A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Biochimica et Biophysica Acta, 1010 (1989) 78-87 Elsevier

78

BBA 12388

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages Jonny Wijkander and Roger Sundler Department of Physiological Chemistry, University of Lund, Lund (Sweden) (Received 18 July 1988)

Key words: Phorbol ester; Protein phosphorylation; Zymosan; Lipopolysaccharide; Lipocortin; (Mouse macrophage)

Mouse peritoneal macmphages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phusphorylation induced in these cells by 4/~-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyi-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of pbosphorylation differed among the phospboprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the pbosphorylation sufficiently rapid to be involved in the activation of the pbospholipase A pathway. Phosphorylation of lipocortin I or I! could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phospborylation and the mobilization of arachidonic acid in response to PMA or dioetanoyMglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibite~ ~by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidunic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phospborylation pattern different from that induced by PMA, and down-regulation of protein kinase C did no: affect lipopolysaceharide.induced protein phosphorylation. The results indicate (i) that protein kinase C plays a crt~ca| role also in zymosan-induced activation of the phosphol,.'pase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipoeortins does not explain the kinase C-dependent activation.

Introduction Monocytes and macrophages represent a widely dispersed cellular system that takes part in tissue homeostasis and the defense against microbial invasio~a by a variety of mechanisms [1]. These cells recognize microbial cell wall components such as zymosan via cell surface receptors [2] and their response to such agents includes a rapid mobilization of phospholipid-bound arachidonic acid, accompanied by release of prosta~andins and ieukotrienes [3,4]. Resident mouse peritoneal macrophages have been shown to respond to direct activators of protein kinase

Abbreviations: PMA, 4/3-phorbol 12-myristate 13-acetate; DOG, dioctanoyigiyceml; LPS, lipopolysaccharide. Correspondence: J. Wijkander, Department of Physiological Chemistry, University o~ Lund, Box o4, S 221 00 Lund, Sweden.

C, such as phorbol diesters and diacylglycerol, as well as to zymosan by enhanced deacylation of phosphatidylinositol and mobilization of arachidonic acid from the major phospholipids [5,6]. While this strongly suggests a role for kinase C-induced protein phosphorylation in the sequence of events leading t~ enhanced phospholipid deacylation, current knowledge about the signal-transducing pathway subsequent to kinase Cactivation in this as well as in many other cellular systems is linfited [7]. It has been proposed earlier that certain proteins that express phospholipase A 2-inhibitory properties in vitro (now named lipocortin [8]) might play such a role also in vivo [9,10] and that their inhibitory effect could be reduced by phosphorylation on serine or tyrosine residues [11,12]. We, therefore, compared the phosphorylation of proteins, including iipocortin, with the mobilization of arachidonic acid in macroDhages responding to zymosan, calcium ionophore and activators of kinase C.

0157-48q9/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical P , ision)

79

Experimental procedures Isolation and labeling of macrophages Resident cells were harvested from the peritoneal cavity of outbre,d female NMRI mice (Alab, Sweden) in 4 ml of Medium 199 containing heparin (20 units/ml) and 1% heat-inactivated fetal calf serum (Flow Laboratories). The cells were plated (approx. 4-106 ceils/35mm well) onto 6-well Costar tissue culture dishes and incubated at 37 °C in an atmosphere of 5~ CO 2 in air. Nonadherent cells were removed 2-3 h after plating and to each dish was added 1 ml of Medium 199 (Earle's balanced salt solution supplemented with 10 mM Hepes) containing 10% fetal calf serum. On the second day of culture, cells were washed with PO4-free Earle's balanced salt solution supplemented with 10 mM Hepes (buffer A) and were then labeled for 1 h with 0.2 mCi carrier-free 32po4 (Amersham International, U.K.) dissolved in 1 ml of buffer A . When protein phosphorylation experiments were conducted in calcium-free medium, 3 2 p o 4 was added in Ca2+-free buffer A. Labeling of macrophages with 3 #Ci of [5,6,8,9,11, 12,14,15-3H]arachidonic acid or 10 #Ci of myo-[23H]inositol (Amersham International, U.K.) was carried out for 24 h as described [6]. At the end of the experiment, the release of radioactivity into culture medium and the polar phase of the lipid extract (arachidonic acid-labeled cells) or the generation of lysophosphatidylinositol, glycerophosphoinositol and inositol mono-, bis- and trisphosphate (inositoi-labeled cells; see Refs. 5 and 6) was determined. In some experiments, macrophages were preincubated with 50 nM PMA for 24 h, either prior to labeling with 32PO4 or during the labeling period in the case of [3H]arachidonic acid or [3H]inositol. After preincubation, cells were washed three times with either PO4-free buffer A or, in the case of tritium-labeled cells, with phosphate-buffered saline. In one series of experiments (data not shown), specific pathogen-free Swiss Webster mice (Bantin & Kingman Ltd., Hull, U.K.) were used as the source of peritoneal macrophages. Results on protein phosphorylation and mobilization of ar~chidonic acid in response to all agents were indistinguishable from those presented in this paper.

