Biochimica et Biophysica Acta, 1055 (1990) 55-62
55
Elsevier BBAMCR 12782
Utility of staurosporine in uncovering differences in the signal transduction pathways for superoxide production in neutrophils John M. Robinson
3,
Paul G. Heyworth 4 and John A. Badwey 1,2
J Department of Cell Physiology, Boston Biomedical Research Institute, 2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 3 Department of Anatomy and Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH and 4 Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA (U.S.A.)
(Received 22 March 1990)
Key words: Neutrophil;' Superoxide generation; Signal transduction; Protein kinase; (Guinea pig)
Neutrophils exhibit an intense phosphorylation of a 47 kDa protein and release large quantities of superoxide (O2-) upon stimulation with phorbol 12-myristate 13-acetate (PMA) or fMet-Leu-Phe (fMLP). Antagonists of protein kinases (e.g., 200 pM l-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7); 15 nM staurosporine) inhibited these phenomena when the stimulus was PMA (Badwey, J.A. et al. (1989) J. Biol. Chem. 264, 14947-14953). In this paper, we now report that while neutrophils treated with 15 nM staurosporine and PMA release little 02-, cells in the presence of these compounds can be stimulated to release near normal quantities of 02- by the subsequent addition of fMLP. Surprisingly, staurosporine (15 nM) reduced the incorporation of 32p into the 47 kDa protein in fMLP stimulated cells at least as effectively as H-7, yet, while the staurosporine treated cells released substantial amounts of O2-, the cells treated with H-7 did not. These data suggest that a stimulatory pathway exists in neutrophils that contains a protein kinase 'distinct' from that which is activated when PMA is the stimulus and that this pathway may enable the O 2producing system to become functional with little or no phosphorylation of the 47 kDa protein. They further suggest that the steps which are sensitive to H-7 in the signal-transduction pathways utilized by PMA and fMLP may be different.
Introduction Neutrophils produce large quantities of superoxide (O2), a cytocidal substance, upon perturbation of their plasmalemma by a variety of agents. The stimuli include certain tumor-promoters (e.g., PMA) and chemoattractants (e.g., fMLP) [1,2]. The former activate PKC by substituting for the endogenous regulator of this enzyme, sn-l,2-diacylglycerol ([3] for review, see Ref. 4). Several changes occur in the phosphoprotein pattern of neutrophils upon stimulation. The most prominent alterations are an enhanced incorporation of 32p into two proteins with molecular masses of approx. 47 and
Abbreviations: O~-, superoxide; PKC, protein kinase C; PMA, 4flphorbol 12-myristate 13-acetate; fMLP, N-formyl-Met-Leu-Phe; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; H-7, 1-(5isoquinolinylsulfonyl)-2-methylpiperazine; HA1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; Me2SO, dimethylsulfoxide. Correspondence: J.A. Badwey, Department of Cell Physiology, Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114, U.S.A.
49 kDa (for review, see Refs. 5, 6). The 47 kDa protein has been linked to O f production by the observation that it is absent from the neutrophils of most patients with the autosomal recessive/cytochrome b positive form of chronic granulomatous disease [7,8]. This disease is an inherited syndrome in which the patient's phagocytic leukocytes fail to produce 0 2 upon stimulation (e.g., Ref. 2). Moreover, recombinant 47 kDa protein can restore the ability to generate 0 2 to a cell-free system derived from the neutrophils of these patients which is deficient in this activity [9]. While the 47 kDa protein has been characterized [8], cloned and its sequence predicted [9,10] its function remains obscure. The 47 kDa protein is a substrate for PKC (e.g., Refs. 11, 12). No similar genetic data exist which tie the 49 kDa protein to 0 2 production, although the kinetics of phosphorylation for this entity are compatible with this possibility (e.g., Refs. 13-16). Several antagonists of PKC are nearly equally effective in blocking 0 2 release from neutrophils stimulated with either PMA or fMLP [17-21]. Results obtained with these inhibitors are partly responsible for a hypothesis in which the stimulation by fMLP is thought to
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
56 require both the activation of PKC and a separate, uncharacterized signal [21-24]. Staurosporine, however, is an exception. Low concentrations of this substance, which is a potent nonspecific inhibitor of PKC [25,26], are far more effective in reducing 0 2 release from neutrophils stimulated with PMA than with fMLP [18,27]. To our knowledge, this is the only drug reported to date that behaves in this manner (cf. Ref. 17 for discussion of the isoquinolinesulfonamide H-7). In this paper, we present a far more detailed analysis of the inhibition of 0 2 release by staurosporine. In particular, the effects of this drug and H-7 on the phosphorylation of the 47 and the 49 kDa proteins are now reported for neutrophils stimulated with fMLP. The data indicate that fMLP can utilize a stimulatory pathway that contains a protein kinase 'distinct' from that which is activated when PMA is the stimulus and that this pathway may function with little or no phosphorylation of the 47 kDa protein (cf. Ref. 28). The sensitivity of this pathway to these drugs provides criteria for uncovering the relevant kinase(s). Moreover, differential sensitivity to staurosporine can clearly distiguish the stimulatory pathways in neutrophils that are functional with PMA, sn-l,2-dioctanoylglycerol and fMLP (see Discussion).
Experimental procedures Materials The isoquinolinesulfonamides H-7 and HA1004 were obtained from Seikagaku America, St. Petersburg, FL. Staurosporine was purchased from Kamiya, Thousand Oaks, CA. Sources of all other materials used have been described elsewhere [14,29].
Methods Preparation of neutrophils. Guinea-pig peritoneal neutrophils were prepared as described previously [30]. Cell preparations contained not less than 90% neutrophils with viabilities always of 90% or more. Superoxide release. Superoxide release was measured in disposable 1 cm plastic cuvettes at 3 7 ° C by the continuous spectrophotometric measurement of the superoxide dismutase-inhibitable reduction of ferricytochrome c at 550 nm (e.g., Ref. 29). The standard assay mixture consisted of a modified Dulbecco's phosphate buffered saline medium (138 mM NaC1, 2.7 mM KC1, 16.2 mM Na2HPO 4, 1.47 mM KH2POa, 0.90 mM CaC12 and 0.50 mM MgC12 (pH 7.35)) [31] containing 7.5 mM D-glucose, 0.075 mM ferricytochrome c and 1.0 tO 1 . 5 . 1 0 6 cells/ml. The blank contained all of these components plus 20/~g/ml superoxide dismutase. Cells were incubated in the reaction mixture for 3 min at 37 ° C before 0 2 release was initiated by the addition of PMA (50 nM) or fMLP (1.0/xM).
