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The Journal of Immunology, 1998, 161: 4375-4381.
Copyright © 1998 by The American Association of Immunologists

Products of Sphingolipid Catabolism Block Activation of the p21-Activated Protein Kinases in Neutrophils1

Jian P. Lian{ddagger}, RiYun Huang*, Dwight Robinson* and John A. Badwey2,{ddagger},{dagger}

* Arthritis Unit, Massachusetts General Hospital, Boston, MA 02114; {dagger} Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and {ddagger} Boston Biomedical Research Institute, Boston, MA 02114


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neutrophils stimulated with the chemoatttractant FMLP are known to exhibit a rapid and transient activation of two p21-activated protein kinases (Paks) with molecular masses of approximately 63 and 69 kDa. Paks can be detected by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 to 331 of the 47-kDa subunit of the nicotinamide-adenine dinucleotide phosphate-oxidase complex (p47-phox) fixed within a gel. In this study, we demonstrate that N-acetylsphingosine (C2-ceramide) and a variety of sphingoid bases (e.g., D-erythrosphingosine) block activation of the 63- and 69-kDa Paks in neutrophils. The concentrations of these lipids that were effective in blocking Pak activation were similar to those that inhibit a variety of neutrophil responses. Activation of the 63- and 69-kDa Paks was also markedly reduced in neutrophils treated with sphingomyelinase before stimulation. Moreover, we report that addition of C2-ceramide or D-erythrosphingosine to neutrophils after stimulation with FMLP markedly enhances the rate of Pak inactivation. These effects were not mimicked by arachidonate, which is a potent disorganizing agent of neutrophil membranes. These data support and extend the proposal that sphingoid bases may establish a set point in neutrophils for positive stimuli.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neutrophils stimulated with the chemoattractant FMLP exhibit a rapid and transient activation of four renaturable protein kinases with molecular masses of approximately 69, 63, 49, and 40 kDa (1, 2, 3). Activation of all four of these kinases appears to be under the control of a single stimulatory pathway that is sensitive to antagonists of heterotrimeric G proteins, type 1 and/or 2A protein phosphatases, tyrosine kinases, and phosphatidylinositol 3-kinase (1, 2, 3, 4, 5, 6). The 63- and 69-kDa enzymes were subsequently identified as p21-activated kinases (Paks)3 (7, 8). Paks undergo autophosphorylation/activation upon binding the active (GTP-bound) forms of the small GTPases (p21) Rac or Cdc42 (9). Paks can catalyze the phosphorylation of the 47-kDa subunit of the nicotinamide-adenine dinucleotide phosphate-oxidase complex (p47-phox) (7) and certain heavy and light chains of myosin (e.g., 10, 11). Paks are also involved in the activation/potentiation of several distinct MAP kinase cascades. Transfection of constitutively active Pak or overexpression of wild-type Pak in certain cells is sufficient to activate the c-jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and, to a lesser extent, p38 MAP kinase (e.g., 12–14; for review, see 15 . Moreover, activated Pak can potentiate the ability of wild-type Raf-1 or growth factors to stimulate ERKs and MAP-kinase kinases in numerous cell types (14, 16). Selective antagonists of MAP-kinase kinase and p38 MAP kinase inhibit chemotaxis, phagocytosis, degranulation, and superoxide production by neutrophils (17, 18, 19, 20). Thus, Paks may participate in a variety of neutrophil responses.

A variety of studies over the past decade strongly suggest that certain sphingolipids may function as second messengers or modulators of cellular responses (e.g., for review, see Refs. 21–23). Cell-permeable derivatives of ceramide (C2-ceramide) and sphingoid bases (e.g., D-erythrosphingosine) inhibit phagocytosis, degranulation, and O2- production by neutrophils (17, 24, 25, 26, 27, 28). Interestingly, C2-ceramide also inhibits activation of ERK1 and ERK2 in neutrophils (17) and blocks translocation of certain small GTPases (Cdc42) to the membrane of these cells (29). Sphingoid bases inhibit PKC (25) and phosphatidic acid phosphatase in neutrophils (30). Increased amounts of ceramide and sphingosine have been observed in FMLP-stimulated neutrophils and neutrophils undergoing IgG-dependent phagocytosis (17, 28, 31). The cellular concentrations of these lipids also undergo marked increases in certain genetic diseases (e.g., Farber’s lipogranulomatosis) and during various infections (e.g., Refs. 32–34).

