes, and probed for PKC. Fig. 5 gives representative Western blots; Figs. 6 and 7 show response kinetics and dose dependence, respectively, as judged by densitometric analyses of these blots. AA stimulated transient (<= 8 min) falls in cytosol and rises in membrane PKCalpha , beta II, and delta . Granule fraction PKC did not change (data not shown). PKCdelta responses showed the least percentage change and sensitivity to the fatty acid, translocating only at >= 100 nM AA. Ca2+-chelated PMN exhibited some reduction in the speed and extent of PKCalpha and beta II, but not PKCdelta , responses. In studies not shown, 11,14,17-eicosatrienoic (3 µM) induced PKCalpha , beta II, and delta  translocations, arachidic acid (30 µM) lacked this activity, and nordihydroguaiaretic acid (10 µM), when incubated with PMN for 30 min, did not alter the response to 1 µM AA. Thus, AA, at 10-100 nM, stimulates PDB binding and the translocation of three PKC isoforms. The two effects have similar kinetics, Ca2+-independence, and resistance to an AA antimetabolite. The data support the idea that PDB binding responses to AA reflect the movements of cytosolic PKCalpha , beta II, and delta  to plasmalemma.


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Fig. 5.   PKCalpha , beta II, and delta  in PMN. Cells were Ca2+-manipulated with Quin2, challenged with 1 µM AA for 0-4 min (left two columns) or 0-1000 nM AA for 2 min (right two columns), and fractionated. Fractions were loaded (20 and 60 µg of protein for cytosol and membrane fractions, respectively) and resolved on SDS-polyacrylamide gels, transferred to membranes, reacted with antibodies, and visualized by enhanced chemiluminescence. All bands migrated with PKC standards; doublet bands, seen mostly with PKCdelta , may reflect differences in phosphorylation (44, 48). Ethanol (1 µl/ml) did not alter the distribution of PKC. Each column of panels gives results from the PMN of one donor and is representative of at least four experiments.


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Fig. 6.   Kinetics of AA-induced PKC translocation. PMN were Ca2+-manipulated with Quin2 and processed as in Fig. 5 to obtain Western blots that were quantified by densitometry. Data are mean ± S.E. (4-7 experiments) percentage changes in band density, relative to PMN challenged with ethanol for 0-5 min. Asterisks indicate values significantly (p < 0.05, Student's paired t test) above (plasma membrane) or below (cytosol) ethanol-challenged cells. By itself, ethanol did not alter the location of any PKC isoform.


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Fig. 7.   PKC translocation at different AA concentrations. PMN were Ca2+-manipulated with Quin2; challenged by 0 (1 µl/ml ethanol), 10, 100, or 1000 nM AA for 2 min; processed; and analyzed as in Fig. 6. Data (mean ± S.E. for 3-6 experiments) are percentage changes in band density relative to PMN challenged by ethanol. Asterisks indicate values significantly (p < 0.05, Student's paired t test) above (plasma membrane (solid lines)) or below (cytosol (interrupted lines)) those for ethanol-challenged cells.

HEK 293 Cells-- HEK 293 cells expressing EGFP-PKCbeta presented homogenous fluorescence in the cytosol; nuclei lacked fluorescence. On exposure to AA, cytosol fluorescence fell, whereas fluorescence at the cell periphery rose (Fig. 8, top two rows). Nomarsky optical analysis localized the latter fluorescence to surface membrane (Fig. 9, A-C). These responses occurred at 30, 10, 1, and 0.5 µM AA but not at 0.1 µM AA. Cells exposed to 30 µM AA exhibited visible changes at <= 8 min; maximal changes occurred by 20 min. At 10, 1, and 0.5 µM, just detectable and maximal effects occurred by ~20 and 30, 30 and 40, or 30 and 45 min, respectively (Fig. 10). In all cases, maximal responses followed detectable responses within 10 min and did not reverse over 60 min. Cells challenged with arachidic acid (30 µM) had no change in fluorescence pattern over 60 min (data not shown). Finally, addition of an antioxidant, ascorbic acid (100 µM), to the cell cultures for 10 min did not alter the EGFP-PKCbeta response to 1 or 10 µM AA (data not shown).


