299
Biochimica et Biophysica Acta, 1052 (1990) 299-305 Elsevier BBAMCR 12672
Continuous phosphorylation of both the 47 and the 49 kDa proteins occurs during superoxide production by neutrophils P a u l G. H e y w o r t h a n d J o h n A. B a d w e y Department of Cell Physiology, Boston Biomedical Research Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA and Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA (U.S.A.) (Received 29 August 1989)
Key words: Phosphorylation; Proteins, 47 an 49 kDa; Superoxide; Neutrophil
Neutrophils stimulated with 4fl-phorbol 12-myristate 13-acetate release large quantities of superoxide (02-) and exhibit an intense phosphorylation of two proteins with molecular masses of approx. 47 and 49 kDa. Treatment of unstimulated cells with antagonists of protein kinase C (e.g., staurosporine; 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) is known to inhibit both of these phenomena upon stimulation. These antagonists of PKC also cause a rapid cessation of 02- release when added to calls that are already stimulated. In this paper, we report that the addition of staurosporiue or H-7 to stimulated ueutrophils resulted in a rapid loss of 32p from both the 47 and the 49 kDa phosphoprotein bands, as detected by autoradiography. This suggests that these two proteins may be regulated by a continual cycle of phosphorylation and dephosphorylation in the stimulated cell, with the phosphorylation reactions predominating, or undergo a rapid degradation subsequent to phosphorylation. Either explanation is consistent with the view that protein kinase C activity is necessary to both innitiate and maintain 02- production in neutrophils stimulated with tumor promoters.
Introduction
Superoxide ( 0 2 ) is a major component of the oxygen-dependent antimicrobial mechanisms of phagocytic leukocytes. Neutrophils produce substantial amounts of that anion during phagocytosis or upon perturbation of their plasmalemma by a variety of agents (for review see Refs. 1 and 2). Some of the most potent stimulating agents are certain tumor promoters (e.g., phorbol esters) which activate protein kinase C (PKC). These compounds substitute for the endogenous cellular activator of this kinase, sn-l,2-diacylglycerol (for review see Ref. 3). Neutrophils produce increased amounts of this neutral lipid during cell-stimulation (e.g. Refs. 4-6). Phosphorylation of several proteins accompanies the stimulation of neutrophils with tumor-promoters or syn-
Abbreviations: O2-, superoxide; PKC, protein kinase C; PMA, 4/3phorbol 12-myristate 13-acetate; Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; H-7, 1-(5-isoquinolinylsulfonyl)-2methylpiperazine; Me2SO , dimethyl sulfoxide. Correspondence: J.A. Badwey, Ph.D., Department of Cell Physiology, Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114, U.S.A.
thetic diacylglycerols (e.g., Ref. 7). In particular, two proteins with molecular masses of approx. 47 and 49 kDa exhibit a dramatic incorporation of 32p, with kinetics compatible with their involvement in O~- production (e.g., Refs. 8 and 9). The 47 kDa protein is absent from the neutrophils of most patients with the autosomal recessive/cytochrome b-positive form of chronic granulomatous disease [10,11]. This disease is an inherited syndrome in which the patient's phagocytic leukocytes fail to produce 0 2 upon stimulation [2]. The 47 kDa protein is a substrate for PKC in vitro (e.g., Refs. 12 and 13). We have previously reported that antagonists of PKC block O~- release from neutrophils when added to the cells either prior to or during O~- release instigated by tumor promoters. These effects were observed with all of the inhibitors of PKC tested: certain phenothiazines (trifluoperazine, chlorpromazine) [14,15], the naphthalene sulfonamide W7 [14,15], the isoquinolinesulfonamide H-7 [16] and staurosporine [17]. This suggests that the activity of PKC is necessary both to initiate and maintain this response. Treatment of unstimulated neutrophils with these antagonists is known to inhibit phosphorylation of the 47 and the 49 kDa proteins upon subsequent stimulation (e.g., Refs. 12,13,17-19).