Cell stimulation The 1,2-sn-DOG and 1,3-DOG were prepared and purified as described earlier [6]. PMA, 4fl-phorbol 12,13-dibutyrate, chlorpromazine (Sigma), A23187 (Boehfinger, Mannheim) and DOG were all dissolved in dimethylsulfoxide while LPS from Salmonella minnesota Re 595 (Calbiochem) was dissolved in distilled water. These stimuli were added in 5-10 #1 to ceil cultures. Phospholipase C from Clostridium t,~rfingens (40 /zg)

and zymosan (Sigma) were added to cultures in 50 #l of buffer A. Addition of the various agents were made during the last part of the labeling period. At the end of the treatment, the cells were washed three times with buffer A and then scraped off the dish with a teflon policeman in 200 #1 of electrophoresis sample buffer containing 0.625 M Tris-HCl (pH 6.8)/2~ SDS/10% glycerol/5% 2-mercaptoethanol/0.002~ Bromophenol blue. Samples were boiled for 5 rain prior to electrophoresis of 50 #1 aliquots.

Fractionation and gel chromatography Labeled cells were washed with ice-cold buffer A and scraped off the dish in 1 ml of 10 mM Hepes, 1 mM EGTA and 10 mM NaF (pH 7.4). Cells were homogenized using a tight-fitting teflon pestle with 10 full strokes at 1500 rpm. Rapidly sedimenting material was removed by centrifugation at 700 x g for 5 min and the supernatant was then subjected to ultracentrifugation at 100000 x g for 1 h. Proteins in the soluble fraction were subjected to gel chromatography on a column of Sephadex G-200 superfine (1 × 47 cm). The column was eluted with a buffer consisting of 0.1 M NaCI/10 mM Hepes/1 mM EGTA/10 mM NaF (pH 7.4). Proteins in the collected fractions were precipitated by the addition of 6 vol. of ice-cold acetgne/13 M NH4OH (30:1.7, v/v) as described [13]. The samples were allowed to stand for 10 rain and were then centrifuged for 10 min at 16000 ×g. Samples were kept at 4°C during the whole fractionation procedure. Precipitated proteins from gel chromatography and the pellet from ultracentrifugation were suspended in electrophoresis sample buffer and boiled for 5 min. The void volume of ~he Sephadex G-200 column (13.2 ml) was determined w~th Blue De×tran 2000 and the proteins used for calibration of the column were bovine serum albumin, ovalbumin and soya bean trypsin inhibitor (K.~v= 0.30, 0,.43 and 0.62, respectively).

Isolation of ribosomes Labeled cells were washed with buffer A and scraped off the dish in 0.6 ml of a detergent buffer containing 1% Triton X-100/l% sodium deoxycholate/10 mM N a F / 5 mM dithioerythritol/1.5 mM MgCI2/1 mM EGTA/10 mM Tris-HCl (pH 7.0). After centrifugation at 3000 rpm for 10 rain, the supernatant was layered on a 1.6 M sucrose cushion in a buffer containing 500 mM KCI/5 mM MgCl2/1 mM dithJoerythritol/20 mM Tris-HCl (pH 7.4) and centrifuged at 120000 × g for 20 h at 4 ° C [14]. The supernatant was removed and the ribosomal p e l l ~ •as dissolved in electrophoresis sample buffer. Proteins .~n the upper part of the supernatant (containing detergent buffer) were i,recipitated with acetone/13 M NtI4OH and dissolved in electrophoresis sample buffer.