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Fig. 8.   EGFP-PKC in HEK 293 cells. Cells expressing EGFP-PKCbeta (top panels) or delta  (bottom panels) were exposed to 1 µM AA for 0-39 min and monitored for fluorescence every 60 s by confocal laser microscopy. EGFP-PKCbeta fluorescence began to fall in the cytosol and rise at the cell periphery by 31 min. The change peaked at ~39 min and did not reverse over the ensuing 21 min. EGFP-PKCdelta fluorescence began to fall in the cytosol and rise at the nuclear membrane by 2 min. Changes peaked at 4-8 min and returned to baseline in 12 min. Results are representative of 10 experiments done on at least two separate clones of cells.


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Fig. 9.   Location of EGFP-PKCs in HEK 293 cells. Cells expressing EGFP-PKCbeta were exposed to 10 µM AA for 0 (A) or 31 (B and C) min and examined by fluorescent (A and B) or Nomarski (C) optics. Resting cells had homogenous fluorescence throughout the cytosol; nuclei lacked fluorescence. After 22 min of challenge, fluorescence in the cytosol shifted to silhouette plasmalemma. Cells expressing EGFP-PKCdelta were exposed to 10 µM AA for 0 (D) and 5 (E and F) min or 50 nM AA for 0 (G) and 8 (H and I) min. Cells were examined by fluorescent (D, E, G, and H) or Nomarski (F and I) optics. Resting cells had fluorescence throughout the cytosol and, to a lesser extent, nucleus. Within 2 min of challenge, fluorescence in the cytosol fell, and that at the nuclear perimeter rose. Nucleoplasm fluorescence was unaltered. Changes peaked at 5 and reversed by 12 min. E, F, H, and I show perinuclear fluorescence-silhouetted nuclear membrane, e.g. at the indentation of the right nucleus in E and F. AA also caused irregular rises in plasmalemma fluorescence, at e.g. 1-3 o'clock in the cell shown in E, 2-4 o'clock in the upper cell shown in H, and the contact interface between the two cells in H.


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Fig. 10.   Effect of varying AA concentrations on the location of EGFP-PKCbeta . HEK 293 cells expressing EGFP-PKCbeta were stimulated and monitored for fluorescence as in Fig. 8. The two cell clumps in the top row showed falls in cytosolic and rises in cell periphery fluorescence by 21 min of challenge with 30 µM AA. Cells in the second row had similar changes in response to 10 µM AA but only after 21 min. The two adjacent cells in the third row had more modest fluorescent changes after 39 min of exposure to 0.5 µM AA, whereas cells in the bottom rows had no fluorescence change after challenge with the AA vehicle, ethanol (1 µl/ml).

Cells expressing EGFP-PKCdelta displayed homogeneous fluorescence in cytosol and, to a lesser extent, nucleus. AA caused declines in cytosolic and rises in perinuclear fluorescence but little or no change in nucleoplasm fluorescence (Fig. 8, bottom two rows). Concurrently with these changes, localized areas of the cell periphery gained fluorescent intensity. Fig. 9, E, F, H, and I, indicates that rises in the fluorescence intensity occurred specifically at nuclear and plasma membranes. Cells exposed to 0.1-30 µM AA initiated, maximized, and reversed these changes by ~2, 8, and 14 min, respectively (Fig. 11). For cells treated with a 30 or 50 nM concentration of the fatty acid, fluorescence increases in plasma and nuclear membrane occurred 2-4 min after challenge (Fig. 9, G-I). Again, 30 µM arachidic acid did not translocate EGFP-PKCdelta , and 100 µM ascorbic acid, when added to cultures for 10 min, did not alter the translocation response of EGFP-PKCdelta to 0.1 or 1 µM AA (data not shown).