Stock solutions of PMA (2.0 mg/rnl), fMLP (4.0 mM) and staurosporine (1 mg/ml) were prepared in Me2SO. PMA and fMLP were stored in the dark at - 2 0 ° C, whereas staurosporine was stored in the dark at 4 ° C. These compounds were diluted with Me2SO so that the final concentration of solvent in the assays was 0.25 or 0.50% (v/v) in all cases. Such amounts of solvent were shown not to cause any of the effects noted. Stock solutions (10 mM) of H-7 and HA1004 were prepared in water and stored at 4 ° C in the dark. Labeling of neutrophils with 32Pi. Neutrophils (108/ml) were resuspended at 30 ° C in a Hepes-buffered saline solution (10 mM Na-Hepes, 138 mM NaC1, 2.7 mM KC1, and 7.5 mM D-glucose (pH 7.35)) containing 400 /~Ci 32pi/ml. A f t e r 30 rain the cells were placed on ice and used in subsequent experiments without washing. The rates of O z- release from 32p-labeled neutrophils stimulated with PMA (50 nM) or fMLP (1.0/~M) were 65 + 9% (S.D., n = 10) and 81 +_ 18% (S.D., n =- 5), respectively, of the analogous value for unlabeled cells, i.e., cells maintained on ice and not subjected to a preliminary incubation at 3 0 ° C with 32pi. Thus, the labeling procedure per se only moderately affected 0 2 release.
Stimulation of ~:P-labeled cells for autoradiography. 32p-labeled neutrophils (1.5. 106/ml) were stimulated in plastic cuvettes under the same conditions utilized for measuring 0 2 release, except that cytochrome c was omitted from the reaction mixture. After the appropriate time, the entire contents of the reaction mixture (1.0 ml) were transferred to a microcentrifuge tube containing 0.25 ml of ice-cold 50% (w/v) trichloroacetic acid and rapidly mixed. Samples were stored on ice until prepared for electrophoresis.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. These procedures are described elsewhere in detail [14].
Results Differential effects of staurosporine on the release of Ojfrom neutrophils stimulated with PMA or fMLP Neutrophils stimulated with optimal amounts of PMA (50 nM) or fMLP (1.0 btM) release large quantities of 0 2. The maximal rates were 5 4 + 13 (S.D., n = 39) and 4 6 _ 9 (S.D., n---21) n m o l / m i n per 10 7 cells, respectively (Fig. 1; [32]). The progress curves for 0 2 release instigated by these agents were different (Fig. 1A). The response with fMLP was immediate and transient (Fig. 1A, curve b) whereas that with PMA occurred after a short lag-period (approx. 30 s) and was prolonged (Fig. 1A, curve a). As noted above, staurosporine inhibits a variety of protein kinases (e.g., Refs. 25, 26, 33, 34). Nevertheless, it is valuable in comparative studies to help determine
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Fig. 2. Effects of staurosporine on superoxide release by neutrophils. Dose-response curves demonstrate the inhibition of 0 2 release by different concentrations of staurosporine. The data shown are for cells stimulated with 50 n M P M A (e) or 1.0 laM f M L P (o). Cells were incubated with staurosporine for 3 min at 37 ° C in the standard assay mixture prior to the initiation of the reactions by the addition of the stimulus. The inset shows the Hill plots of these data, where V is the activity without inhibitor, and v is the activity with inhibitor. These concentrations of staurosporine did not affect ceU viability, as measured by the exclusion of T r y p a n blue, nor did they affect 02- release in an 0 2-generating system (i.e., xanthine oxidase plus purine).
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200 /~M) of H-7 have previously b e e n employed to s u b s t a n t i a l l y i n h i b i t 0 2 release from n e u t r o p h i l s [17,18]. C o m p l e t e dose-response curves are n o w presented in Fig. 3. H a l f - m a x i m a l i n h i b i t i o n with either P M A or f M L P occurred at approx. 60 # M H7. A s u b o p t i m a l a m o u n t of H-7 (40 ttM) i n h i b i t e d O 2 release instigated b y P M A or f M L P b y 42 ± 16% a n d 42 + 10%, respectively. Thus, u n l i k e staurosporine, low a m o u n t s of H-7 were equally effective in blocking 0 2 release instaged b y these agents. I n contrast, HA1004 was largely ineffective with either stimulus (Fig. 3).
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Fig. 4 c o m p a r e s the p h o s p h o p r o t e i n s i n u n s t i m u l a t e d n e u t r o p h i l s (lane a) a n d n e u t r o p h i l s s t i m u l a t e d with 1.0 ffM f M L P (lane b) or 50 n M P M A (lane c). T h e levels of i n c o r p o r a t i o n of 32p i n t o the 47 k D a p r o t e i n (unb r o k e n arrow) a p p e a r e d similar with either stimulus. This p a t t e r n was observed in five separate experiments. T h e reaction times utilized in Fig. 4 represent periods at which the labeled cells exhibited m a x i m a l rates of 0 2 release (see Fig. 1).
Fig. 3. Effects of isoquinolinesulfonamidies (H-7 and HA1004) on superoxide release by neutrophils. Dose-response curves for inhibition of O~- release by H-7 (circles) and HA1004 (squares) for cells stimulated with PMA (O, m) or fMLP (o, []). Cells were incubated with these compounds for 3 min at 37°C in the standard assay mixture prior to the initiation of the reactions by the addition of the stimulus. The inset shows the Hill plots of the data for H-7. These concentrations of H-7 and HA1004 did not affect cell viability, as measured by the exclusion of trypan blue, nor did they affect 0 2 release in an 0 2-generating system (i.e., xanthine oxidase plus purine). A a
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Fig. 4. Phosphorylation of neutfophil proteins during cell stimulation. (A) compares the complete autoradiograms obtained from 3gp-labeledcells treated with: (a) 0.25% (v/v) Me2SO for 1 rain (unstimulated cells); (b) 1.0 #M fMLP for 30 s; and (c) 50 nM PMA for 1 min. The reaction times represent periods at which the 32p-labeledcells exhibited maximal rates of O~- release under the conditions specified. Stimulation of Of release was accompanied by an enhanced incorporation of 32p into two proteins with molecular masses of approx. 47 kDa (unbroken arrow) and 49 kDa (broken arrow). Peak V (right margin) is present in both unstimulated and stimulated cells and is thus useful for orientation purposes. Peak I (right margin) exhibited a partial loss of 32p in neutrophils stimulated with fMLP. This overall pattern of changes was observed in five separate experiments performed on different preparations of cells. (B) presents the densitometric scans of the 47 and the 49 kDa proteins, along with Peak V. (C) presents the densitometric scans of Peak I.
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Fig. 5. Effects of staurosporine on the phosphorylation of the 47 and 49 kDa proteins of neutrophils stimulated with fMLP. 32p-labeled neutrophils (1.5-106/ml) were treated with 15 nM staurosporine for 3 rain at 37 ° C prior to stimulation with fMLP (1.0/tM) for 30 s. The autoradiograms in (A) were from: (a) unstimulated cells (i.e., treated with 0.25% (v/v) Me2SO for 30 s); (b) stimulated cells; and (c) stimulated cells treated with staurosporine (I). The 47 and 49 kDa proteins are indicated by the unbroken and broken arrows, respectively. (B) shows the densitometric scans of those proteins, along with Peak V, which is shown for the purpose of orientation. This pattern was observed in three separate experiments performed on different preparations of cells.