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
300 In this paper, we now report that the addition of staurosporine or H-7 to neutrophils that are already 3-~ stimulated resulted in a loss of "P from the 47 and the 49 kDa bands. This indicates that these proteins may be continually phosphorylated and dephosphorylated throughout the period of O~- release, with the phosphorylation reactions predominating. Alternatively, these proteins may undergo a rapid degradation upon increased phosphorylation. Implications of these data to the mechanism of O 2 production and the known reversibility of the stimulated state of neutrophils are discussed. These data provide additional support for a role for the 49 kDa protein in the stimulation of these cells (cf. Ref. 9). Experimental
in a Hepes-buffered saline solution (10 mM sodium Hepes, 138 mM NaC1, 2.7 mM KCI, and 7.5 mM D-glucose (pH 7.35)) containing 400 #Ci 32p i p e r ml. After 30 min, the cells were placed on ice and used in subsequent experiments without washing. The rate of O~- release from 32P-labeled neutrophils stimulated with PMA (50 nM) was 65 _+ 9% (SD, n = 10) of the analogous value for unlabeled cells, i.e., cells maintained on ice and not subjected to a preliminary incubation at 3 0 ° C w i t h 32p i. Thus, the labeling procedure per se only moderately affected O~ release.
A
procedures
B a
Materials
b
I
Staurosporine was purchased from Kamiya Biochemical Company, Thousand Oaks, CA. Sources of all other materials used have been described elsewhere [9,15,16].
t Cl
Methods
94
Preparation of neutrophils Guinea pig peritoneal neutrophils were prepared as described [20], Cell preparations contained > 90% neutrophils with viabilities always >_ 90%.
67 a3 U ¢-. v
13n
Superoxide release Superoxide release was measured in disposable 1-cm plastic cuvettes at 37 ° C by the continuous spectrophotometric measurement of the superoxide dismutase-inhibitable reduction of ferricytochrome c at 550 nm (e.g., Ref. 15). The standard assay mixture consisted of a modified Dulbecco's phosphate-buffered saline medium (138 mM NaC1, 2.7 mM KC1, 16.2 mM NazHPO4, 1.47 mM KHzPO4, 0.90 mM CaCI2, and 0.50 mM MgC12 (pH 7.35)) [21] containing 7.5 mM D-glucose, 0.075 mM ferricytochrome c and 1.0 to 1.5-10 6 cells/ml. The blank contained all of these components plus 2 0 / ~ g / m l superoxide dismutase. Cells were incubated in the reaction mixture for 3 min at 37 ° before 0 2 release was initiated by the addition of PMA (50 nM). Stock solutions of P M A (2 m g / m l ) and staurosporine (1 m g / m l ) were prepared in Me2SO and stored in the dark at - 2 0 ° C and 4 ° C , respectively. 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 solvents were shown not to cause any of the effects noted. Stock solutions (10 mM) of H-7 were prepared in water and stored at 4 ° C in the dark.
Labeling of neutrophils with 32Pi Neutrophils (108 per ml) were resuspended at 30 ° C
c
°~
43--
.w
-V 13
13 I--
N
f_
E ,-
O ::31 I,i
o
30
:E C
20 o
~
H
n
Distance Fig. 1. Effects of s t a u r o s p o r i n e on the p h o s p h o r y l a t i o n of the 47 and
the 49 kDa proteins of neutrophils. (A) 32p-labeled neutrophils (1.510 6 per ml) were treated with 15 nM staurosporine for 3 min prior to stimulation with PMA (50 nM) for 1 min. The autoradiograms shown are from: (a) unstimulated cells (i.e., treated with 0.25% (v/v) Me2SO for 1 minute); (b) stimulated cells; and (c) stimulated cells treated with staurosporine (I). The 47 and the 49 kDa proteins are indicated by the unbroken and broken arrows, respectively. Peak V (right margin) is present in both unstimulated and stimulated cells and is useful for orientation purposes. Staurosporine (15 nM) did not affect cell viability, as measured by the exclusion of Trypan blue, nor did it affect 0 2 release in an 0 2 generating system (i.e., xanthine oxidase plus purine). (B) The densitometric scans of the 47 and 49 kDa proteins, and Peak V. This pattern was observed in three separate experiments performed on different preparations of cells (a-c).
