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* This work was supported by Grant AI23323 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Several novel protein kinases are known to be rapidly activated in neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP). These kinases include a histone H4 protein kinase and several renaturable kinases with molecular masses of about 69, 63, 49, and 40 kDa. The renaturable kinases can catalyze the phosphorylation of a peptide that corresponds to residues 297–331 of the 47-kDa subunit of the NADPH-oxidase system (p47-phox). Previous studies have indicated that the activation of all of these protein kinases involves an uncharacterized stimulatory pathway and/or novel second messenger. The studies reported herein were undertaken to determine if phosphatidylinositol 3-kinase (PI3-K) is a component of this pathway. We report that certain chromone derivatives (e.g. 2-(4-morpholinyl)-8-phenylchromone (LY294002)) and wortmannin, which inhibit PI3-K by distinct mechanisms, blocked activation of all of these novel kinases. These antagonists also inhibited the phosphorylation of p47-phox (about 50%) and O2⨪ release (about 80%) in cells stimulated with fMLP, but not with 4β3-phorbol 12-myristate 13-acetate. A strong correlation exists between the amounts of these antagonists required to produce 50% inhibition of PI3-K in vitro and O2⨪ release in vivo. In contrast, a single atom substitution of LY294002 produced a compound (LY303511) that did not inhibit PI3-K. Compound LY303511 did not appreciably inhibit the activation of the novel protein kinases or OT2 generation. These data strongly suggest that PI3-K is involved in the activation of several novel protein kinases in neutrophils, one or more of which may be involved in O2⨪ release.
The abbreviations used are: fMLP, N-fMet-Leu-Phe; O2, superoxide; PI3-K, phosphatidylinositol 3-kinase; PMA, 4β-phorbol 12-myristate 13-acetate; Ptdlns(3,4,5)3, phosphatidylinositol 3,4,5-trisphosphate; PIP3, phosphatidylinositol trisphosphate; p47-phox, the 47-kDa protein component of the phagocyte oxidase; PKC, protein kinase C; MLCK, myosin light chain kinase; LY294002, 2-(4-morpholinyl)-8-phenylchromone; LY292223, 2-(4-morpholinyl)-chromone; LY303511, 2-(4-piperazinyl)-8-phenyl-4H-1-benzopyran-4-one; H-7, 1-(5-isoquinolinylsulfonyl)-2-meth-ylpiperazine; ML-7, 1-(5-iodonaphthalene-1-sulfonyl)-lH-hexahydro-1,4-diazepine hydrochloride; ML-9, l-(5-chloronaphthalene-l-sulfonyl)-lH-hexahydro-l,4-diazepine hydrochloride; IC50, concentration of drug that produces 50% inhibition; Me2SO, dimethyl sulfoxide.
1The abbreviations used are: fMLP, N-fMet-Leu-Phe; O2, superoxide; PI3-K, phosphatidylinositol 3-kinase; PMA, 4β-phorbol 12-myristate 13-acetate; Ptdlns(3,4,5)3, phosphatidylinositol 3,4,5-trisphosphate; PIP3, phosphatidylinositol trisphosphate; p47-phox, the 47-kDa protein component of the phagocyte oxidase; PKC, protein kinase C; MLCK, myosin light chain kinase; LY294002, 2-(4-morpholinyl)-8-phenylchromone; LY292223, 2-(4-morpholinyl)-chromone; LY303511, 2-(4-piperazinyl)-8-phenyl-4H-1-benzopyran-4-one; H-7, 1-(5-isoquinolinylsulfonyl)-2-meth-ylpiperazine; ML-7, 1-(5-iodonaphthalene-1-sulfonyl)-lH-hexahydro-1,4-diazepine hydrochloride; ML-9, l-(5-chloronaphthalene-l-sulfonyl)-lH-hexahydro-l,4-diazepine hydrochloride; IC50, concentration of drug that produces 50% inhibition; Me2SO, dimethyl sulfoxide.