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Fig. 6. Effects of isoquinolinesulfonamides (H-7 and HA1004) on the phosphorylation of the 47 and 49 kDa proteins of neutrophils stimulated with fMLP. 32p-labeled neutrophils (1.5 × 106/ml) were treated with 200 p M H-7 or HA1004 for 3 rain at 37 ° C prior to stimulation with fMLP (1.0 p M ) for 30 s. The autoradiograms in ( A ) were from: (a) unstimulated cells (i.e., treated with 0.25% (v/v) Me2SO for 30 s); (b) stimulated cells; (c) stimulated cells treated with H-7; and (d) stimulated cells treated with HA1004. The 47 and 49 kDa proteins are indicated by the unbroken and broken arrows, respectively. (B) presents the densitometric scans of those proteins, along with Peak V, which is shown for the purpose of orientation. This pattern was observed in three separate experiments performed on different preparations of cells.
60 The 49 kDa protein (broken arrow) exhibits a variable amount of labeling in unstimulated neutrophils (e.g., Ref. 14, 15). Increased incorporation of 32p over the basal level was generally observed upon stimulation of the cells with fMLP (i.e., 4 out of 5 experiments). However, the increases varied widely between experiments (i.e., compare lane b in Figs. 4 and 6) and were always less than that seen with PMA. Neutrophils stimulated with fMLP also exhibited a partial diminution (approx. 50%) of 32p from a band with a molecular mass of approx. 21 kDa (peak I, Fig. 4; [14,36]), which may represent proteolysis of the light chains of myosin [37]. This alteration was not observed in cells stimulated with PMA for 1 rain (Fig. 4, lane c). Treatment of neutrophils with staurosporine (15 nM) or H-7 (200 #M) for 3 rain at 37°C sign!ficantly reduced the phosphorylation of the 47 kDa protein upon stimulation with fMLP (Figs. 5 and 6). The degree of inhibition caused by staurosporine was always greater than or comparable to that seen with H-7 (e.g., compare lane c in Figs. 5 and 6). These data were confirmed in three separate experiments. These drugs also inhibited the enhanced phosphorylation of several other proteins during cell stimulation and reduced the background. A more exact quantitative analysis of these changes was hampered by the large increase in the background phosphorylation which accompanied cell stimulation. HA1004 (200/~M) did not affect the phosphorylation of the 47 kDa protein (Fig. 6). H-7 and staurosporine also appeared to be effective in reducing the phosphorylation of the 49 kDa protein (Figs. 5 and 6) although the results were not always as clear here due to the low amount of labeling of that protein which sometimes occurred (e..g, Fig. 6). Discussion
Two significant observations are developed in this paper. First, fMLP may stimulate neutrophils by a signal transduction pathway that involves a protein kinase 'distinct' from what which is activated when PMA is the stimulus. Second, this pathway can function in parallel with the route mediated by PKC and involves little or no phosphorylation of the 47 kDa protein. The NADPH-oxidase system which is responsible for generating O~- is disassembled in unstimulated cells and consists of both cytoplasmic and membrane-bound components (e.g., Refs. 38-41). Upon stimulation, the cytoplasmic components undergo translocation to the plasmalemma, where the oxidase system is assembled (e.g., Refs. 38, 42, 43). Phosphorylation of certain components may play a role in this assembly (e.g., Refs. 6, 44). Staurosporine (15 nM) or H-7 (200/~M) dramatically inhibited both the phosphorylation of the 47 kDa pro-
tein and 0 2 release in neutrophils stimulated with PMA ([18]; Figs. 1-3). Phosphorylation of this protein may thus be reuqired in the pathway by which PMA elicits O~- release (see Scheme I; for review, see Ref. 6). In contrast, fMLP may employ both the pathway utilized by PMA and a second route of signal transduction (see Scheme II). Low concentrations of staurosporine (i.e., 15 nM) would primarily block the former, enabling O 2 production to continue by the alternate route (i.e., pathway b). This concept is strongly supported by the observation that cells treated with 15 nm staurosporine and PMA released little O2, yet could be stimulated to produce near normal quantities of 0 2 upon the subsequent addition of fMLP (Fig. 1, curve e). The second pathway intimated above (i.e., route b) is also likely to involve a protein kinase as O~- release with fMLP is substantially blocked by antagonists of these enzymes ([17-21]; Figs. 1-3). It is particularly noteworthy that the concentrations of staurosporine required to inhibit 0 2 release initiated with fMLP are significantly higher than those needed with PMA (Fig. 2). Staurosporine and its analogs have recently been shown to differentially inhibit the CaE+-responsive and Ca2+-unresponsive forms of PKC and certain tyrosinespecific protein kinases, with the former being the most sensitive [33,34]. All three of these kinases are present in neutrophils [45-50]. A role for a protein tyrosine kinase in O~- production has been intimated [45,48] and is consistent with data presented here. Our studies further show that a pathway containing such a kinase is likely to function in parallel with the route mediated by PKC and involves little or no phosphorylation of the 47 kDa protein (see below). Staurosporine (15 nM) markedly reduced the phosphorylation of the 47 kDa protein in fMLP-stimulated cells (Fig. 5) and this inhibition was greater than or comparable to that observed with 200 #M H-7 (Fig. 6). This was surprising, since the cells treated with staurosporine (15 nM) released large amounts of O 2 (Fig. 1), whereas cells treated with H-7 (200 /~M) did not (Fig. 3). At present, a method by which the postulated kinase may allow for substantial production of O 2 with little phosphorylation of the 47 kDa protein is
Stimulotion by phorbol 12-myristote 15-acetate (PMA) Low Staurosporine (15nM) or H-7 (2001,JM) i PKC G)! )D 47 kDa Protein (membrane~ , \ insetled ] r
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61
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Scheme II. This scheme suggests an overall mechanism of stimulation for neutrophils treated with fMLP. Pathway b, (bold arrows) may involve a protein kinase distinct from that activated by PMA. This kinase may phosphorylate the 47 kDa protein on a site distinct from those which are phosphorylated when PMA is the stimulus a n d / o r may phosphorylate a yet unidentified component (protein X) that enables the oxidase to become functional with little or no phosphorylation of the 47 kDa protein. Stimulation may also occur by pathway a. Phosphatidate may also function in a stimulatory pathway that does not involve phosphorylation of the 47 kDa protein (pathway c). However, that pathway is largely insensitive to H-7 (200/~M) and staurosporine (150 nM) and is thus not likely to be functional with fMLP (see Refs. 18, 32). Neutrophils stimulated with PMA or fMLP exhibit a redistribution of the activity of PKC from a soluble to a particulate fraction that is tight ('membrane inserted') or loose (' membrane elutable'), respectively [52,53]. The significance of these types of associations are discussed elsehwere [54].