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Fig. 11.   Effect of varying AA concentrations on the location of EGFP-PKCdelta . HEK 293 cells expressing EGFP-PKCdelta were stimulated and monitored as in Fig. 8. The cell in the top row showed gains in perinuclear fluorescence. These peaked at 2-4 min and fell to baseline by 14 min of challenge with 33 µM AA. Similar but progressively less intense changes occurred with the cell in the second row challenged with 300 nM AA and the cell in the third row challenged with 100 nM AA. The two cells in the fourth row showed subtle changes (better depicted in Fig. 9, bottom three panels) in response to 50 nM AA. The two cells in the bottom row had no change in fluorescence after challenge with 1 µl/ml ethanol. Associated with rises in perinuclear fluorescence, fluorescence in the cytosol showed partial declines (best seen at the 2 and 4 min time points for the 30 and 0.3 µM AA challenge) and irregular gains in fluorescence at the cell periphery (see Fig. 9).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

UFAs directly activate PKC and, when added to hepatocytes (23), platelets (24, 25), myocytes (26), cultured monocytes (27, 31), macrophages (27, 28, 31), endothelial cells (30), or PMN (29), induce PKC to translocate. Although the latter effect may be consequential to activating these enzymes, the two responses are separable. For example, a low level of phosphatidylserine or Ca2+ causes PKC to attach to membranes in vitro whereas a higher level of either agent also activates the membrane-attached PKC (1-4, 50, 51). The activating effect of AA on most PKC isoforms, including alpha , beta II, and delta , reportedly requires at least 20-100 µM. Such extreme levels of the fatty acid can occur in vivo; microparticles shed by perturbed cells may stimulate PKC and PKC-dependent functions by delivering up to 100 µM free AA to various target cells (31, 52). Under most physiological conditions, however, AA occurs at levels below this. Cells responding to receptor agonists, in particular, are unlikely to propel cytosolic AA to levels engendering PKC activation. We find that >= 10-100 nM AA causes PMN and HEK 293 cells to bind [3H]PDB and translocate PKC.

PDB binding responses to AA reflected an increase in the number of receptors available to the ligand (Tables I and II). In studies not shown, PMN were challenged for 2 min with 3 µM AA or arachidic acid, diluted with 9 volumes of 4 °C buffer, disrupted, and fractionated on Percoll gradients. The former but not the latter fatty acid caused significant falls in cytosol and rises in plasmalemma PDB receptors but no such change in granule, endoplasmic reticulum, or heavy Golgi fractions. Thus, AA-induced PDB binding responses involve the movement of PDB receptors from cytosol to plasmalemma. In sucrose gradient experiments, AA, but not arachidic acid, similarly caused PMN to decrease cytosol and increase membrane PKCalpha , beta II, and delta  (Fig. 5). Although membranes isolated from the latter gradients contain not only plasmalemma but also Golgi, endoplasmic reticulum, and other organelles (36), PKC translocation paralleled [3H]PDB binding. We accordingly interpret sucrose gradient data as indicating that PKC moved mainly if not exclusively from cytosol to plasmalemma. We stress, however, that other PDB receptor-bearing proteins (PKCbeta I, chimerin, etc.) may and likely do contribute to PDB binding (41, 42).

PDB binding responses to CFs were slowed in Ca2+-fixed and absent in Ca2+-chelated PMN (34, 35) (Fig. 1). CFs, including FMLP, likewise have reduced ability to translocate various PKC isoforms in PMN deprived of Ca2+ (43). In marked contrast, AA caused prominent PBD binding and PKCbeta II, alpha , and delta  translocation (Figs. 3 and 5-7) responses regardless of PMN Ca2+ status. Thus, CFs need a minimal level of cell Ca2+ to affect PKC, whereas UFAs have far less such a requirement. Because PKCalpha and beta II, but not PKCdelta , exhibited reduced responses to AA in Ca2+-chelated PMN (Figs. 6 and 7), [Ca2+]i rises likely contribute to PDB binding (34, 35) (Fig. 1) responses to CFs by enhancing the movement of Ca2+-sensitive PKC isoforms. On the other hand, the effect of AA on PDB proved largely indifferent to PMN Ca2+ status (Fig. 3). The relatively slow-paced [Ca2+]i rise initiated by AA may explain this anomaly: Ca2+-actuated PDB binding response to AA overlap and may be obscured by Ca2+-insenstive PDB binding response. In any case, studies clearly show that Ca2+-sensitive PKC isoforms translocate in the absence of Ca2+ transients in PMN challenged with AA (Figs. 5-7), as well as CFs (43) or other receptor agonists (32). It will be important to determine whether endogenous AA has a role in these receptor-driven translocations. Relevant to this, agents that block AA-releasing enzymes, phospholipases A2, have recently been found to inhibit interleukin 2 in stimulating T lymphocytes to translocate PKC activity (53).