80

Immunoprecipitation of lipocortin I and H Recombinant human lipocortin I protein and antisera against lipocortin I and II was provided by Dr. R.B. Pepinsky, Biogen Research Corporation, U.S.A. Macrophages labeled with 32po4 were washed with buffer A and scraped off the dishes in a detergent buffer containing 50 mM NaCI/20 mM Tris-HCl (pH 7.3)/4 mM iodoacetic acid/1 mM ammonium vanadate/0.5% Nonidet P-40/0,5% sodium deoxycholate. After centrifugation for 5 min at 5000 rpm the supernatant was subjected to immunoprecipitation with antisera followed by adsorption onto protein A-Sepharose as described by Pepinsky and Sinclair [15]. The immunoprecipitate was washed four times with detergent buffer and solubilized in electrophoresis sample buffer. Electrophoresis and autoradiography Samples (approx. 20 ~ag of protein) were subjected to SDS-polyacrylamide gel electrophoresis according to the method of Laemmli [16], using 1.5-ram slab gels. Separation and stacking gels contained 10 and 3~ polyacrylamide, respectively. After electrophoresis at constant current (20 mA) for about 14 h, the gels were fixed, stained with 0.15~ Coomassie blue R250, destained and dried under vacuum onto filter papers. Gels with immunoprecipitated proteins were stained with silver according to a method described by Wayne et al. [17]. The dried gels were exposed to X-ray film (Hyperfilm-~max, Amersham) at - 6 0 ° C for 20-30 h except for gels from immunoprecipitation where exposure time was 160 h with an intensifying screen. Scanning of the autoradiographs was conducted with a Joyce-Loebl Chromoscan 3 with a slit width of 0.05 mm and an absorbance range of 1.0 or 2.0 ram. The apparent molecular weight of the phosphoproteins was determined using a mixture of reference proteins (MWSDS-200, Sigma) containing myosin (205 kDa), /~galactosidase (116 kDa), phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin 45 kDa) and carbonic anhydrase (29 kDa). The mixture was loaded separately onto each gel. Results

P,ct¢i;; phosphorylation in response to protein kinase C activators The pattern of protein subunit phosphorylation in macrophages exposed for 5 rain to PMA (10 nM) in comparison to untreated cells, is shown in Fig. 1 (lanes A and B). The most conspicious increase in phosphorylation in response to PMA occurred in bands of approx. 31 and 45 kDa. A comparison of autoradiogra~hs with their parent gels, stained for protein, showed that the 45 kDa phosphoprotein was well separated from a heavily stained 43 kDa band that most likely represents actin and did not comigrate with any major protein band.

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Fig. 1. Protein phosphorylation in response to activation of protein kinase C. Mouse peritoaeal macrophages were labeled with 32p o 4 and treated with the indicated agents. Whole cell samples were subjected to electrophoresis followed by autoradiography (see Experimental procedures). Bars to the left mark the position of reference proteins (kDa) and the position of the 29, 31 and 45 kDa bands are indicated to the fight. Lane A, control; lane B, 10 nM PMA for 5 min; lane C, 50 ~M chlorpromazine for 10 rain prior to addition of 10 nM PMA for 5 min; lane D, 20 /~M 1,2-sn-DOG for 5 min; lane E, 20 ~M 1,3-DOG for 5 min; lane F, 40/~g of phospholipase C for 15 min.

Increases in phosphorylation were also seen in bands of 29, 54, 63, 78 kDa and to a minor extent, in 88 and 103 kDa bands. This pattern of PMA-induced protein phosphorylation, complemented with a decrease in the phosphorylation of a 95 kDa band, has been consistently observed in more than ten separate experiments. No additional phosphoproteins responding to PMA could be detected by analyzing samples on either 7.5~ or 12.5~ polyacrylamide gels (not shown). Neither was any difference in the PMA-induced phosphorylation pattern observed when cells were labeled with 32po4 for 3 h instead of 1 h. Other experiments showed that the 4-a-isomer of phorbol 12,13-didecanoate, in contrast to the r-isomer, did not enhance protein phosphorylation. Fig. 1 also demonstrates that the same phosphorylation pattern was induced by another direct activator of protein kinase C [18], 1,2-sn-DOG (lane D), but not by the inactive 1,3-isomer (lane E), and by treatment of the cells with phospholipase C from Clostridium perfringens (lane F). This enzyme generates endogenous diacylglycerol from cellular phospho!,_'p,_'ds,primarily from phosphatidylcholine [19]. The phosphorylation induced by PMA was partially inhibited by 50 #M chlorpromazine (lane C), which is known to inhibit protein kinase C in vitro [20]. However, further experiments showed that both chlorpromazine and trifluoperazine were less potent inhibitors of PMA-induced protein phosphoryla-

81

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duced phosphorylation after removal of the phorbol ester, but not to the same extent as the 45 kDa protein. Upon a second addition of phorbol dibutyrate the original phosphorylation pattern reappeared within minutes.