a matter of conjecture. However, a modest amount of speculation may be appropriate. Perhaps it catalyzes the phosphorylation of the 47 kDa protein on a unique site so that it becomes activated with comparatively little phosphorylation or catalyzes the phosphorylation of a yet unidentified component (component X in Scheme II) that obviates or reduces the necessity for phosphorylation of the 47 kDa protein. As noted above, neutrophils which lack the 47 kDa protein do not produce 0 2 in response to any stimulus [2]. Thus, this protein must be a critical component of all the stimulatory pathways, even if its phosphorylation is not always required. In contrast, several sites on the 47 kDa protein are phosphorylated when PMA is the stimulus [8] and the extent of this phosphorylation correlates with the quantities of 0 2 released (e.g., Refs. 8, 11, 14). H-7 inhibits 0 2 release from neutrophils stimulated with either PMA or fMLP with nearly identical kinetics (Fig. 3) and must therefore inhibit both pathways a and b in Scheme II. This antagonist exhibits very similar K i values towards certain protein kinases [35]. The inhibition of 0 2 release from fMLP stimulated cells by H-7 has a large temperature dependence, whereas that with PMA does not [17], which is consistent with the critical targets for this drug being different in cells stimulated with these agents. Two related studies have been published [28,51]. One reported that H-7 inhibits the phosphorylation of the 47 kDa protein, but not O{ release in neutrophils stimulated with fMLP [28]. Whether the failure to block 02release in that report was due to the temperature-depen-
dence cited above a n d / o r the concentration of H-7 employed (25 /~M) is unknown. However, these data again indicate that phosphorylation of the 47 kDa protein is not the critical target for 0 2 release in cells stimulated with this agent. The second report noted that 100 nM staurosporine inhibited both the phosphorylation of the 47 kDa protein and 0 2 release in neutrophils stimulated with either PMA or fMLP [51]. It was suggested that these results were due to the blocking of a common step in the mechanism of signal transduction with these stimuli; i.e., phosphorylation of the 47 kDa protein by PKC [51]. However, the effects at lower concentrations of staurosporine, where a dissociation between these phenomena is evident, were not examined. Neutrophils stimulated with retinal or high amounts of sn-l,2-dioctanoylglycerol can also produce large amounts of 0 2 without any detectable phosphorylation of the 47 kDa protein [18,32]. However, the O{ released under those circumstances was largely insensitive to 200/~M H-7 or 150 nM staurosporine [18,32], which clearly indicates that certain aspects of the pathway(s) involved with those stimuli are different from those functional with fMLP. Thus, staurosporine is a very useful drug for uncovering differences in the signaltransduction pathways of neutrophils.
Acknowledgements These studies were supported by grants AI-23323, AI-24321 and AI-28342 from the National Institutes of
62 Health, Council of Tobacco Research 2065, and the Medical Research Council of Great Britain. J.A.B. is the recipient of a Research Career Development Award (AI-00672) from the National Institute of Allergy and Infectious Diseases. J.M.R. was also supported by a Bremer Foundation Grant and a University Seed Grant from Ohio State University. References 1 Badwey, J.A. and Karnovsky, M.L. (1980) Annu. Rev. Biochem. 49, 695-726. 2 Curnutte, J.T. and Babior, B.M. (1987) Adv. Human Genet. 16, 229-297. 3 Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851. 4 Badwey, J.A. and Karnovsky, M.L. (1986) Curr. Top. Cell. Regul. 28, 183-208. 5 Babior, B.M. (1988) Arch. Biochem. Biophys. 264, 361-367. 6 Heyworth, P.G. and Badwey, J.A. (1990) J. Bioenerg. Biomembr. 22, 1-26. 7 Segal, A.W., Heyworth, P.G., Cockcroft, S. and Barrowman, M.M. (1985) Nature 316, 547-549. 80kamura, N., Curnutte, J.T., Roberts, R.L. and Babior, B.M. (1988) J. Biol. Chem. 263, 6777-6782. 9 Lomax, K.J., Leto, T.L., Nunoi, H., Gallin, J.I. and Malech, H.L. (1989) Science 245, 409-412. 10 Volpp, B.D., Nauseef, W.M., Donelson, J.E., Moser, D.R. and Clark, R.A. (1989) Proc. Natl. Acad. Sci. USA 86, 9563. 11 White, J.R., Huang, C.-K., Hill, J.M., Naccache, P.H., Becker, E.L. and Sha'afi, R.I. (1984) J. Biol. Chem. 259, 8605-8611. 12 Kramer, I.M., Verhoeven, A.J., Van der Bend, R.L., Weening, R.S. and Roos, D. (1988) J. Biol. Chem. 263, 2352-2357. 13 Schneider, C., Zanetti, M. and Romeo, D. (1981) FEBS Lett. 127, 4-8. 14 Badwey, J.A., Heyworth, P.G. and Karnovsky, M.L. (1989) Biochem. Biophys. Res. Commun. 158, 1029-1035. 15 Heyworth, P.G., Karnovsky, M.L. and Badwey, J.A. (1989) J. Biol. Chem. 264, 14935-14939. 16 Heyworth, P.G. and Badwey, J.A. (1990) Biochim. Biophys. Acta 1052, 299-305. 17 Nath, J., Powledge, A. and Wright, D.G. (1989) J. Biol. Chem. 264, 848-855. 18 Badwey, J.A., Horn, W., Heyworth, P.G., Robinson, J.M. and Karnovsky, M.L. (1989) J. Biol. Chem. 264, 14947-14953. 19 Wilson, E., Olcott, M.C., Bell, R.M., Merrill, Jr., A.H. and Lambeth, D.H. (1986) J. Biol. Chem. 261, 12616-12623. 20 Lambeth, J.D., Burnham, D.N. and Tyagi, S.R. (1988) J. Biol. Chem. 263, 3818-3822. 21 Kramer, I.M., Van der Bend, R.L., Tool, A.T.J., Van Blitterswijk, W.J., Roos, D. and Verhoeven, A.J. (1989) J. Biol. Chem. 264, 5876-5884. 22 Dewald, B., Thelen, M. and Baggiolini, M. (1988) J. Biol. Chem. 263, 16179-16184. 23 Korchak, H.M., Vosshall, L.B., Haines, K.A., Wilkenfeld, C., Lundquist, K.F. and Weissmann, G. (1988) J. Biol. Chem. 263, 11098-11105. 24 Wyman, M.P., Von Tscharner, V., Deranleau, D.A. and Baggiolini, M. (1987) J. Biol. Chem. 262, 12048-12053.