AA did not trigger PKC translocation as a result of its oxygenation. 5-HETE and LTB4, the major oxygenation products formed from AA by PMN, did not mimic the action of AA. Moreover, LTB4 down-regulated PMN to itself but not AA (data not shown), nordihydroguaiaretic acid did not alter the action of AA (Table I), and other UFAs stimulated PDB binding (Table II and Fig. 4) and PKC translocation (data not shown). These results contrast with those found with many AA activities. For example, AA stimulates PMN to activate cytosolic phospholipase A2. The effect is sensitive to nordihydroguaiaretic acid and produced by LTB4 and 5-HETE but not other UFAs. Here, then, PMN convert AA to LTB4 and 5-HETE, which bind respective receptors to initiate signal pathways activating the enzyme (54, 55). Our data exclude this route for AA-induced PKC translocation. An alternate metabolic route, however, could explain PKC translocation responses to UFAs. Cells acylate UFAs to CoA, transfer the UFA moiety of UFA-CoA to glycerolipids, and shuttle glycerolipid-bound UFAs to other lipids. PMN conduct these steps rapidly even if overloaded with Ca2+-chelators (56). Because UFA-CoA alters PKC activity and because glycerolipids bearing UFAs have higher affinity for PKC than their saturated fatty acid-containing counterparts (57-59), UFA-CoA or UFA-glycerolipids may influence the dispositions of PKC and thereby the translocation response to UFAs. The same events could operate in cells stimulated to release endogenous AA. Our studies also do not address other indirect means for translocating PKC, e.g. by stimulating formation of phosphatidylinositol phosphates, phosphorylation of PKC, changes in PKC docking proteins, or issuance of various other signals, although these events may not occur in Ca2+-chelated cells challenged by low levels of AA. Studies with pharmacological inhibitors offer a first step to determine whether AA acts directly, upon esterification to CoA or glycerolipids, or through signal pathways.

Fluorescent fusion products have been used to monitor the movements of various PKC isoforms. In CHO cells, EGFP-PKCdelta localizes to cytosol and nucleoplasm (60, 61); ATP stimulates the label to shift to plasmalemma, whereas phorbol esters cause it to move to plasma or nuclear membranes (61, 62). LLCPK-1 cells localize EGFP-PKCdelta to plasmalemma and do not translocate it in response to dopamine, although dopamine translocates other PKC isoforms (63). EGFP-PKCbeta II localizes exclusively to cytosol in HEK 293 cells and moves to plasmalemma in less than 1 min of challenge by G protein-coupled receptor agonists (64). These reports evidence that the location, as well as the direction and kinetics of EGFP-PKCdelta and beta II movements, varies with cell, isoform, and stimulus type. Other PKC isoforms show similar variability (60, 65). Most relevant here, however, are reports on UFAs. In CHO cells, >= 200 µM AA and oleic acid cause rapid, transient shifts in EFFP-PKCgamma from cytosol to plasma membrane, whereas 50 µM AA causes a slow, sustained shift in cytosol EGFP-PKCepsilon to the perinuclear area (66, 67). We are unaware of reports on the effect of UFAs on other EGFP-PKC isoforms. We show here that AA caused HEK 293 cells to translocate EGFP-PKCdelta from cytosol to the entire nuclear and limited portions of the plasma membrane (Fig. 9, D-I). Responses occurred and reversed within minutes of exposure to >= 30 nM AA (Fig. 8, bottom two rows, and Fig. 11). AA affected EGFP-PKCbeta differently, causing it to move from cytosol to plasma membrane (Fig. 9, A-C). Unlike the EGFP-PKCdelta response to AA here or the EGFP-PKCgamma responses to >= 200 µM AA in CHO cells (66), EGFP-PKCbeta movements evolved slowly, did not reverse for 60 min, and occurred at >= 0.5 µM AA in HEK 293 cells (Fig. 8, top two rows, and Fig. 10). As found elsewhere (60-68), then, the kinetics and direction of PKC translocation responses to a given agent are isoform-specific and may relate to the unique functions of each PKC isoform (1-4). Interestingly, resting HEK 293 cells localized EGFP-PKCdelta to both the cytosol and nucleoplasm but localized EGFP-PKCbeta only to the cytosol. This allows for the possibility that PKCdelta may have a nuclear membrane-targeting motif that over longer periods aids its penetration into nuclei.