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TIME train) Fig. 2. Time-courseof PMA-induced protein phosphorylation. Macrophages were labeled with -~2PO, and stimulated with 10 nM PMA for the time indicated (0-5 min). Whole cell samples were then subjected to electrophoresis followed by autoradiography and the autoradio. graph was scanned as described in Experimental procedures. Relative absorbance was determined by integration of peak areas. (a) 65 kDa band; (b) 45 kDa band; (c) 31 kDa band; (d) 29 kDa band.

tion (virtually no inhibition at 20 /~M) than of the mobilization of arachidonic acid induced by PMA in the same cell type (half-maximal inhibition at 10-15 ~M, Ref. 5). The phosphorylation induced by PMA occurred rapidly and the pattern was fully developed within 5 min, although it was not synchronous for the different phosphoproteins (Fig. 2). Thus, the 45 kDa band reached its final level of phosphorylation within 1 min. while the increase in the 29 and 31 kDa bands showed a lag period. The 65 kDa band followed a similar time course as the 45 k D a band, while other changes, including the reduction in phosphorylation of the 95 kDa band, occurred later. The dose-response relationship for both PMA and 1,2-sn-DOG showed partial phosphorylation of the 45 k D a band, but not of the 29 and 31 kDa bands at 0.2-1 nM PMA or 0.2-2 # M 1,2-sn-DOG, while 10 nM PMA or 20 #M i,2-sn-DOG was required for the phosphorylation pattern to develop fully within 5 rain (not shown). Reversibility of phorbol diester-induce, d protein phosphotylation The v e ~ fast phosphorylation of the 45 k D a protein was found to be rapidly reversible, by using phorbol dibutyrate instead of PMA as slimulus. When macrophages were first stimulated and then incubated for 5 min in the absence of phorbol cster, the phosphorylation of the 45 kDa band returned to almost the control level. Other phosphorylated proteins also showed re-

Protein phosphorylation in response to zymosan, Ca 2 + ionophore and L P S It is known from previous studies that zymosan particles and the Ca 2÷ ionophore A23187 in macrophages cause activation of both phospholipase C-type degradation of inositol phospholipids and mobilization of arachidonic acid [5,6]. It was, therefore, of interest to assess whether they enhanced protein phosphorylation in a manner similar to that of direct activators of protein kinase C. As shown in Fig. 3 (lanes B and D) zymosan and ionophore induced a phosphorylation pattern quite similar to that induced by PMA, although the increase in the 45 kDa band was not quite as large as that seen with 10 nM PMA. The 45 and 65 kDa bands, that were rapidly phosphorylated in response to PMA, were also rapidly phosphorylated in response to zymosan and seen already at 1 min (not shown). Although stimulation with 1 m g / m l of zymosan was used in this study, the same phosphorylation response was seen even at 0.01 mg/ml. LPS has previously been shown to induce protein phosphorylation in thioglycollate-elicitated mouse mac-

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Fig. 3. Zymosan-, LPS- and ionophore A23187-indu:ed protein phosphorylation and effect of pretreatment with PMA. Mouse peritoneal macrophages were labeled with 3Zpo4 and treated with the indicated agents. Whole cell samples were subjected to electrophoresis followed by autoradiography. Bars to the left mark the position of reference proteins (kDa) and the position of the 29, 31 and 45 kDa bands are indicated to the right. Lanes A an( E, control; lanes B and F, zymosan (1 mg/ml) for 15 min; lanes C and G, 10 pg LPS from Salmonella minnesota for 15 min; lanes D and H, 1/tM A23187 for 5 min. Lanes E-H were subjected to pretreatment with 50 nM PMA for 24 h prior to labeling and stimulation.