25 Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M. and Tomita, F. (1986) Biochem. Biophys. Res. Commun. 135, 397-402. 26 Kase, H., Iwahasi, K., Nakanishi, S., Matsuda, Y., Yamada, K., Takahashi, M., Murakata, C., Sato, A. and Kaneko, M. (1987) Biochem. Biophys. Res. Commun. 142, 436-440. 27 Sako, T., Taber, A.I., Jeng, A.Y., Yuspa, S.H. and Blumberg, P.M. (1988) Cancer Res. 48, 4646-4650. 28 Sha'afi, R.I., Molski, T.F.P., Gomez-Cambronero, J. and Huang, C.-K. (1988) J. Leukocyte Biol. 43, 18-27. 29 Robinson, J.M., Badwey, J.A., Karnovsky, M.L. and Karnovsky, M.J. (1985) J. Cell. Biol. 101, 1052-1058. 30 Badwey, J.A. and Karnovsky, M.L. (1986) Methods Enzymol. 132, 365-368. 31 Dulbecco, R. and Vogt, M. (1954) J. Exp. Med. 98, 167-199. 32 Badwey, J.A., Robinson, J.M., Heyworth, P.G. and Curnutte, J.T. (1989) J. Biol. Chem. 264, 20676-20682. 33 Gschwendt, M., Leibersperger, H. and Marks, F. (1989) Biochem. Biophys. Res. Commun. 164, 974-982. 34 Fujita-Yamaguchi, Y. and Kathuria, S. (1988) Biochem. Biophys. Res. Commun. 157, 955-962. 35 Hidaka, H., Inagaki, M., Kawamoto, S. and Sasaki, Y. (1984) Biochemistry 23, 5036-5041. 36 Andrews, P.C. and Babior, B.M. (1983) Blood 61, 333-340. 37 Pontremoh, S., Melloni, E., Michetti, M., Sparatore, B., Salamino, F., Sacco, O. and Horecker, B.L. (1987) Proc. Natl. Acad. Sci. USA 84, 398-401. 38 McPhail, L.C., Shirley, P.S., Clayton, C.C. and Snyderman, R. (1985) J. Clin. Invest. 75, 1735-1739. 39 Curnutte, J.T. (1985) J. Clin. Invest. 75, 1740-1743. 40 Volpp, B.D., Nauseef, W.M. and Clark, R.A. (1988) Science 242, 1295-1297. 41 Nunoi, H., Rotrosen, D., Gallin, J.l. and Malech, H.L. (1988) Science 242, 1298-1301. 42 Ligeti, E., Doussiere, J. and Vignais, P.V. (1988) Biochemistry 27, 193-200. 43 Clark, R.A., Leidal, K.G., Pearson, D.W. and Nauseef, W.M. (1987) J. Biol. Chem. 262, 4065-4074. 44 Heyworth, P.G., Shrimptom, C.F. and Segat, A.W. (1989) Biochem. J. 260, 243-248. 45 Berkow, R.L., Dodson, R.W. and Kraft, A.S. (1989) Biochim. Biophys. Acta 997, 292-301. 46 Huang, C.-K., Laramee, G.F. and Casnellie, J.F. (1988) Biochem. Biophys. Res. Commun. 151,794-801. 47 Gomez-Cambronero, J., Huang, C.-K., Bonak, V.A., Wang, E., Casnellie, J.E., Shiraishi, T. and Sha'afi, R.I. (1989) Biochem. Biophys. Res. Commun. 162, 1478-1485. 48 Grinstein, S., Furuya, W., Lu, D.J. and Mills, G.B. (1990) J. Biol. Chem. 265, 318-327. 49 Nasmith, P.E., Mills, G.B. and Grinstein, S. (1989) Biochem. J. 257, 893-897. 50 Phillips, W.A., Fujiki, T., Rossi, M.W., Korchak, H.M. and Johnston, Jr., R.B. (1989) J. Biol. Chem. 264, 8361-8365. 51 Dewald, B., Thelen, M., Wymann, M.P. and Baggiolini, M. (1989) Biochem. J. 264, 879-884. 52 Wolfson, M., McPhail, L.C., Nasrallah, V.N. and Snyderman, R. (1985) J. Immunol. 135, 2057-2062. 53 Horn, W. and Karnovsky, M.L. (1986) Biochem. Biophys. Res. Commun. 139, 1169-1175. 54 Bazzi, M.D. and Nelsestuen, G.L. (1988) Biochemistry 27, 75897593.
55
Elsevier BBAMCR 12782
Utility of staurosporine in uncovering differences in the signal transduction pathways for superoxide production in neutrophils John M. Robinson
3,
Paul G. Heyworth 4 and John A. Badwey 1,2
J Department of Cell Physiology, Boston Biomedical Research Institute, 2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 3 Department of Anatomy and Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH and 4 Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA (U.S.A.)
(Received 22 March 1990)
Key words: Neutrophil;' Superoxide generation; Signal transduction; Protein kinase; (Guinea pig)
Neutrophils exhibit an intense phosphorylation of a 47 kDa protein and release large quantities of superoxide (O2-) upon stimulation with phorbol 12-myristate 13-acetate (PMA) or fMet-Leu-Phe (fMLP). Antagonists of protein kinases (e.g., 200 pM l-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7); 15 nM staurosporine) inhibited these phenomena when the stimulus was PMA (Badwey, J.A. et al. (1989) J. Biol. Chem. 264, 14947-14953). In this paper, we now report that while neutrophils treated with 15 nM staurosporine and PMA release little 02-, cells in the presence of these compounds can be stimulated to release near normal quantities of 02- by the subsequent addition of fMLP. Surprisingly, staurosporine (15 nM) reduced the incorporation of 32p into the 47 kDa protein in fMLP stimulated cells at least as effectively as H-7, yet, while the staurosporine treated cells released substantial amounts of O2-, the cells treated with H-7 did not. These data suggest that a stimulatory pathway exists in neutrophils that contains a protein kinase 'distinct' from that which is activated when PMA is the stimulus and that this pathway may enable the O 2producing system to become functional with little or no phosphorylation of the 47 kDa protein. They further suggest that the steps which are sensitive to H-7 in the signal-transduction pathways utilized by PMA and fMLP may be different.
Introduction Neutrophils produce large quantities of superoxide (O2), a cytocidal substance, upon perturbation of their plasmalemma by a variety of agents. The stimuli include certain tumor-promoters (e.g., PMA) and chemoattractants (e.g., fMLP) [1,2]. The former activate PKC by substituting for the endogenous regulator of this enzyme, sn-l,2-diacylglycerol ([3] for review, see Ref. 4). Several changes occur in the phosphoprotein pattern of neutrophils upon stimulation. The most prominent alterations are an enhanced incorporation of 32p into two proteins with molecular masses of approx. 47 and
Abbreviations: O~-, superoxide; PKC, protein kinase C; PMA, 4flphorbol 12-myristate 13-acetate; fMLP, N-formyl-Met-Leu-Phe; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; H-7, 1-(5isoquinolinylsulfonyl)-2-methylpiperazine; HA1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; Me2SO, dimethylsulfoxide. Correspondence: J.A. Badwey, Department of Cell Physiology, Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114, U.S.A.