In conclusion, AA causes PMN and HEK 293 cells to translocate PKCalpha , beta I, beta II, and delta  at concentrations 2-4 orders of magnitude below those reportedly activating the enzymes. Because PKCs must be correctly compartmentalized in order to engage their cofactors and substrates, AA, as presented to cells by, for example, microparticles or released during stimulation, may serve to direct Ca2+-sensitive and Ca2+-insensitive PKC isoforms to proper membrane targets and thereby assist, modulate, or (when no Ca2+ transient occurs) replace [Ca2+]i signals. Indeed, exogenous AA and DG operate synergistically to stimulate PKC and PKC-dependent responses in whole cells (1-4) and may similarly cooperate when formed endogenously. The effect of AA on the translocation of PKC, at least as it occurs in PMN, proceeds independently of [Ca2+]i, DG production, and oxygenation to eicosanoids. The fatty acid may act directly, after acylation to CoA or glycerolipids, or by stimulating PKC phosphorylation, PKC binding proteins, or other signal pathways.

    ACKNOWLEDGEMENT

We thank Prof. N. Saito from the Biosignal Research Center, Kobe University (Kobe, Japan) for his generous gift of pPKCdelta -EGFP.

    FOOTNOTES

* This work was supported by National Institute of Health, NHLBI, Grant 1 RO1 HL56710.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Internal Medicine, Section on Infectious Diseases, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27156. Tel.: 336-716-6039; Fax: 336-716-3825; E-mail: joflaher@wfubmc.edu.