301 neutrophils (lane a) and neutrophils stimulated with an optimal amount of PMA (50 nM) for 1 minute (lane b). As previously reported (e.g., Ref. 9), the most striking changes upon stimulation were a marked incorporation of 32p into a 47 kDa proteins and an enhanced incorporation of 32p into a 49 kDa species. These proteins are indicated by the unbroken and broken arrows, respectively. 1 min represents a time at which the maximal rate of 0 2 release was occurring (Fig. 2A, curve b / e ; see below). This rate for 32p-labeled cells was 36 + 5 nmol 0 2 per min per 107 cells (SD, n = 10). Treatment of the cells with the PKC antagonist staurosporine (15 nM) [22] for 3 min at 37 o C prior to stimulation reduced this value to 2.5 + 2.0 nmol O~- per min per 10 7 cells (SD, n = 4). This treatment with staurosporine also dramatically inhibited the enhanced incorporation of 32p into the 49 kDa protein and reduced the labeling of the 47 kDa protein (Fig. 1, lane c; Ref. 17). It is interesting to note here that the staurosporine-treated cells did exhibit an increased labeling of the 47 kDa
Stimulation of 32p-labeled cells for autoradiography 32P-labeled neutrophils (1.5 • 106/ml) were stimulatd in plastic cuvettes under the same conditions utilized for measuring O 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 [9]. Results
Effects of staurosporine on neutrophils Fig. 1 compares the phosphoproteins in unstimulated A
I
I
i
i
i
E 0.2
I
i
0.3
E 0
I
_
i
i
1
g L 0 0.1 J~
Biochimica et Biophysica Acta, 1052 (1990) 299-305 Elsevier BBAMCR 12672
Continuous phosphorylation of both the 47 and the 49 kDa proteins occurs during superoxide production by neutrophils P a u l G. H e y w o r t h a n d J o h n A. B a d w e y Department of Cell Physiology, Boston Biomedical Research Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA and Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA (U.S.A.) (Received 29 August 1989)
Key words: Phosphorylation; Proteins, 47 an 49 kDa; Superoxide; Neutrophil
Neutrophils stimulated with 4fl-phorbol 12-myristate 13-acetate release large quantities of superoxide (02-) and exhibit an intense phosphorylation of two proteins with molecular masses of approx. 47 and 49 kDa. Treatment of unstimulated cells with antagonists of protein kinase C (e.g., staurosporine; 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) is known to inhibit both of these phenomena upon stimulation. These antagonists of PKC also cause a rapid cessation of 02- release when added to calls that are already stimulated. In this paper, we report that the addition of staurosporiue or H-7 to stimulated ueutrophils resulted in a rapid loss of 32p from both the 47 and the 49 kDa phosphoprotein bands, as detected by autoradiography. This suggests that these two proteins may be regulated by a continual cycle of phosphorylation and dephosphorylation in the stimulated cell, with the phosphorylation reactions predominating, or undergo a rapid degradation subsequent to phosphorylation. Either explanation is consistent with the view that protein kinase C activity is necessary to both innitiate and maintain 02- production in neutrophils stimulated with tumor promoters.