Paks and Pak-related kinases may be regulated by various lipids (35, 36). In particular, a recent study has demonstrated that high concentrations of sphingoid bases (50–200 µM) and certain other lipids can activate Pak 1 in vitro in a manner similar to that observed with Cdc42-GTP (36). COS-7 cells expressing Pak 1 exhibit enhanced activity of this kinase when treated with agents that elevate the content of sphingosine (36). T cells also exhibit an enhanced activity of Pak when treated with C2-ceramide through an unknown mechanism (37). In this study, we report that products of sphingolipid catabolism block activation of the 63- and 69-kDa Paks in FMLP-stimulated neutrophils. The effective concentrations of these lipids are in the range of those that can be achieved under various physiologic/pathologic situations. The significance of these results to neutrophil function and Pak activation under various circumstances is discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

D-Erythro-C2-ceramide (N-acetylsphingosine), D-erythrodihydro-C2-ceramide (N-acetyldihydrosphingosine), D-erythrosphingosine, D-erythrodihydrosphingosine, and sphingosine-1-phosphate were purchased from Calbiochem (La Jolla, CA). D,L-threodihydrosphingosine was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). An affinity-purified, rabbit polyclonal Ab raised against a peptide corresponding to residues 525–544 of rat Pak 1 (Pak(C-19) Ab) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anhydrous sodium sulfate, o-phthalaldehyde, arachidonate, and sphingomyelinase (520 U/mg) from Bacillus cereus were obtained from Sigma (St. Louis, MO). Sources of all other materials are described elsewhere (1, 2, 8).

Methods

Preparation of neutrophils. Guinea pig peritoneal neutrophils were prepared as described previously (38). These preparations contained >90% neutrophils with viabilities always >90%.

Detection of renaturable protein kinases in polyacrylamide gels. Paks and certain other protein kinases were detected directly in gels by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed within a gel that corresponds to residues 297–331 of p47-phox. This technique was performed as described previously (1, 2).

Stock solutions of FMLP (4 mM), PMA (2 mg/ml), C2-ceramide (16 mM), D-erythrosphingosine (16 mM), and D,L-threodihydrosphingosine (8 mM) were prepared in DMSO. D-erythrodihydroceramide (12 mM) and D-erythrodihydrosphingosine (16 mM) were prepared in ethanol. Sphingosine-1-phosphate (1.25 mM) was prepared in methanol. All stock solutions were stored at -20°C and diluted with DMSO or ethanol so that the final amount of solvent in each assay did not exceed 0.50% (v/v) (this includes the 0.25% added with the stimulus). These amounts of solvent did not cause any of the effects noted.

Miscellaneous procedures. Procedures for immunoprecipitating Paks from neutrophil lysates with the Pak(C-19) Ab and methods for detecting these kinases in immune complexes by autophosphorylation are described elsewhere (7, 39). Sphingosine was measured by the procedure of Merrill and coworkers (31, 40).

Analysis of data. Unless otherwise noted, all of the autoradiographic observations were confirmed in at least three separate experiments performed on different preparations of cells. The number of observations (n) is based on different cell preparations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of various sphingolipids on the activation of the renaturable protein kinases in neutrophils

Neutrophils stimulated with FMLP exhibit rapid activation of four renaturable protein kinases with molecular masses of approximately 69, 63, 49, and 40 kDa (2, 3) (Fig. 1Go). The 63- and 69-kDa enzymes have been identified as Paks (8). Recent studies indicate that these kinases may be forms of Pak 1 that differ in their content of phosphate (39). Paks can be detected directly in gels by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate uniformly fixed within a gel. The positions of these kinases are visualized by autoradiography after exposure of the gel to [{gamma}-32P]ATP (2, 3). The peptide substrate utilized corresponds to amino acid residues 297–331 of p47-phox, which contains several of the phosphorylation sites of this protein (41).