Published, JBC Papers in Press, April 27, 2001, DOI 10.1074/jbc.M101093200

    ABBREVIATIONS

The abbreviations used are: DG, diacylglycerol; PKC, protein kinases C; EGFP, enhanced green fluorescent protein; UFA, unsaturated fatty acid; AA, arachidonic acid; PDB, phorbol dibutyrate; PMA, phorbol 12-myristate 13-acetate; FMLP, N-formyl-methionyl-leucyl-phenylalanine; 5-HETE, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoate; LTB4, leukotriene B4; PMN, polymorphonuclear neutrophils; BSA, bovine serum albumin; DMEM, Dulbecco's modified essential medium; CF, chemotactic factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Peterson, R. T., and Schreiber, S. L. (1999) Curr. Biol. 9, R521-R524[CrossRef][Medline] [Order article via Infotrieve]
2. Oancea, E., and Meyer, T. (1998) Cell 95, 307-318[Medline] [Order article via Infotrieve]
3. Newton, A. C., and Johnson, J. E. (1998) Biochim. Biophys. Acta 1376, 155-172[Medline] [Order article via Infotrieve]
4. Parekh, D. B., Ziegler, W., and Parker, P. J. (2000) EMBO J. 19, 496-503[Free Full Text]
5. Hayashi, A., Seki, N., Hattori, A., Lozuma, S., and Saito, T. (1999) Biochim. Biophys. Acta 1450, 99-106[Medline] [Order article via Infotrieve]
6. Matthews, S. A., Iglesias, T., Rozengurt, E., and Cantrell, D. (2000) EMBO J. 19, 2935-2945[Abstract/Free Full Text]
7. Sturany, S., Van Lint, J., Müller, F., Wilda, M., Hameister, H., Höcker, M., Brey, A., Gern, U., Vandenheede, J., Gress, T., Adler, G., and Seufferlein, T. (2001) J. Biol. Chem. 276, 3310-3318[Abstract/Free Full Text]
8. Moriya, S., Kazlauskas, A., Akimoto, K., Hirai, S.-I., Mizuno, K., Takenawa, T., Fukui, Y., Watanabe, Y., Ozaki, S., and Ohno, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 151-155[Abstract/Free Full Text]
9. Akimoto, K., Takahashi, R., Moriya, S., Nishioka, N., Takayanagi, J., Kimura, K., Fukui, Y., Osada, S., Mizuno, K., Hirai, S., Kazlauskas, A., and Ohno, S. (1996) EMBO J. 15, 788-798[Abstract]
10. Wang, Y.-X., Dhulipala, P. D. K., Li, L., Benovic, J. L., and Kotlikoff, M. I. (1999) J. Biol. Chem. 274, 13859-13864[Abstract/Free Full Text]
11. Limatola, C., Schaap, D., Moolenaar, W. H., and van Blitterswijk, W. J. (1994) Biochem. J. 304, 1001-1008[Medline] [Order article via Infotrieve]
12. Sasaki, Y., Asaoka, Y., and Nishizuka, Y. (1993) FEBS Lett. 320, 47-51[CrossRef][Medline] [Order article via Infotrieve]
13. Murray, N. R., and Fields, A. P. (1998) J. Biol. Chem. 273, 11514-11520[Abstract/Free Full Text]
14. Mochly-Rosen, D., and Gordon, A. S. (1998) FASEB J. 12, 35-42[Abstract/Free Full Text]
15. Mellor, H., and Parker, P. J. (1998) Biochem. J. 332, 281-292[Medline] [Order article via Infotrieve]
16. Newton, A. C. (1997) Curr. Opin. Cell Biol. 9, 161-167[CrossRef][Medline] [Order article via Infotrieve]
17. Ron, D., Napolitano, E. W., Voronova, A., Vasquez, N. J., Roberts, D. N., Calio, B. L., Caothien, R. H., Pattiford, S. M., Wellik, S., Mandac, J. B., and Kauvar, L. M. (1999) J. Biol. Chem. 274, 19003-19010[Abstract/Free Full Text]
18. Sanchez, P., De Carcer, G., Sandoval, I. V., Moscat, J., and Diaz-Meco, M. (1998) Mol. Cell Biol. 18, 3069-3080[Abstract/Free Full Text]
19. Edwards, A. S., Faux, M. C., Scott, J. D., and Newton, A. C. (1999) J. Biol. Chem. 274, 6461-6468[Abstract/Free Full Text]
20. Feng, X., Becker, K. P., Stribling, S. D., peters, K. G., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 17024-17034[Abstract/Free Full Text]