binds to specific receptors on neutrophils and triggers a myriad of phenomena which include shape changes, directed migration, degranulation, and the release of large quantities of O2⨪. Superoxide is a major component of the oxygen-dependent, antimicrobial mechanisms of phagocytic leukocytes (
). The latter kinases can be detected by a procedure based on their ability to undergo renaturation and autophosphorylation in a gel, or to catalyze the phosphorylation of a protein or peptide substrate fixed in the gel (
). Interestingly, the renaturable kinases can catalyze the phosphorylation of peptides that correspond to the sites of phosphorylation in p47-phox with kinetics virtually identical to the phosphorylation of this protein in intact neutrophils (
). Thus, this compound affects a very specific stimulatory pathway. Whether this pathway may contain the novel protein kinases and involves phosphorylation of p47-phox has not been investigated until now.
In this paper, we have compared the abilities of a far more extensive series of antagonists of PI3-K on O2⨪ release from neutrophils than hitherto examined. We report that these antagonists block activation of the histone H4 protein kinase. Effects of a number of chromones on the activation of the renaturable 69-, 63-, 49-, and 40-kDa kinases are also described. Evidence against a role for MLCK or phospholipase D in the activation of these kinases is provided. Finally, we report that wortmannin or LY294002 inhibit phosphorylation of p47-phox in neutrophils stimulated with fMLP, but not with PMA. The data indicate PI3-K plays a critical role in the activation of several novel protein kinases in neutrophils, one or more of which may be involved in O2⨪ production,
Compound LY294002, LY303511, and all of the other chromone derivatives employed were synthesized as described in Ref.
. Wortmannin was purchased from Kamiya Biomedical Co., Thousand Oaks, CA. Rabbit antiserum to the p85-kDa subunit of rat PI3-K that precipitates both the 85- and 110-kDa subunits of this enzyme was obtained from UBI, Lake Placid, NY. Phosphatidylinositol (
)-bisphosphate, p-nitrophenyl phosphate, and phenylmethylsulfonyl fluoride were purchased from Sigma. Histone H4 was obtained from Calbiochem. K6 Silica Gel 60 A TLC plates (20 × 20 cm) and p81 filter paper were obtained from Whatman. Sources of all other materials are described elsewhere (
). These preparations contained >90% neutrophils with viabilities always >90%.
Superoxide release was measured in disposable 1-cm plastic cuvettes at 37 °C by the continuous spectrophotometric measurement of superoxide dismutase-inhibitable reduction of ferricytochrome c at 550 nm (e.g. Ref.
). The standard assay mixture consisted of a modified Dulbecco's phosphate-buffered saline medium (135 mM NaCl, 2.7 mM KCl, 16.2 mM Na2P04, 1.47 mM KH2P04, 7.5 mM D-glucose, 0.90 mM CaCl2, and 0.50 mM MgCl2, pH 7.35) containing 0.075 mM ferricytochrome c and 1.0 to 3.0 × 106 cells/ml. The blank contained all of these components plus 20 µg/ml superoxide dismutase. Cells were incubated in the reaction mixture for 3–5 min at 37 °C before O2⨪ release was initiated by the addition of 1.0 μΜ fMLP or 50 nM PMA.
Stock solutions of PMA (2.0 mg/ml), fMLP (10 mM), wortmannin (4.0 mM), LY294002 (40 mM), LY303511 (40 mM), and the various chromones (40–60 mM) utilized in Fig. 2 were prepared in Me2SO and stored at-20 °C. These compounds were diluted with Me2SO so that the final amount of solvent in each assay was either 0.25 or 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.
Labeling of Neutrophils with 32Pi and SDS-Polyacrylamide Gel Electrophoresis for Studies on Protein Phosphorylation—These techniques were performed as described previously (
Neutrophils were labeled with *2Ρ, as described above. Cells (107/ml) were incubated in phosphate-buffered saline for 5 min at 37 °C and then stimulated with 1.0 μΜ fMLP for 30 s. The lipids were extracted, washed, dried, and separated on K6 Silica Gel 60A TLC plates as described in Refs.
. Separated [32P]phosphoIipids were visualized by autoradiography.