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  		FIGURE 1. Effects of C2-ceramides on the activation of the 63- and 69-kDa Paks in neutrophils. The renaturable protein kinases in neutrophils were assayed directly in gels containing the fixed peptide substrate, as referenced under Materials and Methods. A, Cells were incubated in the absence (lanes a and b) and presence of 40 µM C2-ceramide (lanes c and d) or 40 µM C2-dihydroceramide (lanes e and f) for 5 min prior to treatment for 15 s with 0.25% (v/v) DMSO (lanes a, c, and e; unstimulated cells) or 1 µM FMLP (lanes b, d, and f; stimulated cells). B, Shows the complete profile for the renaturable kinases from the same experiment, except the time of development of the autoradiograph was increased from 18 to 72 h. The concentrations of C2-ceramide and C2-dihydroceramide utilized in these studies did not affect cell viability, as measured by the exclusion of trypan blue. The positions of the 63- and 40-kDa kinases are designated by unbroken arrows, whereas the 69- and 49-kDa kinases are marked by arrowheads. *, Denotes a 38/39-kDa kinase that was frequently observed to undergo activation in cells stimulated with FMLP. Molecular mass markers are provided on the left.

 
Treatment of neutrophils with 40 µM C2-ceramide for 5 min before stimulation with 1 µM FMLP for 15 s blocked activation of the 63- and 69-kDa Paks along with the 49- and 40-kDa kinases (Fig. 1Go). C2-ceramide also reduced the basal activities of these kinases in unstimulated neutrophils (Fig. 1GoC). The decreases in Pak activity were estimated by comparing the heights of the bands in lane b with those in lane d by densitometry. Treatment of neutrophils with 40 µM C2-ceramide for 5 min before stimulation with 1 µM FMLP for 15 s reduced the content of 32P in the 63- and 69-kDa bands by 84 ± 15% and 83 ± 14% (SD, n = 6), respectively. In contrast, similar treatment of the cells with 40 µM C2-dihydroceramide reduced the content of 32P in these bands only 15 ± 11% and 23 ± 18% (SD, n = 4), respectively (Fig. 1Go). Neither increasing the incubation time of the cells with C2-dihydroceramide to 15 min, nor increasing the concentration of C2-dihydroceramide to 80 µM altered these results (data not shown).

Effects of the various sphingoid bases on the activation of the 63- and 69-kDa Paks are summarized in Figure 2Go. Treatment of neutrophils with D-erythrosphingosine, D,L-threodihydrosphingosine, or D-erythrodihydrosphingosine, each at 10 µM, for 5 min before stimulation with 1 µM FMLP for 15 s reduced the content of 32P in the 63- and 69-kDa bands by 82 ± 14% and 77 ± 18% (SD, n = 6), 85 ± 5% and 76 ± 8% (SD, n = 3), and 85 ± 2% and 85 ± 4% (SD, n = 3), respectively. The complete gel profiles showing the effects of D-erythrosphingosine on the renaturable protein kinases in unstimulated and stimulated neutrophils are presented in Figure 4Go. It is noteworthy that the effects of D-erythrosphingosine and D,L-threodihydrosphingosine were very similar in these studies (Fig. 2Go), even though these compounds differ in their stereochemistry at carbon atoms 2 and 3 and in the presence of a trans-double bond between carbon atoms 4 and 5. The significance of these data to the specificity of the relevant target(s) of the sphingoid bases is dealt with in Discussion. The concentrations of sphingolipids utilized in these studies did not affect cell viability, as measured by the exclusion of trypan blue or the release of lactate dehydrogenase. Previous studies monitoring Ca2+ fluxes in neutrophils have established that C2-ceramide and sphingosine do not block the binding of FMLP to its receptor (24, 27).



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  		FIGURE 2. Effects of various sphingoid bases on the activation of the 63- and 69-kDa Paks in neutrophils. Autoradiographs demonstrate the effects of D-erythrosphingosine (A), D,L-threodihydrosphingosine (B), and D-erythrodihydrosphingosine (C) on the activation of the 63- and 69-kDa Paks in neutrophils. Intact cells were treated with: lane a, 0.25% (v/v) DMSO for 5 min, followed by an additional 0.25% (v/v) DMSO for 15 s (unstimulated cells); lane b, 0.25% (v/v) DMSO for 5 min, followed by 1 µM FMLP for 15 s; and lane c, 10 µM sphingoid base for 5 min, followed by 1 µM FMLP for 15 s. The concentrations of sphingoid bases utilized in these studies did not affect cell viability, as measured by the exclusion of trypan blue. Paks were monitored after renaturation by their ability to catalyze the phosphorylation of the p47-phox peptide fixed within a gel, as described in Materials and Methods. The positions of the 69- and 63-kDa Paks are designated by an arrowhead and arrow, respectively.