21. McPhail, L. C., Clayton, C. C., and Snyderman, R. (1984) Science 224, 622-625[Medline] [Order article via Infotrieve]
22. Khan, W. A., Blobe, G. C., and Hannun, Y. A. (1995) Cell. Signal. 7, 171-184[CrossRef][Medline] [Order article via Infotrieve]
23. Diaz-Guerra, M. J. M., Junco, M., and Bosca, L. (1991) J. Biol. Chem. 266, 23568-23576[Abstract/Free Full Text]
24. Khan, W. A., Blobe, G., Halpern, A., Taylor, W., Wetsel, W. C., Burns, D., Loomis, C., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 5063-5068[Abstract/Free Full Text]
25. Hii, C. S. T., Huang, Z. H., Bilney, A., Costabile, M., Murray, A. W., Rathjen, D. A., Der, C. J., and Ferrante, A. (1998) J. Biol. Chem. 273, 19277-19282[Abstract/Free Full Text]
26. Huang, X. P., Pi, Y., Lokuta, A. J., Greaser, M. L., and Walker, J. W. (1997) J. Cell Sci. 110, 1625-1634[Abstract/Free Full Text]
27. Huang, Z. H., Hii, C. S. T., Rathjen, D. A., Poulos, A., Murray, A. W., and Ferrante, A. (1997) Biochem. J. 325, 553-557[Medline] [Order article via Infotrieve]
28. Padma, M., and Das, U. N. (1999) Prostaglandins Leukotrienes Essent. Fatty Acids 60, 55-63[CrossRef][Medline] [Order article via Infotrieve]
29. Hii, C. S. T., Huang, Z. H., Bilney, A., Stacey, K., Murray, A. W., Rathjen, D. A., and Ferrante, A. (1999) Adv. Exp. Med. Biol. 469, 365-370[Medline] [Order article via Infotrieve]
30. Hii, C. S. T., Ferrante, A., Edwards, Y. S., Huang, Z. H., Hartfield, P. J., Rathjen, D. A., Poulos, A., and Murray, A. W. (1995) J. Biol. Chem. 270, 4201-4204[Abstract/Free Full Text]
31. Barry, O. P., Kazanietz, M. G., Praticò, D., and FitzGerald, G. A. (1999) J. Biol. Chem. 274, 7545-7556[Abstract/Free Full Text]
32. Lennartz, M. R. (1999) Int. J. Biochem. Cell Biol. 31, 415-430[CrossRef][Medline] [Order article via Infotrieve]
33. O'Flaherty, J. T., Kuroki, M., Nixon, A. B., Wijkander, J., Yee, E., Lee, S. L., Smitherman, P. K., Wykle, R. L., and Daniel, L. W. (1996) J. Biol. Chem. 271, 17821-17828[Abstract/Free Full Text]
34. O'Flaherty, J. T., Jacobson, D. P., Redman, J. F., and Rossi, A. G. (1990) J. Biol. Chem. 265, 9146-9152[Abstract/Free Full Text]
35. O'Flaherty, J. T., Redman, J. F., Jacobson, D. P., and Rossi, A. G. (1990) J. Biol. Chem. 265, 21619-21623[Abstract/Free Full Text]
36. Kent, J. D., Sergeant, S., Burns, D. J., and McPhail, L. C. (1996) J. Immunol. 157, 4641-4647[Abstract]
37. Sergeant, S., and McPhail, L. C. (1997) J. Immunol. 159, 2877-2885[Abstract]
38. Brown, J. H., Trilivas, J., and Martinson, E. A. (1990) Symp. Soc. Exp. Biol. 44, 147-156[Medline] [Order article via Infotrieve]
39. Rais, S., Combadiere, C., Hakim, J., and Perianin, A. (1994) Biochem. Pharmacol. 47, 1797-1804[Medline] [Order article via Infotrieve]
40. Combadière, C., Pedruzzi, E., Hakim, J., and Perianin, A. (1993) Biochem. J. 289, 695-701[Medline] [Order article via Infotrieve]
41. Kazanietz, M. G. (2000) Mol. Carcinog. 28, 5-11[CrossRef][Medline] [Order article via Infotrieve]
42. Majumdar, S., Rossi, M. W., Fujiki, T., Phillips, W. A., Disa, S., Queen, C. F., Johnston, R. B., Jr., Rosen, O. M., Corkey, B. E., and Korchak, H. M. (1991) J. Biol. Chem. 266, 9285-9294[Abstract/Free Full Text]
43. Dang, P. M. C., Rais, S., Hakim, J., and Périanin. (1995) Biochem. Biophys. Res. Commun. 212, 664-672[CrossRef][Medline] [Order article via Infotrieve]
44. Laudanna, C., Mochly-Rosen, D., Liron, T., Constantin, G., and Butcher, E. C. (1998) J. Biol. Chem. 273, 30306-30315[Abstract/Free Full Text]