Detection of Renaturable Protein Kinases in Polyacrylamide Gels
In this procedure, protein kinases are detected directly in gels by their ability to undergo renaturation and catalyze the phosphorylation of histone H4 or a peptide substrate that corresponds to residues 297 to 331 of p47-phox “fixed” in the gel. This technique is presented in detail in Refs.
). Neutrophils (107 cells) were incubated in 0.50 ml of phosphate-buffered saline at 37 °C for 5 min and then stimulated with 1.0 μΜ fMLP for various periods of time. Reactions were terminated by the addition of 0.50 ml of ice-cold “lysis buffer” and the samples were placed in wet ice for 10–15 min. The lysis buffer consisted of 1.0% (v/v) Triton X-100, 50 mM Tris (pH 7.2), 33.0 mM p-nitrophenyl phosphate, 0.133 mM sodium orthovanadate, 1.33 mM phenylmethylsulfonyl fluoride, and 66.7 mM mercaptoethanol. Samples were subjected to microcentrifugation for 5 min at 15,000–20,000 rpm and 0.03 ml of the supernatant was assayed for kinase activity. The assay mixture (0.10 ml) for measuring histone H4 protein kinase contained histone H4 (20 µg/0.10 ml), 40 mM Hepes (pH 7.2), 10.0 mM MgCl2, 1.0 mM EDTA, 50 μΜ [γ-32P]ATP (1.0 U.Ci), and all of the components of the lysis buffer described above diluted by one-third. Reactions were initiated by the addition of [γ-32P]ATP and run for 7 min at 37 °C. Aliquots (0.03 ml) of the reaction mixture were spotted on Whatman p81 filter paper and the samples washed, dried, and counted for 32Pi as described in Ref.
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) are also based on different cell preparations.
Effects ofLY294002, LY303511, and Various Antagonists of PI3-K on Superoxide Release by Neutrophils
Neutrophils stimulated with 1.0 μΜ fMLP release O2⨪ at a rate of about 45 nmol of O2⨪/min/107 cells (e.g. Ref.
). This response was immediate and transient with the majority of the event occurring in the first 3 min (Fig. 1, curve a). Treatment of the cells with different concentrations of LY294002 for 5 min prior to stimulation with fMLP resulted in a dose-dependent inhibition of O2⨪ release (Fig. L4). Half-maximal inhibition occurred at 6.4 ± 1.2 μΜ LY294002 (S.D., n = 3) (Fig. LS, open circles). The Hill coefficient for these data was about 1.0 (0.95 ± 0.11 (S.D., n = 3); Fig. 1B, inset) which is consistent with this drug interacting with a single target. In contrast, LY294002 (50 μΜ) did not appreciably block O2⨪ release from neutrophils stimulated with 50 nM PMA (Fig. LS, triangles). LY294002 inhibited O2⨪ release from neutrophils stimulated with 1.0 μΜ fMLP or 50 nM PMA by 83 ± 10 and 20 ± 10% (S.D., n = 3), respectively. PMA bypasses surface receptors and directly activates PKC (
Compound LY303511 is a single atom substitution of LY294002 that has little activity against PI3-K and thus provides an excellent control for evaluating possible nonspecific effects of chromones on cells (
). This analog involves the simple replacement of the oxygen atom in the morpholine ring of LY294002 with an N-H group. Compound LY303511 (50 μΜ) did not appreciably inhibit O2⨪ release (7 ± 9%, S.D., n = 3) from fMLP-stimulated cells (Fig. 1B, closed circles).
Fig. 2 compares the abilities of a number of chromone derivatives to inhibit O2⨪ release from fMLP-stimulated neutrophils. The structures of these molecules are presented in Ref.
. All of these analogs blocked O2⨪ release from neutrophils in a dose-dependent manner (Fig. 2A). A striking correlation (r = 0.997) was observed between the amounts of these antagonists required to produce 50% inhibition of PI3-K in vitro and O2⨪ release in vivo (Fig. 2B). In contrast, optimal amounts of these derivatives did not appreciably inhibit (s20%) O2⨪ release from cells stimulated with 50 nM PMA (data not shown).