49 kDa (for review, see Refs. 5, 6). The 47 kDa protein has been linked to O f production by the observation that it is absent from the neutrophils of most patients with the autosomal recessive/cytochrome b positive form of chronic granulomatous disease [7,8]. This disease is an inherited syndrome in which the patient's phagocytic leukocytes fail to produce 0 2 upon stimulation (e.g., Ref. 2). Moreover, recombinant 47 kDa protein can restore the ability to generate 0 2 to a cell-free system derived from the neutrophils of these patients which is deficient in this activity [9]. While the 47 kDa protein has been characterized [8], cloned and its sequence predicted [9,10] its function remains obscure. The 47 kDa protein is a substrate for PKC (e.g., Refs. 11, 12). No similar genetic data exist which tie the 49 kDa protein to 0 2 production, although the kinetics of phosphorylation for this entity are compatible with this possibility (e.g., Refs. 13-16). Several antagonists of PKC are nearly equally effective in blocking 0 2 release from neutrophils stimulated with either PMA or fMLP [17-21]. Results obtained with these inhibitors are partly responsible for a hypothesis in which the stimulation by fMLP is thought to
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
56 require both the activation of PKC and a separate, uncharacterized signal [21-24]. Staurosporine, however, is an exception. Low concentrations of this substance, which is a potent nonspecific inhibitor of PKC [25,26], are far more effective in reducing 0 2 release from neutrophils stimulated with PMA than with fMLP [18,27]. To our knowledge, this is the only drug reported to date that behaves in this manner (cf. Ref. 17 for discussion of the isoquinolinesulfonamide H-7). In this paper, we present a far more detailed analysis of the inhibition of 0 2 release by staurosporine. In particular, the effects of this drug and H-7 on the phosphorylation of the 47 and the 49 kDa proteins are now reported for neutrophils stimulated with fMLP. The data indicate that fMLP can utilize a stimulatory pathway that contains a protein kinase 'distinct' from that which is activated when PMA is the stimulus and that this pathway may function with little or no phosphorylation of the 47 kDa protein (cf. Ref. 28). The sensitivity of this pathway to these drugs provides criteria for uncovering the relevant kinase(s). Moreover, differential sensitivity to staurosporine can clearly distiguish the stimulatory pathways in neutrophils that are functional with PMA, sn-l,2-dioctanoylglycerol and fMLP (see Discussion).
Experimental procedures Materials The isoquinolinesulfonamides H-7 and HA1004 were obtained from Seikagaku America, St. Petersburg, FL. Staurosporine was purchased from Kamiya, Thousand Oaks, CA. Sources of all other materials used have been described elsewhere [14,29].
Methods Preparation of neutrophils. Guinea-pig peritoneal neutrophils were prepared as described previously [30]. Cell preparations contained not less than 90% neutrophils with viabilities always of 90% or more. Superoxide release. Superoxide release was measured in disposable 1 cm plastic cuvettes at 3 7 ° C by the continuous spectrophotometric measurement of the superoxide dismutase-inhibitable reduction of ferricytochrome c at 550 nm (e.g., Ref. 29). The standard assay mixture consisted of a modified Dulbecco's phosphate buffered saline medium (138 mM NaC1, 2.7 mM KC1, 16.2 mM Na2HPO 4, 1.47 mM KH2POa, 0.90 mM CaC12 and 0.50 mM MgC12 (pH 7.35)) [31] containing 7.5 mM D-glucose, 0.075 mM ferricytochrome c and 1.0 tO 1 . 5 . 1 0 6 cells/ml. The blank contained all of these components plus 20/~g/ml superoxide dismutase. Cells were incubated in the reaction mixture for 3 min at 37 ° C before 0 2 release was initiated by the addition of PMA (50 nM) or fMLP (1.0/xM).
Stock solutions of PMA (2.0 mg/rnl), fMLP (4.0 mM) and staurosporine (1 mg/ml) were prepared in Me2SO. PMA and fMLP were stored in the dark at - 2 0 ° C, whereas staurosporine was stored in the dark at 4 ° C. These compounds were diluted with Me2SO so that the final concentration of solvent in the assays was 0.25 or 0.50% (v/v) in all cases. Such amounts of solvent were shown not to cause any of the effects noted. Stock solutions (10 mM) of H-7 and HA1004 were prepared in water and stored at 4 ° C in the dark. Labeling of neutrophils with 32Pi. Neutrophils (108/ml) were resuspended at 30 ° C in a Hepes-buffered saline solution (10 mM Na-Hepes, 138 mM NaC1, 2.7 mM KC1, and 7.5 mM D-glucose (pH 7.35)) containing 400 /~Ci 32pi/ml. A f t e r 30 rain the cells were placed on ice and used in subsequent experiments without washing. The rates of O z- release from 32p-labeled neutrophils stimulated with PMA (50 nM) or fMLP (1.0/~M) were 65 + 9% (S.D., n = 10) and 81 +_ 18% (S.D., n =- 5), respectively, of the analogous value for unlabeled cells, i.e., cells maintained on ice and not subjected to a preliminary incubation at 3 0 ° C with 32pi. Thus, the labeling procedure per se only moderately affected 0 2 release.
Stimulation of ~:P-labeled cells for autoradiography. 32p-labeled neutrophils (1.5. 106/ml) were stimulated in plastic cuvettes under the same conditions utilized for measuring 0 2 release, except that cytochrome c was omitted from the reaction mixture. After the appropriate time, the entire contents of the reaction mixture (1.0 ml) were transferred to a microcentrifuge tube containing 0.25 ml of ice-cold 50% (w/v) trichloroacetic acid and rapidly mixed. Samples were stored on ice until prepared for electrophoresis.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. These procedures are described elsewhere in detail [14].
Results Differential effects of staurosporine on the release of Ojfrom neutrophils stimulated with PMA or fMLP Neutrophils stimulated with optimal amounts of PMA (50 nM) or fMLP (1.0 btM) release large quantities of 0 2. The maximal rates were 5 4 + 13 (S.D., n = 39) and 4 6 _ 9 (S.D., n---21) n m o l / m i n per 10 7 cells, respectively (Fig. 1; [32]). The progress curves for 0 2 release instigated by these agents were different (Fig. 1A). The response with fMLP was immediate and transient (Fig. 1A, curve b) whereas that with PMA occurred after a short lag-period (approx. 30 s) and was prolonged (Fig. 1A, curve a). As noted above, staurosporine inhibits a variety of protein kinases (e.g., Refs. 25, 26, 33, 34). Nevertheless, it is valuable in comparative studies to help determine
57 B PMA
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Fig. 2. Effects of staurosporine on superoxide release by neutrophils. Dose-response curves demonstrate the inhibition of 0 2 release by different concentrations of staurosporine. The data shown are for cells stimulated with 50 n M P M A (e) or 1.0 laM f M L P (o). Cells were incubated with staurosporine for 3 min at 37 ° C in the standard assay mixture prior to the initiation of the reactions by the addition of the stimulus. The inset shows the Hill plots of these data, where V is the activity without inhibitor, and v is the activity with inhibitor. These concentrations of staurosporine did not affect ceU viability, as measured by the exclusion of T r y p a n blue, nor did they affect 02- release in an 0 2-generating system (i.e., xanthine oxidase plus purine).
