Protein kinase activity on the cell surface of a macrophage-like cell line, J774.1 cells

Biochimica etBiophysica Acta, 803 (1984) 163-173

163

Elsevier BBAl1261

PROTEIN KINASE ACTIVITY ON THE CELL SURFACE OF A MACROPHAGE-LIKE CELL LINE, J774.1 CELLS FUMIO AMANO, TAKAYUKI KITAGAWA and YUZURU AKAMATSU

Department of Chemistry, National Institute of Health, 10-35 Kamioosaki 2, Shinagawa-ku, Tokyo 141 (Japan) (Received September 14th, 1983)

Key words: Protein kinase; Phosphorylation; (J774.1 cell)

Protein kinase activity was demonstrated on the cell surface of a murine macrophage-like cell line, J774.1 cells, and was characterized in detail. When intact cells were incubated with [V-32p]ATP, a transfer of [32p]phosphate into acid-insoluble materials of the cells occurred. This reaction was Mg2+-dependent but cAMP-independent, and Mg 2+ could be substituted for by Mn 2÷. The reaction products were found to he proteins, as revealed by SDS-polyacrylamide gel electrophoresis and autoradiography, with phosphomonoester linkages to serine and threonine residues, but not to tyrosine. The results of experiments with chemical and enzymatic treatments as well as Con A-Sepharose column chromatography ruled out the possibility that an acyl-phosphate linkage or phosphomannosylglycopeptide was present in the reaction products. The protein kinase(s) and the reaction products were located on the cell surface of the cells, as shown by the fact that the products were removed by mild trypsinization of cells carefully controlled so that the cells remained in an intact state. Phosphorylation of exogenous proteins (phosvitin and casein) by intact cells further supported the location of the enzyme. The phosphorylated proteins of the cells were found to be metabolically stable and remained on the cell surface even at 120 min after the phospborylation reaction. Possible roles of ecto-protein kinase activity in macrophage functions and macrophage-activation are also discussed.

Macrophages are known to play important roles in host-defense mechanisms through a variety of their functions such as phagocytosis, antigenprocessing, and secretion of enzymes and chemical mediators to attack parasites, to activate lymphocytes and to kill bacteria and tumor cells [1,2]. These macrophage functions have been studied usually with murine or guinea pig peritoneal macrophages, rabbit alveolar macrophages and human peripheral blood monocytes. However, there are a number of inherent limitations such as heterogeneous populations of cells and difficulty in continuous cell cultures in studies with these cells. In this sense, the availability of monoclonal macroAbbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid. 0167-4889/84/$03.00 © 1984 Elsevier Science Publishers B.V.

phage-like cell lines offers a unique and useful tool for studying macrophage functions. A macrophage-like cell line, J774.1, has been shown to carry Fc and C3 receptors, to respond to bacterial lipopolysaccharides, and to act in phagocytosis and the cytotoxic reaction [3,4]. Moreover, genetic studies with this cell line are also possible [5,6]. In many of the macrophage functions, the plasma membrane, especially the cell surface of the membrane, seems to be of primary importance and enzymes on its cell surface could regulate the macrophage functions. Recently, protein phosphorylation has been recognized as a widespread mechanism by which many essential metabolic pathways and physiological processes are regulated [7,8]. There have been several reports in recent years demonstrating

164 the presence of protein kinases on the cell surface of a variety of mammalian cells, including Ehrlich ascites tumor cells [9], mouse 3T3 cells [10], human skin fibroblasts [11], rat fat cells [12,13], C-6 glioma cells [14] and HeLa cells [15], as well as guinea pig peritoneal macrophages [16]. However, their characteristics and biological functions are largely unknown. In the present study, we demonstrated protein kinase activity and the phosphorylated products on the cell surface of a macrophage-like cell line, J774.1, and characterized it in detail. Possible roles of these protein phosphorylations in macrophage functions are also discussed. Materials and Methods