Effects of Various Compounds on PIP3 Production
PI3-K was immunoprecipitated from neutrophils with an antibody to the 85-kDa subunit of this kinase and the effects of various compounds on the activity of this enzyme were measured in the immunoprecipitates (Fig. 3A). LY294002 (25 and 50 μΜ) and wortmannin (200 nM) markedly inhibited the enzyme, whereas compound LY303511 (25 and 50 μΜ) did not. Human neutrophils and U937 cells contain different isozymes of PI3-K, some of which do not react with the antibodies available to the 85-kDa subunit (
). The location of PIP3 in the chromatogram was confirmed by comparison with PIP3 generated enzymatically as in Fig. 3A. Synthesis of PIP3 in stimulated neutrophils was inhibited by 25 μΜ LY294002 (lane c) and 200 nM wortmannin (lane f), but not by 25 μΜ 303511 (lane d). An antagonist of PKC, H-7 (
) (Fig. 4). These kinases can be detected directly in gels after renaturation by their ability to catalyze the phosphorylation of a peptide substrate uniformly distributed and fixed in the gel. The positions of the protein kinases are visualized by autoradiography after exposure of the gel to [γ-32P]ATP (
). However, in those experiments in which significant increases in activity were observed, the activation of this enzyme exhibited the same sensitivity to inhibitors as the 63-, 49-, and 40-kDa kinases (e.g.Fig. 4; Ref.
). Treatment of neutrophils with 50 μΜ LY294002 for 5 min prior to stimulation with 1.0 μΜ fMLP significantly reduced the activation of the 69-, 63-, 49-, and 40-kDa protein kinases (Fig. AB). Some inhibition of these protein kinases was observed at 15 s after stimulation (e.g.Fig. 4, panels C and D) and this inhibition increased at later time points (e.g. 30 s; compare lane c in Fig. 4, A and B). These decreases were estimated by densitometry by comparing the heights of the bands in lane c of Fig. 4A with those in lane c of Fig. 4B. Treatment of the cells with 50 μΜ LY294002 for 5 min prior to stimulation with 1.0 μΜ fMLP for 30 s reduced the content of 32P in the 63-, 49-, and 40-kDa bands by 89 ± 9, 82 ± 8, and 86 ± 10% (S.D., n = 3), respectively. In contrast, similar treatment of the cells with 50 μΜ LY303511 reduced the content of 32P in these bands by only 16 ± 5, 21 ± 8, and 11 ± 8%, respectively (mean ± range, n = 2).
The effects of LY294002 on the activation of the 69-, 63-, 49-, and 40-kDa protein kinases appeared specific since this compound did not effect the renaturable kinases in unstimulated cells (Fig. 4, compare lane a in parts A and B) nor did it block the activation of the 96-kDa kinase (Fig. 4, broken arrow). Moreover, neither 200 μΜ H-7 nor 50 μΜ ML-7 had any inhibitory effects on the activation of these kinases (
) and also blocks activation of the renaturable protein kinases at a concentration of 100 μΜ (Fig. 5). Incubation of neutrophils with 100 μΜ LY292223 for 5 min prior to stimulation with 1.0 μΜ fMLP for 30 s reduced the content of 32P in the 63-, 49-, and 40-kDa bands by 76 ± 6, 79 ± 9, and 71 ± 7% (mean ± range, n = 2), respectively. Neutrophils stimulated with fMLP can also exhibit a variable activation of 3 or 4 uncharacterized protein kinases in the 45- to 40-kDa range (
) (Fig. 5). LY292223 also blocked the activation of these kinases (Fig. 5). In contrast, addition of 50 μΜ LY294002 or 100 μΜ LY292223 to the assay mixture employed to detect the renaturable protein kinases in the gel did not effect the activity of any of these enzymes (data not shown). This suggests that these compounds do not interact with the 69-, 63-, 49-, and 40-kDa protein kinases themselves but on an upstream component involved in the activation of these enzymes.