 


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  		FIGURE 4. Effects of different concentrations of C2-ceramide and D-erythrosphingosine on the activation of the renaturable protein kinases in neutrophils. Neutrophils were treated with various amounts of C2-ceramide (A) or D-erythrosphingosine (B) for 5 min at 37°C before stimulation with 1 µM FMLP for 15 s. The renaturable kinases were monitored by their ability to catalyze the phosphorylation of the p47-phox peptide fixed within the gel. A, The concentrations of C2-ceramide utilized were: lane b, 0 µM; lane c, 50 µM; lane d, 40 µM; lane e, 20 µM; and lane f, 10 µM. B, The concentrations of D-erythrosphingosine utilized were: lane b, 0 µM; lane c, 15 µM; lane d, 10 µM; lane e, 5 µM; and lane f, 2.5 µM. Lane a in A and B is for unstimulated cells not treated with C2-ceramide or D-erythrosphingosine. The positions of the 63- and 40-kDa kinases are designated by unbroken arrows, whereas the 69- and 49-kDa kinases are marked by arrowheads.

 
Kinetics of the inhibition of Pak activation by sphingolipids

The 63- and 69-kDa Paks exhibited maximal activation within 15 s of cell stimulation, followed by significant inactivation at 3 min (2) (Fig. 3Go). Optimal amounts of C2-ceramide and the sphingoid bases were effective at blocking activation of the 63- and 69-kDa Paks at all time points examined (Fig. 3Go, II, IV, and V), whereas C2-dihydroceramide was ineffective at all periods tested (Fig. 3GoIII). C2-ceramide and D-erythrosphingosine blocked activation of the 63- and 69-kDa Paks in a dose-dependent manner, with the effective concentrations being 20 to 40 µM and 5 to 10 µM, respectively (Fig. 4Go).



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  		FIGURE 3. Effects of various sphingolipids on the time course for activation of the 63- and 69-kDa Paks in stimulated neutrophils. Neutrophils were incubated with 0.25% (v/v) DMSO (I; control cells), 40 µM C2-ceramide (II), 40 µM C2-dihydroceramide (III), 10 µM D-erythrosphingosine (IV), and 10 µM D,L-threodihydrosphingosine (V) for 5 min prior to stimulation with 1 µM FMLP. The cells were treated with: lane a, 0.25% (v/v) DMSO for 15 s (unstimulated cells); lane b, FMLP for 15 s; lane c, FMLP for 30 s; lane d, FMLP for 1 min; lane e, FMLP for 3 min; and lane f, 0.25% (v/v) DMSO for 3 min. Paks were monitored by their ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed within a gel. The positions of the 69- and 63-kDa Paks are designated by an arrowhead and arrow, respectively.

 
Sphingosine-1-phosphate can function as a second messenger that modulates certain cellular responses (e.g., 22). Exposure of neutrophils to this lipid (10 µM) for 1 to 10 min did not trigger activation of the 63- and 69-kDa Paks, nor did it block activation of these kinases upon subsequent stimulation of the cells with FMLP (n = 2) (data not shown).

Activation of the 63- and 69-kDa Paks can also be monitored by the ability of these kinases to undergo autophosphorylation after immunoprecipitation from lysates of stimulated neutrophils (7, 39) (Fig. 5Go). Treatment of neutrophils with 40 µM C2-ceramide or 10 µM D-erythrosphingosine for 5 min before stimulation with 1 µM FMLP for 15 s reduced this autophosphorylation reaction (Fig. 5Go, lanes c and e). The amounts of 32P in the 63- and 69-kDa bands from stimulated cells treated with 40 µM C2-ceramide or 10 µM D-erythrosphingosine were reduced by 78 ± 8% and 84 ± 6% (SD, n = 3) and 73 ± 3% and 78 ± 4% (range, n = 2), respectively. Western blotting was performed to establish that equal amounts of Pak were immunoprecipitated in these experiments (data not shown). In contrast, addition of these lipids to the immunoprecipitated kinases during the autophosphorylation assay (Fig. 5Go, lanes d and f) or to the phosphorylation step of the "in gel" renaturation assay with the p47-phox peptide substrate did not affect these reactions (n = 2; data not shown). These data strongly indicate that sphingolipids at these concentrations do not interact with the 63- and 69-kDa Paks themselves, but on (an) upstream component(s) involved in the activation of these kinases (see Discussion).