82

rophages with a pattern bearing similarity to that induced by PMA [21]. It has also been suggested that LPS (or lipid A) might act as an activator of protein kinase C in vitro [22] and in mouse macrophages [23]. Also in our study, treatment with LPS either from Salmonella minnesota or Escherichia coil induced phosphorylation of a set of proteins that overlapped with that seen in response to PMA, but there were large differences in intensity for certain proteins (Fig. 3, lane C). Compared to PMA and other stimuli known to cause activation of protein kinase C, LPS induced virtually no increase in the 45 kDa band, while the enhanced phosphorylation of the 29 and 31 kDa bands was comparable to that seen with PMA. The phosphorylation of the 88 and 103 kDa bands was more pronounced with LPS than with PMA, while the degree of reduction in phosphorylation of the 95 kDa band was similar. Stimulation for 60 min caused no further change in the pattern of phosphorylation observed at 15 min. Lipid A (0.1-2 ~tg/ml), which is considered to be the biologically active part of LPS with regard to effects on macrophages [22], caused phosphorylation of the same set of proteins as LPS.

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Protein phosphorylation and arachidonic acid mobilization in kinase C.depleted cells Treatment with phorbol diester for extended periods of time has been shown to reduce greatly the protein kinase C activity in several cell types, including macrophages [24]. When cells had been pretreated with either 50 nM PMA, 200 nM 4,8-phorbol uidecanoate (but not the 4a-isomer) or 300 nM phorbol dibutyrate for 24 h, specific binding of [3H]phorbol dibutyrate was reduced by 94-100~$. Stimulation of such cells with either a second dose of PMA or with 1,2-sn-DOG, resulted in almost no increase in phosphorylation compared to control cells (Fig. 4). When kinase C-depleted cells were stimulated with either zymosan (FIB. 3 lane F) or A23187 (Fig. 3 lane H) the phosphorylation of the 45 kDa band was virtually completely inhibited, while the 29 and 31 kDa bands increased to approximately the same extent as those without pretreatment with PMA, indicating that these proteins are phosphorylated by kinase(s) other than kinase C. The phosphorylation pattern induced by LPS appeared to be unaffected by pretteatment with PMA (Fig. 3 lane (3). On the basis of this result and the very weak phosphorylation of the 45 kDa protein, we conelude that LPS-induced phosphorylation is not mediated via kinase C. This is of special interest, since we found LPS, in contrast to PMA and other stimuli that activate protein kinase C, to be a very poor activator of araehidonie acid mobiliTation (not shown), in agreement with the results of Aderem et al. [25]. We also determined how the ability of PMA,! 1,2-snDOG, zymosan and ionophore A23187 to induce

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Scan length Fig. 4. Effect of pretreatment with PMA on protein phosphorylation induced by PMA and 1,2-sn-DOG. Macrophages were either pretreated with 50 nM PMA for 24 h (B, D and F) or not subjected to pretreatment (A, C and E). This w~s followedby labelingwith 32pot and stimulation with the indicated agertts. Cell samples were then subjected to electrophoresis followed by ~utoradiography and scannin8 of the autoradiograph.The position and molecularmass (kDa) of the reference proteins are marked at the top. Bars on the scans mark the position of specific phosphoprotein bands with apparent molecular masses of (from left to right); 103, 95, 88, 78, 65, 54, 45, 31 and 29 kDa. A and B, control; C and D, 50 nM PMA for 5 min; E and F, 20 FM 1,2-sn-DOGfor 5 min.

83 TABLE I

Effect of down-regulation of protein kinase C on arachidonic acid mobilization, deacylation of phosphatidylinositol and generation of inositol phosphates Macrophage cultures were prelabeled for 24 h with either [ 3H]arachi. donic acid or [3H]inositol in the presence c,r absence of 50 nM PMA (see Experimental procedures). The presence of PMA did not significantly affect the incorporation of radiolabeled arachidonic acid (mean incorporation 1.5.106 dpm), nor its distribution among individual phospholipids. Parallel cultures, with and without PMA-pretreatment, were incubated in fresh medium containing either no addition, PMA (50 or 500 nM), 1,2-sn-DOG (20 laM), ionophore A23187 (1/tM) for 15 rain or zymosan (1 mg/ml) for 30 rain, and the mobilization of arachidonic acid and the generation of iysophosphatidylinositol plus glycerophosphoinositol (phosphatidylinositol deacylation) and of inositol phosphates above that in control cultures was determined. In non-pretreated cultures, the stimulus-induced release of [ 3H]arachidonic acid ranged between 90 and 120.10 3 dpm, and the generation of water-soluble compounds was as previously reported [5,6,26]. The data shown represent PMA-pretreated cultures and are expressed as a percentage of the response in non-pretreated cultures (mean + S.D.) Stir,talus