It is noteworthy that neutrophils treated with a high concentration of LY294002 (50 μΜ) still exhibited a low rate of O2⨪ release (about 20%) that lasted for about 45 to 60 s (Fig. 1A, curve e) along with some activity of the 69-, 63-, 49-, and 40-kDa protein kinases at 15 and 30 s (Fig. 4B, lanes b and c). This short burst of O2⨪ release and residual kinase activity were not eliminated by doubling the concentration of LY294002 and/or by increasing the time of exposure of the cells to this antagonist to 15 min (data not shown). These data provide additional support for a link between one or more of the novel protein kinases and O2⨪ production. Moreover, they indicate that the stimulatory pathway that triggers activation of the 69-, 63-, 49-, and 40-kDa protein kinases may contain a component that is refractory to inhibition by LY294002.
Studies on the Histone H4 Protein Kinase
Neutrophils contain a histone H4 protein kinase of about 70 kDa that is rapidly activated in fMLP-stimulated cells (
). We investigated the effects of antagonists of PI3-K on this activity in both a direct biochemical phosphotransferase assay (Fig. 6A) and in the renaturation assay (Fig. 6B). In the direct biochemical assay it was necessary to rapidly lyse the cells with 0.50% (v/v) Triton X-100 after stimulation since this enzyme undergoes rapid and transient activation (
). The levels of histone H4 protein kinase activity in unstimulated cells and cells treated with 1.0 μΜ fMLP for 10 s were 8.8 ± 5 and 43 ± 2 (S.D., n = 4) picomoles of Ρ incorporated/min/107 cell equivalents, respectively (Fig. 6A).
The histone H4 kinase activity in the Triton X-100 extracts exhibited properties very similar to those of the renaturable 69- and 63-kDa kinases, which suggests that these activities may represent the same enzyme(s) (see Ref.
). Most importantly, activation of the histone H4 protein kinase in neutrophils measured by the quantitative phosphotransferase assay was extensively blocked by wortmannin (200 nM) or LY294002 (50 μΜ), but was much less sensitive to LY303511 (50 μΜ) or H-7 (200 μΜ) (Fig. 6A, inset). Similar behavior was observed in the renaturation assay (Fig. 6B, II-V). In contrast, very little inhibition (<20%) occurred when these compounds were added to assay mixtures containing Triton X-100 extracts from stimulated cells (data not shown). This indicates that LY294002 and wortmannin did not inhibit the histone H4 protein kinase(s) directly but blocked an upstream component involved in the activation of this/these enzyme(s).
Wortmannin is also known to inhibit MLCK at micromolar concentrations (Ki = 1.9 μΜ) (
). Incubation of neutrophils with 30 μΜ ML-7 or ML-9 for 5 min prior to stimulation with 1.0 μΜ fMLP for 30 s did not effect the activation of the histone H4 protein kinase(s) (<10% inhibition, n = 2) measured in either the Triton X-100 extracts or in the renaturation assay (data not shown).
Protein Phosphorylation in Neutrophils
As noted above, LY294002 and wortmannin block O2⨪ release from neutrophils stimulated with fMLP but not with PMA (e.g. Ref.
, 18, and 22). The effects of these antagonists on the phosphorylation of p47-phox with these agonists were therefore examined. Stimulation of 32P-labeled neutrophils with fMLP or PMA results in a rapid incorporation of 32P into a 47-kDa protein (p47-phox) (FIG. 7, FIG. 9, lane b, arrow) (e.g. 3). This protein undergoes transient phosphorylation in guinea pig neutrophils stimulated with fMLP, and responds to various agonists and antagonists of 02 release in a manner identical to that of p47-phox in human neutrophils (e.g. Refs.