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  		FIGURE 5. Effects of C2-ceramide and D-erythrosphingosine on the activation of the 63- and 69-kDa Paks in neutrophils monitored by autophosphorylation. Paks were immunoprecipitated from neutrophil lysates with the Pak(C-19) Ab and monitored by their ability to undergo autophosphorylation, as referenced in Materials and Methods. The autoradiograms shown were derived from Paks immunoprecipitated from lysates of: lane a, unstimulated cells; lane b, stimulated cells; lane c, cells treated with 40 µM C2-ceramide for 5 min prior to stimulation; lane d, stimulated cells with 40 µM C2-ceramide added to the immune complex during the autophosphorylation reaction; lane e, cells treated with 10 µM D-erythrosphingosine for 5 min prior to stimulation; and lane f, stimulated cells with 10 µM D-erythrosphingosine added to the immune complex during the autophosphorylation reaction. Cells were stimulated with 1 µM FMLP for 15 s. The 69- and 63-kDa Paks are designated by the arrowhead and solid arrow, respectively.

 
Effects of sphingomyelinase on the activation of the 63- and 69-kDa Paks

Treatment of leukocytes and other cell types with sphingomyelinase increases the cellular content of natural, long chain ceramides and their breakdown products (e.g., sphingosine) (37, 42). For example, incubation of HL-60 cells with sphingomyelinase (0.10–0.38 U/ml) for 15 min increases the content of ceramide from 89 to 445 pmol/106 cells (42). Neutrophils were treated with sphingomyelinase to evaluate possible effects of long chain ceramides and/or endogenously generated sphingosine on the activation of the 63- and 69-kDa Paks (Fig. 6Go). Treatment of neutrophils with sphingomyelinase (2 U/ml) at 37°C for 20 min reduced the amount of 32P in the 63- and 69-kDa bands from neutrophils stimulated with FMLP for 1 min by 66 ± 11% and 74 ± 10% (SD, n = 3), respectively. These results appeared to reflect the catalytic activity of sphingomyelinase because the inhibitory effects observed with this enzyme were both time and dose dependent. For example, blockade of Pak activation was markedly reduced if the incubation time with sphingomyelinase was reduced to 2 to 5 min or if the dose was lowered to 0.03 U/ml. Furthermore, heat inactivation of sphingomyelinase abolished its inhibitory effects on the activation of the 63- and 69-kDa Paks (n = 2; data not shown). Interestingly, treatment of neutrophils with sphingomyelinase alone resulted in the activation of a 45-kDa kinase (Fig. 6Go, lane d, asterisk). While the identity of this kinase is not known, it did not react with the Pak(C-19) Ab during Western blotting.



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  		FIGURE 6. Effects of sphingomyelinase on the activation of the 63- and 69-kDa Paks in neutrophils. Neutrophils (3 x 106/ml) were incubated for 20 min at 37°C in the standard assay medium in the absence (lanes a, b, and c) or presence (lanes d, e, and f) of sphingomyelinase (2 U/ml). The cells were subsequently treated with: lanes a and d, 0.25% (v/v) DMSO for 15 s (unstimulated cells); lanes b and e, 1 µM FMLP for 15 s; and lanes c and f, 1 µM FMLP for 1 min. Paks were monitored by their ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed with a gel. The 69- and 63-kDa Paks are designated by an arrowhead and solid arrow, respectively. The broken arrow indicates the position of a 96-kDa kinase that undergoes activation at time periods >=1 min, and the asterisk (*) indicates a 45-kDa kinase that undergoes activation in sphingomyelinase-treated cells. Treatment of neutrophils with sphingomyelinase did not affect cell viability, as measured by the exclusion of trypan blue.

 
Effects of certain sphingolipids on the rate of inactivation of Paks in stimulated neutrophils