45. Reeves, E. P., Dekker, L. V., Forbes, L. V., Wientjes, F. B., Grogan, A., Pappin, D. J. C., and Segal, A. W. (1999) Biochem. J. 344, 859-866[CrossRef][Medline] [Order article via Infotrieve]
46. Raeder, E. M. B., Mansfield, P. J., Hinkovska-Galcheva, V., Kjeldsen, L., Shayman, J. A., and Boxer, L. A. (1999) Blood 93, 686-693[Abstract/Free Full Text]
47. Khwaja, A., and Tatton, L. (1999) Blood 94, 291-301[Abstract/Free Full Text]
48. Nixon, J. B., and McPhail, L. C. (1999) J. Immunol. 163, 4574-4582[Abstract/Free Full Text]
49. Dekker, L. V., Leitges, M., Altschuler, G., Mistry, N., McDermott, A., Roes, J., and Segal, A. W. (2000) Biochem. J. 347, 285-289[CrossRef][Medline] [Order article via Infotrieve]
50. Wolf, M., Cuatrecasas, P., and Sahyoun, N. (1985) J. Biol. Chem. 260, 15718-15722[Abstract/Free Full Text]
51. Keranen, L. M., and Newton, A. C. (1997) J. Biol. Chem. 272, 25959-25967[Abstract/Free Full Text]
52. Barry, O. P., Praticò, D., Lawson, J. A., and FitzGerald, G. A. (1997) J. Clin. Invest. 99, 2118-2127[Abstract/Free Full Text]
53. Lu, Y., Morley, P., and Durkin, J. P. (1999) Cell Signal 11, 275-285[CrossRef][Medline] [Order article via Infotrieve]
54. Wijkander, J., O'Flaherty, J. T., Nixon, A. B., and Wykle, R. L. (1995) J. Biol. Chem. 270, 26543-26549[Abstract/Free Full Text]
55. Capodici, C., Pillinger, M. H., Han, G., Philips, M. R., and Weissmann, G. (1998) J. Clin. Invest. 102, 165-175[Abstract/Free Full Text]
56. Daniele, J. J., Fidelio, G. D., and Bianco, I. D. (1999) Prostaglandins Other Lipid Mediat. 57, 341-350[CrossRef][Medline] [Order article via Infotrieve]
57. Szamel, M., Rehermann, B., Krebs, B., Kurrle, R., and Resch, K. (1989) J. Immunol. 143, 2806-2813[Abstract/Free Full Text]
58. Yaney, G. C., Korchak, H. M., and Corkey, B. E. (2000) Endocrinology 141, 1989-1998[Abstract/Free Full Text]
59. Hodgkin, M. N., Pettitt, T. R., Martin, A., Michell, R. H., Pemberton, A. J., and Wakelam, M. J. O. (1998) Trends Biochem. Sci. 23, 200-204[CrossRef][Medline] [Order article via Infotrieve]
60. Shirai, Y., Sakai, N., and Saito, N. (1998) Jpn. J. Pharmacol. 78, 411-417[CrossRef][Medline] [Order article via Infotrieve]
61. Wang, Q. J., Bhattacharyya, D., Garfield, S., Nacro, K., Marquez, V. E., and Blumberg, P. M. (1999) J. Biol. Chem. 274, 37233-37239[Abstract/Free Full Text]
62. Ohmori, S., Shirai, Y., Sakai, N., Fujii, M., Konishi, H., Kikkawa, U., and Saito, N. (1998) Mol. Cell Biol. 18, 5263-5271[Abstract/Free Full Text]
63. Nowicki, S., Kruse, M. S., Brismar, H., and Aperia, A. (2000) Am. J. Physiol. Cell Physiol. 279, C1812-C1818[Abstract/Free Full Text]
64. Feng, X., Zhang, J., Barak, L. S., Meyer, T., Caron, M. G., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 19755-10762
65. Vallentin, A., Prévostel, C., Fauquier, T., Bonnefont, X., and Joubert, D. (2000) J. Biol. Chem. 275, 6014-6021[Abstract/Free Full Text]
66. Shirai, Y., Kashiwagi, K., Yagi, K., Sakai, N., and Saito, N. (1998) J. Cell Biol. 143, 511-521[Abstract/Free Full Text]
67. Shirai, Y., Segawa, S., Kuriyama, M., Goto, K., Sakai, N., and Saito, N. (2000) J. Biol. Chem. 275, 24760-24766[Abstract/Free Full Text]
68. Maasch, C., Wagner, S., Lindschau, C., Alexander, G., Buchner, K., Gollasch, M., Luft, F. C., and Haller, H. (2000) FASEB J. 14, 1653-1663[Abstract/Free Full Text]


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