). Significantly less phosphorylation of this protein occurred if the cells were treated with 200 nM wortmannin (Fig. 7A, lane c) or 50 μΜ LY294002 (Fig. 8A, lane c) for 5 min prior to stimulation with fMLP. The extent of phosphorylation of p47-phox in the presence of 200 nM wortmannin or 50 μΜ LY294002 was estimated by densitometry (FIG. 7, FIG. 8, part B) and found to be reduced by 54 ± 6% (S.D., n = 3) and 51 ± 6% (S.D., n = 4), respectively. A recent study indicates that more than one protein kinase catalyzes the phosphorylation of p47-phox in stimulated neutrophils (
) which could account for the partial inhibition observed with these antagonists. In contrast, treatment of neutrophils with these inhibitors did not effect the phosphorylation of p47-phox in PMA-stimulated cells (Fig. 9). Wortmannin (200 nM) or LY294002 (50 μΜ) reduced phosphorylation of this protein in PMA-stimulated cells by 5 ± 6 and 0 ± 0% (S.D., n = 3), respectively. The time points utilized for stimulation of neutrophils with fMLP or PMA in FIG. 7, FIG. 8, FIG. 9 represent periods at which the cells exhibited maximal rates of O2 release with these agonists (e.g.Fig. 1A).
Evidence for the Involvement of PI3-K in the Activation of Several Novel Protein Kinases in Neutrophils
As noted above, the stimulatory pathway which triggers activation of the novel protein kinases is likely to be under the control of a novel second messenger and/or stimulatory pathway (
). This pathway is likely to contain PI3-K since activation of the histone H4 kinase and the renaturable kinases was blocked by LY294002 (FIG. 4, FIG. 5, FIG. 6), LY292223 (Fig. 5), and wortmannin (Ref.
) is very similar to those observed for the novel protein kinases (FIG. 4, FIG. 6) which is consistent with PtdIns(3,4,5)P3 serving as a second messenger in this response. Wortmannin and LY294002 block formation of PIP3 in neutrophils (Refs.
). This suggests that a molecule other than phosphatidate is the relevant second messenger. As noted above, PtdIns(3,4,5)P3 production in neutrophils is stimulated by fMLP but not by PMA or an increase in intracellular Ca2+ (
It is likely that the renaturable protein kinases undergo covalent modification during cell stimulation which increases their catalytic activity since the enhanced activity persists even after SDS-polyacrylamide gel electrophoresis and treatment with guanidine. This enhanced activity results, at least in part, from phosphorylation (
). The similar manner in which the novel protein kinases respond to various agonists and antagonists (e.g. LY294002) suggest that all of these enzymes are regulated by a common stimulatory pathway. Earlier studies indicated that this pathway contains a heterotrimeric G-protein and a type 1 and/or 2A protein phosphatase (
). A lipid product of PI3-K may activate a protein kinase that functions upstream of the novel kinases. PtdIns(3,4,5)P3 can activate the Ca2+-insensitive, but not the Ca2+-activated isotypes of PKC under in vivo conditions (
). Alternately, it is possible that the protein Ser kinase activity of PI3-K may directly catalyze the phosphorylation of the novel kinases or a proximal component of the pathway. Only two substrates for this protein Ser kinase activity have been uncovered to date (
). Thus, a general role for PtdIns(3,4,5)P3 in the activation of a variety of protein kinase cascades may be emerging.
Do one or more of the novel protein kinases actually utilize p47-phox as a substrate in vivo? An alternative explanation for the data presented in FIG. 7, FIG. 8 is that PtdIns(3,4,5)P3 activates PKC which catalyzes the phosphorylation of p47-phox at the non-proline directed sites (
). Thus, wortmannin and LY294002 could inhibit the phosphorylation of this protein by indirectly blocking activation of PKC. However, p47-phox is a substrate for the Ca2+-activated β-isoform of PKC, but not the Ca2+-insensitive isoform(s) of this enzyme (
). Similarly PI3-K may function upstream of phospholipase D and products of this phospholipase may activate β-PKC. However, Ca2+-ionophores stimulate very high amounts of phospholipase D activity in neutrophils (
). Thus, the data are consistent with a novel kinase, but not PKC, catalyzing the phosphorylation of p47-phox in fMLP-stimulated neutrophils. Interestingly, antagonists of PI3-K can also inhibit degranulation (