Figure 7Go summarizes the effects of adding D-erythrosphingosine or C2-ceramide to neutrophils shortly after stimulation with FMLP. Neutrophils stimulated with FMLP for 1 or 2 min with D-erythrosphingosine (10 µM) added 15 s after FMLP exhibited significantly less activity for the 63- and 69-kDa Paks than cells stimulated with FMLP alone for 1 or 2 min (Fig. 7GoA). Since these kinases were at maximal activity at the time D-erythrosphingosine was added (i.e., lane b), this lipid must therefore enhance the rate of Pak inactivation under these circumstances. Similar results were observed for the 49- and 40-kDa kinases. The enhanced inactivation of the 63- and 69-kDa Paks with D-erythrosphingosine was estimated by comparing the heights of the peaks in lanes c and d with those in lanes e and f by densitometry. Adding D-erythrosphingosine to stimulated cells reduced the amounts of 32P in the 63- and 69-kDa bands at 1 and 2 min by 83 ± 7% and 85 ± 10% (range, n = 2) and 97 ± 2% and 93 ± 7% (SD, n = 3), respectively. Similarly, addition of C2-ceramide (40 µM) to FMLP-stimulated neutrophils also resulted in a diminution of these activities (Fig. 7GoB). When C2-ceramide was added to neutrophils 15 s after FMLP, the amounts of 32P in the 63- and 69-kDa bands at 1 and 2 min were reduced by 18 ± 6% and 0 ± 0% (SD, n = 3) and 86 ± 9% and 78 ± 9% (SD, n = 6), respectively. The effects with D-erythrosphingosine and C2-ceramide were highly specific for the 69-, 63-, 49-, and 40-kDa kinases, as none of the other renaturable enzymes in unstimulated or stimulated neutrophils exhibited a similar loss in activity. Importantly, addition of arachidonate (10 µM) to neutrophils 15 s after FMLP reduced the content of 32P in the 63- and 69-kDa Paks at 2 min by only 18 ± 4% and 19 ± 8% (SD, n = 3), respectively. Arachidonate is known to disorganize/destabilize the membranes of neutrophils at this concentration and produces striking alterations in the morphology of these cells (43) (see Discussion). Arachidonate alone did not trigger activation of the 63- and 69-kDa Paks (data not shown). Thus, the effects of sphingolipids on Pak cannot be mimicked by an agent that disorganizes the membranes of these cells.



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  		FIGURE 7. Alterations in the activities of the 63- and 69-kDa Paks when D-erythrosphingosine or C2-ceramide was added to neutrophils shortly after stimulation with FMLP. Autoradiographs demonstrate the effects of D-erythrosphingosine (A) and C2-ceramide (B) on the activities of the 63- and 69-kDa Paks when these lipids were added to neutrophils after stimulation with 1 µM FMLP. Paks were monitored by their ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed within a gel, as described in Materials and Methods. A, Neutrophils were treated with: lane a, 0.25% (v/v) DMS0 for 15 s (unstimulated cells); lane b, FMLP for 15 s; lane c, FMLP for 1 min; lane d, FMLP for 2 min; lane e, FMLP for 1 min with 10 µM D-erythrosphingosine added 15 s after FMLP; and lane f, FMLP for 2 min with 10 µM D-erythrosphingosine added 15 s after FMLP. B, Neutrophils were treated with: lane a, 0.25% (v/v) DMS0 for 15 s; lane b, FMLP for 15 s; lane c, FMLP for 2 min; and lane d, FMLP for 2 min with 40 µM C2-ceramide added 15 s after FMLP. The positions of the 69- and 63-kDa Paks are designed by the arrowhead and arrow, respectively. The broken arrow indicates the position of a 96-kDa kinase that undergoes activation at time points >=1 min.

 
It is possible that the effects of C2-ceramide on neutrophils were due to its degradation to D-erythrosphingosine. This possibility was investigated and eliminated by measuring the increase in D-erythrosphingosine in neutrophils (3 x 107/ml) incubated with 40 µM C2-ceramide for 5 min, followed by stimulation with 1 µM FMLP for 15 s (see Materials and Methods). In three experiments, the increases in D-erythrosphingosine were 2.5, 0, and 0%. An earlier study measuring Ca2+ fluxes also concluded that significant conversion of C2-ceramide to D-erythrosphingosine did not occur in human neutrophils over the duration of the experiments (27). Whether C2-ceramide was metabolized to other biologically active lipids (e.g., short chain sphingomyelin, glycosphingolipids) during these experiments is not known.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work, we report that products of sphingolipid catabolism block activation of the 63- and 69-kDa Paks in neutrophils. Moreover, we demonstrate that these compounds can also increase the rate of Pak inactivation when added to cells after stimulation. Paks undergo autophosphorylation/activation upon interacting with the activated forms of Cdc42 or Rac (9). Pak can also undergo a Rac/Cdc42-independent activation upon association with membrane or certain lipids (36, 44). All of these reactions in vitro require several minutes to 1 h for maximal activation of Pak to occur (36, 45, 46). In contrast, optimal activity of the 63- and 69-kDa Paks is observed within 15 s in stimulated neutrophils (2) (Fig. 3Go). The small adaptor protein Nck and the ß-subunit of a heterotrimeric G protein bind specifically to Pak and may mediate the translocation of this kinase to the membrane (44, 47, 48, 49). Pak can also form a tight complex with PIX, a guanine nucleotide exchange factor for Rac (50). Pak, Cdc42, and Nck undergo a very rapid (<=15 s) translocation to the ruffling membranes of stimulated neutrophils (51). The exact events that trigger the rapid activation of the Paks in neutrophils remain unknown.