Arachidonic acid release

Phosphatidylinositol deacylation

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148.7+11.8 ** (n -- 9)

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132.0+18.4 (n =11)

76.7± 8.7 (n = 4)

113.8± 4.3 *** (n = 4)

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cause depletion of intracellular C a 2 + and that are known to lead to considerable inhibition of PMA- and zymosan-induced phosphol/pase A activation and arachidonic acid release in these cells [5], there was no inhibition of the phosphorylation response to PMA (Fig. 5). This is consistent with the finding in human neutrophils that the phosphorylation pattern in response to PMA remains unchanged even at very low (10 nM) intracellular Ca 2+ [27]. Also the 1,2-sn-DOG- and zymosan-induced protein phosphorylations were unaffected by Ca 2+ depletion (not shown). These results indicate that the Ca 2+ dependence for phospholipase A activation and mobilization of araclfidonic acid is greater than that for the activation of protein kinase C.

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mobilization of arachidonic acid was affected by prior depletion of protein kinase C. As shown in Table I, pretreatment with PMA inhibited by 72-100~ the subsequent response to either PMA, 1,2-sn-DOG or zymosan. This strongly supports the proposal that the increase in arachidonic acid release induced by these agents is mediated via protein kinase C. In addition, the inhibition of arachidonic acid release was rough'~y parallel with an inhibition of the deacylation of phosphatidylinositol, determined from the accumulation of glycerophosphoinositol and lysophosphatidylinositol [26] in cells prelabeled with [3H]inositol (Table I). In contrast, the calcium-induced mobilization of arachidonic acid provoked by ionophore A23187 was somewhat enhanced rather than inhibited after pretreatment with PMA, as was the generation of inositolphosphates (via phospholipase C) in response to either zymomm or calcium ionophore (Table I). Ca 2 + dependence of proteirz phosphorylation In Ca2+-free medium containing EGTA and C a 2+ionophore A23187, i.e., conditions that are expected to

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Scan length Fig. 5. Effect of calcium depletion on PMA-induced protein pho:;phorylation. Macrophages were labeled with 32po 4 and treated as indicated. Samples were then subjected to electrophoresis followed by autoradiography. Reference proteins and bars on the scans are as de~':ribed in Fig. 4. A, control; B, 1 ~ M EGTA plus 2 / t M A23187 foz 15 min:; C, 10 nM PMA for 5 min:, D, 1 mM EGTA plus 2 ~M A23187 for 15 rain prior to stimulation with 10 nM PMA for 5 rnin.

84

Cellular localization of phosphoproteins In an attempt to determine the localization and native size of the phosphoproteins, a cell homogenate was fractionated by centrifugation into a 100000 x g pellet and a soluble fraction followed by gel chromatography of the latter fraction. The results showed that the 29 kDa component was recovered exclusively in the soluble fraction, while the 45 kDa band was recovered in both fractions (Figs. 6 and 7). Gel chromatography on Sephadex G-200 of the soluble fraction further showed that the 29 and 45 kDa bands both behaved as monomeric proteins, based on their Kav values (0.50 and 0.39, respectively). PMA has been shown to induce phosphorylation of the ribosomal protein $6 (molecular mass of 30-32 kDa) in various cell types [14,28-30]. When a ribosomal pellet was prepared from macrophages, the 31 kDa band was recovered in this fraction, while it was absent in the supernatant (Fig. 8). The supernatant also contained a closely adjacent phosphoprotein which showed no change in phosphorylation upon stimulation with PMA (see also Fig. 1). The

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Fig. 7. Phosphoproteins in a crude membrane fraction. The particulate fraction from the experiment described in the legend to Fig. 6 was subjected to electrophoresis followed by autoradiography. Lane A, control cells; lane B, cells stimulated with 10 nM PM• :0: ff rain. Bars on the left side mark the pn~ivinr, of refe;ence proteins (kDa) and the position of the 31 and 45 kDa bands are indicated on the right.

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28

Immunot~recipitation of lipocortin ELUTION VOLUME Cml] Fig, 6. Gel chromatography of soluble phosphoproteins. Macrophages labeled with 3ZPO4 received either no further treatment or were treated with 10 nM PMA for 5 rnin. Cells from six cultures were homogenized and fractionated into a crude membrane fraction and a soluble fraction, Soluble proteins were separated on a column of Sephadex G-200 superfine (bed volume 37 ml) and proteins in the collected fractions were precipitated and subjected to electrophoresis followed by autoradiography. The autoradiographs were scanned and relative absorbance was determined by integration of the peak areas. The molecular mass of the proteins used for calibration of the column is indicated at the top. For further details see Exnerimental procedures. Solid and broken lines indicate the phosphorylation of the 45 and 29 kDa proteins, respectively. Control, filled symbols; FMAstimulated cultures, open symbols.