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200 /~M) of H-7 have previously b e e n employed to s u b s t a n t i a l l y i n h i b i t 0 2 release from n e u t r o p h i l s [17,18]. C o m p l e t e dose-response curves are n o w presented in Fig. 3. H a l f - m a x i m a l i n h i b i t i o n with either P M A or f M L P occurred at approx. 60 # M H7. A s u b o p t i m a l a m o u n t of H-7 (40 ttM) i n h i b i t e d O 2 release instigated b y P M A or f M L P b y 42 ± 16% a n d 42 + 10%, respectively. Thus, u n l i k e staurosporine, low a m o u n t s of H-7 were equally effective in blocking 0 2 release instaged b y these agents. I n contrast, HA1004 was largely ineffective with either stimulus (Fig. 3).
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Fig. 4 c o m p a r e s the p h o s p h o p r o t e i n s i n u n s t i m u l a t e d n e u t r o p h i l s (lane a) a n d n e u t r o p h i l s s t i m u l a t e d with 1.0 ffM f M L P (lane b) or 50 n M P M A (lane c). T h e levels of i n c o r p o r a t i o n of 32p i n t o the 47 k D a p r o t e i n (unb r o k e n arrow) a p p e a r e d similar with either stimulus. This p a t t e r n was observed in five separate experiments. T h e reaction times utilized in Fig. 4 represent periods at which the labeled cells exhibited m a x i m a l rates of 0 2 release (see Fig. 1).
Fig. 3. Effects of isoquinolinesulfonamidies (H-7 and HA1004) on superoxide release by neutrophils. Dose-response curves for inhibition of O~- release by H-7 (circles) and HA1004 (squares) for cells stimulated with PMA (O, m) or fMLP (o, []). Cells were incubated with these compounds for 3 min at 37°C in the standard assay mixture prior to the initiation of the reactions by the addition of the stimulus. The inset shows the Hill plots of the data for H-7. These concentrations of H-7 and HA1004 did not affect cell viability, as measured by the exclusion of trypan blue, nor did they affect 0 2 release in an 0 2-generating system (i.e., xanthine oxidase plus purine). A a
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Fig. 4. Phosphorylation of neutfophil proteins during cell stimulation. (A) compares the complete autoradiograms obtained from 3gp-labeledcells treated with: (a) 0.25% (v/v) Me2SO for 1 rain (unstimulated cells); (b) 1.0 #M fMLP for 30 s; and (c) 50 nM PMA for 1 min. The reaction times represent periods at which the 32p-labeledcells exhibited maximal rates of O~- release under the conditions specified. Stimulation of Of release was accompanied by an enhanced incorporation of 32p into two proteins with molecular masses of approx. 47 kDa (unbroken arrow) and 49 kDa (broken arrow). Peak V (right margin) is present in both unstimulated and stimulated cells and is thus useful for orientation purposes. Peak I (right margin) exhibited a partial loss of 32p in neutrophils stimulated with fMLP. This overall pattern of changes was observed in five separate experiments performed on different preparations of cells. (B) presents the densitometric scans of the 47 and the 49 kDa proteins, along with Peak V. (C) presents the densitometric scans of Peak I.
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Fig. 5. Effects of staurosporine on the phosphorylation of the 47 and 49 kDa proteins of neutrophils stimulated with fMLP. 32p-labeled neutrophils (1.5-106/ml) were treated with 15 nM staurosporine for 3 rain at 37 ° C prior to stimulation with fMLP (1.0/tM) for 30 s. The autoradiograms in (A) were from: (a) unstimulated cells (i.e., treated with 0.25% (v/v) Me2SO for 30 s); (b) stimulated cells; and (c) stimulated cells treated with staurosporine (I). The 47 and 49 kDa proteins are indicated by the unbroken and broken arrows, respectively. (B) shows the densitometric scans of those proteins, along with Peak V, which is shown for the purpose of orientation. This pattern was observed in three separate experiments performed on different preparations of cells.
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Fig. 6. Effects of isoquinolinesulfonamides (H-7 and HA1004) on the phosphorylation of the 47 and 49 kDa proteins of neutrophils stimulated with fMLP. 32p-labeled neutrophils (1.5 × 106/ml) were treated with 200 p M H-7 or HA1004 for 3 rain at 37 ° C prior to stimulation with fMLP (1.0 p M ) for 30 s. The autoradiograms in ( A ) were from: (a) unstimulated cells (i.e., treated with 0.25% (v/v) Me2SO for 30 s); (b) stimulated cells; (c) stimulated cells treated with H-7; and (d) stimulated cells treated with HA1004. The 47 and 49 kDa proteins are indicated by the unbroken and broken arrows, respectively. (B) presents the densitometric scans of those proteins, along with Peak V, which is shown for the purpose of orientation. This pattern was observed in three separate experiments performed on different preparations of cells.
60 The 49 kDa protein (broken arrow) exhibits a variable amount of labeling in unstimulated neutrophils (e.g., Ref. 14, 15). Increased incorporation of 32p over the basal level was generally observed upon stimulation of the cells with fMLP (i.e., 4 out of 5 experiments). However, the increases varied widely between experiments (i.e., compare lane b in Figs. 4 and 6) and were always less than that seen with PMA. Neutrophils stimulated with fMLP also exhibited a partial diminution (approx. 50%) of 32p from a band with a molecular mass of approx. 21 kDa (peak I, Fig. 4; [14,36]), which may represent proteolysis of the light chains of myosin [37]. This alteration was not observed in cells stimulated with PMA for 1 rain (Fig. 4, lane c). Treatment of neutrophils with staurosporine (15 nM) or H-7 (200 #M) for 3 rain at 37°C sign!ficantly reduced the phosphorylation of the 47 kDa protein upon stimulation with fMLP (Figs. 5 and 6). The degree of inhibition caused by staurosporine was always greater than or comparable to that seen with H-7 (e.g., compare lane c in Figs. 5 and 6). These data were confirmed in three separate experiments. These drugs also inhibited the enhanced phosphorylation of several other proteins during cell stimulation and reduced the background. A more exact quantitative analysis of these changes was hampered by the large increase in the background phosphorylation which accompanied cell stimulation. HA1004 (200/~M) did not affect the phosphorylation of the 47 kDa protein (Fig. 6). H-7 and staurosporine also appeared to be effective in reducing the phosphorylation of the 49 kDa protein (Figs. 5 and 6) although the results were not always as clear here due to the low amount of labeling of that protein which sometimes occurred (e..g, Fig. 6). Discussion
Two significant observations are developed in this paper. First, fMLP may stimulate neutrophils by a signal transduction pathway that involves a protein kinase 'distinct' from what which is activated when PMA is the stimulus. Second, this pathway can function in parallel with the route mediated by PKC and involves little or no phosphorylation of the 47 kDa protein. The NADPH-oxidase system which is responsible for generating O~- is disassembled in unstimulated cells and consists of both cytoplasmic and membrane-bound components (e.g., Refs. 38-41). Upon stimulation, the cytoplasmic components undergo translocation to the plasmalemma, where the oxidase system is assembled (e.g., Refs. 38, 42, 43). Phosphorylation of certain components may play a role in this assembly (e.g., Refs. 6, 44). Staurosporine (15 nM) or H-7 (200/~M) dramatically inhibited both the phosphorylation of the 47 kDa pro-