The structures of C2-ceramide, C2-dihydroceramide, sphingosine, and dihydrosphingosine are provided in Figure 8Go. It is difficult to envision a single target that is sensitive to all four of these compounds, since the inhibitory effects of C2-ceramide are dependent upon the presence of the double bond between carbon atoms 4 and 5 (Fig. 1Go), whereas the effects with sphingosine are not (Fig. 2Go). Thus, it may be reasonable to assume that C2-ceramide and the sphingoid bases have separate targets in neutrophils. Interestingly, C2-ceramide, but not dihydroceramide or sphingosine, inhibits translocation of Cdc42 to the membrane of FMLP-stimulated neutrophils by 82% (29). Whether C2-ceramide blocks GTP/GDP exchange on Cdc42 or inhibits the interaction of this small GTPase with the membrane after activation is not known. The concentration of C2-ceramide that was effective against Cdc42 (50 µM) (29) was similar to that which inhibits Pak activation (20–40 µM) (Fig. 4Go). Neutrophils treated with 50 µM C2-ceramide for 1 h (versus the 5-min period utilized in these studies) contain 32 ± 6 pmol of this lipid/nmol of phospholipid (29). The concentration of long chain ceramides in unstimulated neutrophils is approximately 2.5 pmol/nmol phospholipid (17, 29), and this value can increase 10-fold during cell stimulation due to the action of a neutral sphingomyelinase (26, 28). Thus, the concentrations of C2-ceramide that are effective against Pak are reasonable physiologically.



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  		FIGURE 8. Structures of C2-ceramide, C2-dihydroceramide, sphingosine, and dihydrosphingosine.

 
C2-ceramide, but not C2-dihydroceramide, can destabilize membranes at ceramide:lipid ratios of 0.2 to 0.3 (52). Data presented above indicate that C2-ceramide blocks neutrophil responses at ratios of <=0.033 (Fig. 4Go and 29 . Even if 100% of the 40 µM C2-ceramide partitioned into the membranes of guinea pig neutrophils, the ceramide:lipid ratio would be only approximately 0.10. Moreover, arachidonate markedly increases the fraction of the membrane in neutrophils which is liquid-cyrstalline (destabilized) as measured in fluorescence polarization studies (43), but did not mimick the effects of sphingolipids on Pak (see Results). These data indicate that the effects of C2-ceramide on the 63- and 69-kDa Paks are not the result of destabilizing the membranes of these cells.

In contrast to the C2-ceramides, the ability of sphingoid bases to block Pak activation was not dependent upon the trans-double bond between carbon atoms 4 and 5, or a particular stereochemistry for the hydroxyl groups at carbon atoms 2 and 3. A similar situation has been reported earlier for the inhibition of PKC and O2- release from neutrophils (25). One explanation for this effect is that positively charged sphingoid bases may partition into regions of the plasmalemma that contain acidic phospholipids, neutralize these molecules, and disrupt complexes required for cell stimulation (e.g., 25). We have reported previously that 3-phosphorylated inositides are involved in the activation of the 63- and 69-kDa Paks (6). D-3-phosphoinositides can activate exchange factors for Rac (e.g., 53). Similarly, Rac forms a stable signaling complex with both a type I phosphatidylinositol-4-phosphate 5-kinase and a diacylglycerol kinase (54). The acidic lipids that are products of these lipid kinases may also function in the activation of Rac (54). It should also be noted that all four stereoisomers of sphingosine provide the same headgroup conformation and vary only in the position of the alkyl chain (25). A specific target for sphingoid bases could therefore exist that recognizes only the headgroup of these molecules and not the hydrophobic region. Thus, few conclusions can be drawn from the data presented herein as to whether sphingoid bases affect a specific target protein that functions in the activation of Pak. Importantly, the amounts of endogenous sphingosine in neutrophils are within an order of magnitude of the exogenous concentrations (5–10 µM) that inhibit O2- release (24, 25) and block activation of Pak (Fig. 4Go) (55).