Lipocortin constitutes a family of proteins that have been showr~ to inhibit phospholipase A 2 and also other phospholipases in vitro [8-11,31,32]. It has also been suggested that this inhibition can be suppressed by phosphorylation of the protein [11,12]. When detergentsolubilized proteins from macrophages were subjected to immunoprecipitation followed by electrophoresis, a single silver-stained protein band (also visible by Coomassie staining), with an apparent molecular mass of 37 kDa and the same electrophoretic mobility as recombinant human lipocortin I, was seen in samples precipitated with antiserum against lipocortin l, but not with antiserum against lipocortin II or non-immune

85

sodium fluoride (10 mM) and EDTA (2 mM) in the detergent buffer, to inhibit protein phosphatase activity during immunoprecipitation [33], did not reveal any deteclable lipocortin phosphorylation.

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Fig. 8. Phosphorylatio~! o[ a ribosomal protein. Macrophages labeled with 32po 4 received ¢:ither no further treatment (lanes A and C) or were treated with IC, nM PMA for 5 min (laaes B and D). A ribosomal pellet ar~fl a ribosome-free supernatant was then prepared from the samples as described in Experimental procedures and subjected to electrophoresi~ and autoradiography. Bars on the left side mark the position of refecence proteins (kDa) and the 31 kDa band is indicated on the right side. Lanes A and B, ribosomal pellet. Lanes C and D, supernatant from ribosome isolation. All of the ribosomal pellet, but only 1/8 of the ribosomal supernatant, was subjected to electrophoresis.

serum (Fig. 9, left). Control experiments showed that the 37 kDz baud was derived from the macrophages and not from the lip¢cortin 1 antiserum. PMA stimulation of 32po~ labeled macrophages for 5 min (Fig. 9, fight) or 15 ]nin (not shown) followed by immunoprecipitation showed, despite a prolonged exposure time during autoradiography, virtually no phosphorylation of the precipitated lipocortin I protein. For comparison, there were traces of other phosphoproteins recovered in the immunoprecipitate that showed increased phosphorylatinn, although they could not be detected by protein staining. These phosphoproteins were not seen (except for the 31 and 205 kDa bands) in samples precipitated with antiserum against lipocortin II or non-immune serum. Inclusion of

The present results define a distinct pattern of protein phosphorylation in mouse macrophages responding to activators of protein kinase C or zymosan. Among the phosphoproteins exhibiting increased phosphorylation, a 45 kDa protein recovered in both the soluble and particulate fraction and a 65 kDa t:ompo,~ent, behave as direct substrates for protein kinase C. Fi~st, they become rapidly phosphorylated when the cells are exposed to phorbol dieslters and are also rapidly phosphorylated in response to dioctanoylglycerol, exogenous phospholipase C, zynlosan or ionophore A23187. Secondly, their phosphorylation is virtually completely inhibited after down-regu~lation of the kinase by prolonged pretreatment with PMA. In contrast, a 29 kDa phosphoprotein recovered exch,,sively as a monomer in the cytosolic fraction from PMA-stimulated cells and a 31 kDa band become labeled more slowly and are still phosphorylated in respon2,e to zymosan and A23187 after down-regulation of protein kinase C The 31 kDa phosphoprotein sediments with ribosomes upon density gradient centrifugation and, in analogy with findings in other cell types [14,28-30,34], we suggest that it represents ribosomal protein $6. Attempts to provoke enhanced phosphorylation of any of the bands described in this paper by dibutyryl cAMP (1 raM) or prostaglandin E2 (1 #M), in the presence or absence of theophylline, have not been successful. This would argue against a role for cAMP-dependent protein kinase. PMA is known to induce phosphorylation of a 40-47 kDa protein in platelets [35,36] and of 43 and 47 kDa components in human neutrophils [37,38]. It is tempting to speculate that the predominant 45 kDa phosphoprotein described here might be related to one or more of these proteins. The 40-47 kDa platelet protein has been shown to be cytosolic, to exist as a monomer and to undergo multisite phosphorylation (predominantly on serine residues), at least in response to thrombin [39]. In comparison, the soluble form of the 45 kDa macrophage phosphoprotein is also monomeric and the heavy 32p-labeling of the protein might well be consistent with multisite phosphorylation, but a considerable part of it is recovered in the particulate fraction. Furthermore, the platelet pho~phoprotein is reported to migrate ahead of actin in the electrophoretic system used here [40], while the macrophage phosphoprotein migrates clearly behind a heavily stained band, tentatively identified as actin. Clearly, a more direct comparison will have to be undertaken to establish a possible relationship between these proteins.