Introduction
Superoxide ( 0 2 ) is a major component of the oxygen-dependent antimicrobial mechanisms of phagocytic leukocytes. Neutrophils produce substantial amounts of that anion during phagocytosis or upon perturbation of their plasmalemma by a variety of agents (for review see Refs. 1 and 2). Some of the most potent stimulating agents are certain tumor promoters (e.g., phorbol esters) which activate protein kinase C (PKC). These compounds substitute for the endogenous cellular activator of this kinase, sn-l,2-diacylglycerol (for review see Ref. 3). Neutrophils produce increased amounts of this neutral lipid during cell-stimulation (e.g. Refs. 4-6). Phosphorylation of several proteins accompanies the stimulation of neutrophils with tumor-promoters or syn-
Abbreviations: O2-, superoxide; PKC, protein kinase C; PMA, 4/3phorbol 12-myristate 13-acetate; Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; H-7, 1-(5-isoquinolinylsulfonyl)-2methylpiperazine; Me2SO , dimethyl sulfoxide. Correspondence: J.A. Badwey, Ph.D., Department of Cell Physiology, Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114, U.S.A.
thetic diacylglycerols (e.g., Ref. 7). In particular, two proteins with molecular masses of approx. 47 and 49 kDa exhibit a dramatic incorporation of 32p, with kinetics compatible with their involvement in O~- production (e.g., Refs. 8 and 9). The 47 kDa protein is absent from the neutrophils of most patients with the autosomal recessive/cytochrome b-positive form of chronic granulomatous disease [10,11]. This disease is an inherited syndrome in which the patient's phagocytic leukocytes fail to produce 0 2 upon stimulation [2]. The 47 kDa protein is a substrate for PKC in vitro (e.g., Refs. 12 and 13). We have previously reported that antagonists of PKC block O~- release from neutrophils when added to the cells either prior to or during O~- release instigated by tumor promoters. These effects were observed with all of the inhibitors of PKC tested: certain phenothiazines (trifluoperazine, chlorpromazine) [14,15], the naphthalene sulfonamide W7 [14,15], the isoquinolinesulfonamide H-7 [16] and staurosporine [17]. This suggests that the activity of PKC is necessary both to initiate and maintain this response. Treatment of unstimulated neutrophils with these antagonists is known to inhibit phosphorylation of the 47 and the 49 kDa proteins upon subsequent stimulation (e.g., Refs. 12,13,17-19).
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
300 In this paper, we now report that the addition of staurosporine or H-7 to neutrophils that are already 3-~ stimulated resulted in a loss of "P from the 47 and the 49 kDa bands. This indicates that these proteins may be continually phosphorylated and dephosphorylated throughout the period of O~- release, with the phosphorylation reactions predominating. Alternatively, these proteins may undergo a rapid degradation upon increased phosphorylation. Implications of these data to the mechanism of O 2 production and the known reversibility of the stimulated state of neutrophils are discussed. These data provide additional support for a role for the 49 kDa protein in the stimulation of these cells (cf. Ref. 9). Experimental
in a Hepes-buffered saline solution (10 mM sodium Hepes, 138 mM NaC1, 2.7 mM KCI, and 7.5 mM D-glucose (pH 7.35)) containing 400 #Ci 32p i p e r ml. After 30 min, the cells were placed on ice and used in subsequent experiments without washing. The rate of O~- release from 32P-labeled neutrophils stimulated with PMA (50 nM) was 65 _+ 9% (SD, n = 10) of the analogous value for unlabeled cells, i.e., cells maintained on ice and not subjected to a preliminary incubation at 3 0 ° C w i t h 32p i. Thus, the labeling procedure per se only moderately affected O~ release.
A
procedures
B a
Materials
b
I
Staurosporine was purchased from Kamiya Biochemical Company, Thousand Oaks, CA. Sources of all other materials used have been described elsewhere [9,15,16].
t Cl
Methods
94
Preparation of neutrophils Guinea pig peritoneal neutrophils were prepared as described [20], Cell preparations contained > 90% neutrophils with viabilities always >_ 90%.