As noted above, Pak 1 can be directly activated in vitro by a variety of sphingoid bases at concentrations of 50 to 400 µM (36). In contrast, neutrophils treated with various sphingoid bases at 10 µM or C2-ceramide for 5 min exhibited a diminution in the basal activities of the 63- and 69-kDa Paks (Figs. 1Go and 3Go, compare lane a in I, IV, and V) and a marked reduction in the activation of these kinases after stimulation with FMLP (Figs. 2Go, 3Go, and 5Go). Sphingoid bases at 10 µM and C2-ceramide (10–50 µM) are known to block a variety of functional responses in neutrophils (24, 25, 26, 27, 28, 29). These data indicate that the effects of sphingolipids on Paks are likely to be dependent upon a number of factors that include the concentration of lipid, cell type employed, and/or the stimulatory pathway utilized to activate these kinases.

Regardless of the exact nature of the upstream events that trigger activation of the 63- and 69-kDa Paks, these kinases must clearly undergo covalent modification during activation (i.e., phosphorylation) (3) since the enhanced activity persists even after SDS-PAGE and the denaturation/renaturation steps (cf 1–3). It is therefore particularly interesting that addition of C2-ceramide or D-erythrosphingosine to neutrophils 15 s after stimulation with FMLP increases the rate of Pak inactivation (Fig. 7Go). Since these kinases were at maximal activation when the sphingolipid was added, the simplest explanation for this effect is that these compounds enhance the dephosphorylation of Pak under these circumstances. There are several possible mechanisms that can explain this effect. First, Paks may undergo both phosphorylation and dephosphorylation in stimulated neutrophils, with the phosphorylation reaction predominating immediately after cell stimulation. Interruption of the phosphorylation reaction by sphingolipids would allow the dephosphorylation reaction to predominate. Second, sphingolipids may disrupt complexes or associations that are necessary for Pak activation (cf 50, 54) or that shield Paks from phosphatases. Third, sphingolipids may activate protein phosphatases that utilize Pak as a substrate (cf 56). In all three of these situations, the same targets for sphingolipids could both block activation of the 63- and 69-kDa Paks (e.g., Figs. 1–5GoGoGoGoGo) and mediate the inactivation of these kinases (Fig. 7Go), depending upon whether these lipids were added to the cells before or after stimulation.

The levels of D-erythrosphingosine and ceramide are increased in neutrophils during a variety of physiologic situations (26, 28, 31). In particular, neutrophils exhibit significant increases in both sphingosine and ceramide at 15 min to 2 h after stimulation with FMLP (26, 31). Since activation of Paks in FMLP-stimulated neutrophils is over by 5 min (Fig. 3Go), it is unlikely that sphingolipids are involved in terminating the activation of these kinases, but rather are active in establishing a set point for a subsequent stimulus. A similar situation has been described earlier for PKC (57). This set point is likely to function to prevent accidental stimulation of neutrophils. Finally, high concentrations of ceramide and sphingoid bases are present in patients with various sphingolipid storage diseases (e.g., Farber’s lipogranulomatosis, type C Niemann-Pick disease) (32, 33) or cells infected with certain mycotoxins (fumonisins) (e.g., 34). The possibility thus exists that inhibition of Pak activation by these lipids may contribute to at least some of the pathologic events associated with these disorders.


   Acknowledgments
 
We are grateful to Ms. Paula Geary for her help with these experiments, and Ms. Angela DiPerri for typing this paper.


   Footnotes
 
1 These studies were supported by National Institutes of Health Grants DK 50015, AI 23323 (to J.A.B.), and AR 43518 (to D.R.). Back

2 Address correspondence and reprint requests to Dr. John A. Badwey, Boston Biomedical Research Institute, 20 Staniford St., Boston, MA 02114. Back

3 Abbreviations used in this paper: Pak, p21-activated protein kinase; ERK, extracellular-regulated kinase; MAP, mitogen-activated protein; p47-phox, the 47-kDa protein component of the phagocyte oxidase; PKC, protein kinase C. Back

Received for publication March 24, 1998. Accepted for publication June 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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