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Fig. 9. [mmunoprecipitation of lipocortin [ and [[ from macrophages. Cells labeled with 32po4 were stimulated and subjected to immunoprecipitation as described in Experimental procedures. (Left) Silver-stained gel showing samples immunoprecipitated with non-immune serum (lane A), lipocortin [ antiserum (lane B) and lipocortin I[ antiserum (lane C). Recombinant human lipocortin [ protein is shown in lane D. The position of reference proteins is indicated on the left side and that of lipocortin ! on the right side. (Right) Scans of an autoradiograph of a gel after exposure to X-ray film for 160 h with an intensifying screen. ]mmunoprecipitation with ]ipocortin I] antiserum of unstimulated cells (E) and cells stimulated with 10 nM PMA for 5 rain (F). Immunoprecipitation with lipocortin I antiserum of unstimulatcd cells (G) and cells stimulated with ]0 nM PMA for 5 rain (H). The bar on the scan mark ttzc position of lipucortin I.

It was recently shown that the platelet .40-47 kDa protein may constit~':t~ iaositol 1,4,5-trisphosphate phosphatase [41], but it has also been suggested that it might be identical to the phospholipase inhibitory protein(s) lipocortin [42], based on the abii~ty of antiserum to a renal lipocortin to immunoprecipitate at least part of the 40 kDa platelet phosphoprotein after stimulation with thtombin or PMA. We have obtained antisera against recombinant human lipocortin I and highly purified human placental lipocortin II that have been shown to recognize proteins of similar size in several other mammalian species, including mouse [32]. However, none of these antisera immunoprecipitated the mouse macrophage 45 kDa phosphoprotein or any other major phosphoprotein labeled in response to PMA, although a 37 kDa protein antigenically related to lipocortin I was recognized by the appropriate antiserum. Neither could we find any significant PMA-induced phosphorylation of this 37 kD~. protein, nor of a protein of that size in immunoprecipitates prepared by antiserum against lipocortin II. Unless a very minor degree of lipocortin phosphorylation would be sufficient for activation of the phospholipase A pathway, it appears unlikely that lipocortin I is involved in the protein kinase C-mediated activation of this pathway. Recent studies from several laboratories have suggested alternative roles for lipocortins. Thus, they have been shown to belong to a family of Ca :+- and membrane-binding

proteins and to be closely related, or identical, to preferred substrates for tyrosine kinases [15,43-45]. It was previously shown that activation of the deacylatioa of phosphatidylinositol in the macrophages occurs within 1-2 min after exposure to PMA or 1,2-sn-DOG [6]. We later found that this is also true for PMA-induced mobilization of arachidonic acid (not shown). As shown here, the phosphorylation of some proteins only is rapid enough to be involved in the activation process. In addition, the parallel inhibition of protein phosphorylation and the mobilization of arachidonic acid after down-regulation of protein kinase C provides farther support for the idea that kinase C constitutes a link in the signal-transducing pathway, not only for PMA or 1,2-sn-DOG, but also for zymosan particles. It is also clear from the present and a previous study [5] that the requirement for calcium is not primarily at the level of protein kinase C activation, since this apparently occurs also after depletion of intracellular calcium, but rather at some later step in the signal pathway - possibly the phospholipase A reaction itself. The ability of the calcium ionophore to provoke mobilization of arachidonic acid also after down-regulation of protein kinase C would be consistentwith such an interpretation. However, zymosan, which would be expected to raise cytosolic calcium via inositol 1,4,5-trisphosphate, is much less efficient, although not completely inactive, after down-regulation of kinase C. Thus,

87 the role of protein kinase C-mediated phosphorylation could be to reduce the calcium requirement for the phospholipase A pathway from a high level towards the level prevailing in the cytosol of resting cells.

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