67 a3 U ¢-. v
13n
Superoxide release Superoxide release was measured in disposable 1-cm plastic cuvettes at 37 ° C by the continuous spectrophotometric measurement of the superoxide dismutase-inhibitable reduction of ferricytochrome c at 550 nm (e.g., Ref. 15). The standard assay mixture consisted of a modified Dulbecco's phosphate-buffered saline medium (138 mM NaC1, 2.7 mM KC1, 16.2 mM NazHPO4, 1.47 mM KHzPO4, 0.90 mM CaCI2, and 0.50 mM MgC12 (pH 7.35)) [21] containing 7.5 mM D-glucose, 0.075 mM ferricytochrome c and 1.0 to 1.5-10 6 cells/ml. The blank contained all of these components plus 2 0 / ~ g / m l superoxide dismutase. Cells were incubated in the reaction mixture for 3 min at 37 ° before 0 2 release was initiated by the addition of PMA (50 nM). Stock solutions of P M A (2 m g / m l ) and staurosporine (1 m g / m l ) were prepared in Me2SO and stored in the dark at - 2 0 ° C and 4 ° C , respectively. 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 solvents were shown not to cause any of the effects noted. Stock solutions (10 mM) of H-7 were prepared in water and stored at 4 ° C in the dark.
Labeling of neutrophils with 32Pi Neutrophils (108 per ml) were resuspended at 30 ° C
c
°~
43--
.w
-V 13
13 I--
N
f_
E ,-
O ::31 I,i
o
30
:E C
20 o
~
H
n
Distance Fig. 1. Effects of s t a u r o s p o r i n e on the p h o s p h o r y l a t i o n of the 47 and
the 49 kDa proteins of neutrophils. (A) 32p-labeled neutrophils (1.510 6 per ml) were treated with 15 nM staurosporine for 3 min prior to stimulation with PMA (50 nM) for 1 min. The autoradiograms shown are from: (a) unstimulated cells (i.e., treated with 0.25% (v/v) Me2SO for 1 minute); (b) stimulated cells; and (c) stimulated cells treated with staurosporine (I). The 47 and the 49 kDa proteins are indicated by the unbroken and broken arrows, respectively. Peak V (right margin) is present in both unstimulated and stimulated cells and is useful for orientation purposes. Staurosporine (15 nM) did not affect cell viability, as measured by the exclusion of Trypan blue, nor did it affect 0 2 release in an 0 2 generating system (i.e., xanthine oxidase plus purine). (B) The densitometric scans of the 47 and 49 kDa proteins, and Peak V. This pattern was observed in three separate experiments performed on different preparations of cells (a-c).
301 neutrophils (lane a) and neutrophils stimulated with an optimal amount of PMA (50 nM) for 1 minute (lane b). As previously reported (e.g., Ref. 9), the most striking changes upon stimulation were a marked incorporation of 32p into a 47 kDa proteins and an enhanced incorporation of 32p into a 49 kDa species. These proteins are indicated by the unbroken and broken arrows, respectively. 1 min represents a time at which the maximal rate of 0 2 release was occurring (Fig. 2A, curve b / e ; see below). This rate for 32p-labeled cells was 36 + 5 nmol 0 2 per min per 107 cells (SD, n = 10). Treatment of the cells with the PKC antagonist staurosporine (15 nM) [22] for 3 min at 37 o C prior to stimulation reduced this value to 2.5 + 2.0 nmol O~- per min per 10 7 cells (SD, n = 4). This treatment with staurosporine also dramatically inhibited the enhanced incorporation of 32p into the 49 kDa protein and reduced the labeling of the 47 kDa protein (Fig. 1, lane c; Ref. 17). It is interesting to note here that the staurosporine-treated cells did exhibit an increased labeling of the 47 kDa
Stimulation of 32p-labeled cells for autoradiography 32P-labeled neutrophils (1.5 • 106/ml) were stimulatd in plastic cuvettes under the same conditions utilized for measuring O 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 [9]. Results
Effects of staurosporine on neutrophils Fig. 1 compares the phosphoproteins in unstimulated A
I
I
i
i
i
E 0.2
I
i
0.3
E 0
I
_
i
i
1
g L 0 0.1 J~