tein and 0 2 release in neutrophils stimulated with PMA ([18]; Figs. 1-3). Phosphorylation of this protein may thus be reuqired in the pathway by which PMA elicits O~- release (see Scheme I; for review, see Ref. 6). In contrast, fMLP may employ both the pathway utilized by PMA and a second route of signal transduction (see Scheme II). Low concentrations of staurosporine (i.e., 15 nM) would primarily block the former, enabling O 2 production to continue by the alternate route (i.e., pathway b). This concept is strongly supported by the observation that cells treated with 15 nm staurosporine and PMA released little O2, yet could be stimulated to produce near normal quantities of 0 2 upon the subsequent addition of fMLP (Fig. 1, curve e). The second pathway intimated above (i.e., route b) is also likely to involve a protein kinase as O~- release with fMLP is substantially blocked by antagonists of these enzymes ([17-21]; Figs. 1-3). It is particularly noteworthy that the concentrations of staurosporine required to inhibit 0 2 release initiated with fMLP are significantly higher than those needed with PMA (Fig. 2). Staurosporine and its analogs have recently been shown to differentially inhibit the CaE+-responsive and Ca2+-unresponsive forms of PKC and certain tyrosinespecific protein kinases, with the former being the most sensitive [33,34]. All three of these kinases are present in neutrophils [45-50]. A role for a protein tyrosine kinase in O~- production has been intimated [45,48] and is consistent with data presented here. Our studies further show that a pathway containing such a kinase is likely to function in parallel with the route mediated by PKC and involves little or no phosphorylation of the 47 kDa protein (see below). Staurosporine (15 nM) markedly reduced the phosphorylation of the 47 kDa protein in fMLP-stimulated cells (Fig. 5) and this inhibition was greater than or comparable to that observed with 200 #M H-7 (Fig. 6). This was surprising, since the cells treated with staurosporine (15 nM) released large amounts of O 2 (Fig. 1), whereas cells treated with H-7 (200 /~M) did not (Fig. 3). At present, a method by which the postulated kinase may allow for substantial production of O 2 with little phosphorylation of the 47 kDa protein is
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Scheme I. An overall m e c h a n i s m of stimulation for neutrophils treated with phorbol esters. Activation of P K C and phosphorylation of the 47 k D a protein are required for O~- generation by this route.
61
Stimulation
by N - f M e t - L e u - P h e
(fMLP)
PKC
~0//membrone~ ~,elutable1 ~ __~__Q. ---~--
/
/
~
"
/
HighStaurosporine (15OHM)
.
?oo,Mi
/~ Pomwoyb~Second Protein fMLP.., • Kinase
®!
f
"
LOWStaurosporine(15nM) or H-7(200pM)
\.
_ ......... ~ • ~ Pr0tem X + 47 kDa Pr0teln
•
Activation of _ ^Oxidase "--~uz
,oo -..
•
"-,4~AdditionalEvents, 1 t t e.g., increased~
phosphatidote/
Scheme II. This scheme suggests an overall mechanism of stimulation for neutrophils treated with fMLP. Pathway b, (bold arrows) may involve a protein kinase distinct from that activated by PMA. This kinase may phosphorylate the 47 kDa protein on a site distinct from those which are phosphorylated when PMA is the stimulus a n d / o r may phosphorylate a yet unidentified component (protein X) that enables the oxidase to become functional with little or no phosphorylation of the 47 kDa protein. Stimulation may also occur by pathway a. Phosphatidate may also function in a stimulatory pathway that does not involve phosphorylation of the 47 kDa protein (pathway c). However, that pathway is largely insensitive to H-7 (200/~M) and staurosporine (150 nM) and is thus not likely to be functional with fMLP (see Refs. 18, 32). Neutrophils stimulated with PMA or fMLP exhibit a redistribution of the activity of PKC from a soluble to a particulate fraction that is tight ('membrane inserted') or loose (' membrane elutable'), respectively [52,53]. The significance of these types of associations are discussed elsehwere [54].
a matter of conjecture. However, a modest amount of speculation may be appropriate. Perhaps it catalyzes the phosphorylation of the 47 kDa protein on a unique site so that it becomes activated with comparatively little phosphorylation or catalyzes the phosphorylation of a yet unidentified component (component X in Scheme II) that obviates or reduces the necessity for phosphorylation of the 47 kDa protein. As noted above, neutrophils which lack the 47 kDa protein do not produce 0 2 in response to any stimulus [2]. Thus, this protein must be a critical component of all the stimulatory pathways, even if its phosphorylation is not always required. In contrast, several sites on the 47 kDa protein are phosphorylated when PMA is the stimulus [8] and the extent of this phosphorylation correlates with the quantities of 0 2 released (e.g., Refs. 8, 11, 14). H-7 inhibits 0 2 release from neutrophils stimulated with either PMA or fMLP with nearly identical kinetics (Fig. 3) and must therefore inhibit both pathways a and b in Scheme II. This antagonist exhibits very similar K i values towards certain protein kinases [35]. The inhibition of 0 2 release from fMLP stimulated cells by H-7 has a large temperature dependence, whereas that with PMA does not [17], which is consistent with the critical targets for this drug being different in cells stimulated with these agents. Two related studies have been published [28,51]. One reported that H-7 inhibits the phosphorylation of the 47 kDa protein, but not O{ release in neutrophils stimulated with fMLP [28]. Whether the failure to block 02release in that report was due to the temperature-depen-
dence cited above a n d / o r the concentration of H-7 employed (25 /~M) is unknown. However, these data again indicate that phosphorylation of the 47 kDa protein is not the critical target for 0 2 release in cells stimulated with this agent. The second report noted that 100 nM staurosporine inhibited both the phosphorylation of the 47 kDa protein and 0 2 release in neutrophils stimulated with either PMA or fMLP [51]. It was suggested that these results were due to the blocking of a common step in the mechanism of signal transduction with these stimuli; i.e., phosphorylation of the 47 kDa protein by PKC [51]. However, the effects at lower concentrations of staurosporine, where a dissociation between these phenomena is evident, were not examined. Neutrophils stimulated with retinal or high amounts of sn-l,2-dioctanoylglycerol can also produce large amounts of 0 2 without any detectable phosphorylation of the 47 kDa protein [18,32]. However, the O{ released under those circumstances was largely insensitive to 200/~M H-7 or 150 nM staurosporine [18,32], which clearly indicates that certain aspects of the pathway(s) involved with those stimuli are different from those functional with fMLP. Thus, staurosporine is a very useful drug for uncovering differences in the signaltransduction pathways of neutrophils.
Acknowledgements These studies were supported by grants AI-23323, AI-24321 and AI-28342 from the National Institutes of
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