Materials. Histone (calf thymus, type II-A), casein (hydrolyzed and partially dephosphorylated), phosvitin (egg yolk), crystalline trypsin, soybean trypsin inhibitor, pronase, nucleases (bovine pancreas DNAase I and RNAase A), alkaline phosphatase (E. coli), standard phosphoamino acids (o-phospho-L-serine, o-phosphoDL-threonine and o-phospho-DL-tyrosine) and all nucleotides were purchased from Sigma (St. Louis, MO, U.S.A.). Endoglycosidase H was obtained from Seikagaku Kogyo (Tokyo, Japan), Sephadex G-25 fine and Con A-Sepharose from Pharmacia (Uppsala, Sweden), and acrylamide and N,N'methylenebisacrylamide from Eastman Kodak (Rochester, NY, U.S.A.). ['/- 32p]ATP was purchased from Amersham (Buckinghamshire, U.K.) as the triethylammonium salt with a specific activity of 3000 Ci/mmol. All other chemicals were of reagent grade. Preparation of cells. A murine macrophage-like cell line, J774.1, originally isolated by P. Ralph [4], was obtained from Dr. T. Tokunaga, National Institute of Health, Tokyo [17]. Cells were cultured in Ham's F12 medium (Flow Laboratories, Rockville, MD, U.S.A.) supplemented with 10% newborn calf serum (Flow Laboratories, Stanmore, NSW, Australia) and antibiotics (100 U / m l penicillin and 100 # g / m l streptomycin) in 90 mm tissue culture dishes at 37°C in 5% CO2/95% air. The cells grown exponentially were suspended by gentle pipetting in ice-cold modified Hanks' balanced salt solution containing 137 mM NaC1, 5.4

mM KC1, 0.34 mM Na2HPO 4, 0.44 mM KHRPO 4, 5.6 mM glucose and 4.4. mM NaHCO3, pH 7.4. These cells were centrifuged at 330 × g for 3 min, and then washed three times with modified Hanks' balanced salt solution. Then the cells were resuspended in modified Hanks' balanced salt solution containing 10 mM MgCI 2 and 10 mM NaF at 2 , 1 0 6 cells/ml for the assay of protein phosphorylation. The cell viability was 90-98%, usually 95%, as tested by Trypan blue exclusion. Phosphorylation conditions. For standard phosphorylation, 1 . 1 0 6 cells in 0.5 ml of modified Hanks' balanced salt solution with 10 mM MgCI z and 10 mM NaF, pH 7.4, were preincubated in a glass test tube at 37°C for 3 min, with reciprocal shaking at 100 strokes/min and phosphorylation was started by addition of [~-32p]ATP (10 /~M, 0.1-0.2 Ci/mmol). The reaction mixture was incubated at 37°C for 5-30 min (usually 15 min) with shaking as above and the reaction terminated with 0.5 N ice-cold perchloric acid containing 1 mM unlabeled ATP. 0.2 mg bovine serum albumin was added to each tube as a carrier and the tubes were kept on ice for 30 min, and then centrifuged at 3000 rpm (1500 × g) for 5 min. The pellet was washed three times with ice-cold 0.5 N HC104, and the final pellet was dissolved in 0.5 ml of 0.4 M NaOH and neutralized with 4 M HCI. An aliquot of the samples was mixed with 5 ml ACS-II (Amersham) and the radioactivity was determined with a Packard Tri-carb scintillation counter (model 3320). For phosphorylation of exogenous proteins, the indicated amounts of proteins were added to a reaction mixture containing 1 - 106 cells and the radioactivity incorporated into acid-insoluble materials was determined after incubation for 15 min at 37°C.

Characterization of the phosphorylated products. To ascertain the nature of phosphorylated products, acid-precipitable material was subjected to chemical and enzymatical treatments as described [10]. The phosphorylated products precipitated with HC104 as above were mixed with 0.5 ml of 1 M KOH, 1 M HCI or 0.1-0.8 M hydroxylamine, pH 5.4, and incubated at 37 or 100°C for 30 min. After incubation, the samples were neutralized and precipitated with HC104 for counting the radioactivity. In other experiments, the phosphorylated and acid-precipitated samples were treated with

165 0.5 ml ethanol/ether (3:1) at 37°C for 30 min, centrifuged at 3000 rpm for 5 min, and washed once with the same organic solvent in order to remove HC104. The resulting precipitates were air-dried and incubated with either 10/~g trypsin in 0.5 ml of modified Hanks' balanced salt solution with 10 mM NaF and 10 mM sodium phosphate buffer, pH 7.4, at 37°C for 15 rain, 98 U DNAase I in 0.5 ml of 20 mM sodium acetate buffer with 2 mM MgC12, pH 5.0, at 37°C for 60 min [18], 4 U RNAase A in 0.5 ml of 20 mM sodium acetate buffer, pH 5.0, at 37°C for 60 min [19], or 0.75 U alkaline phosphatase in 0.5 ml of 20 mM Hepes buffer with 1 mM MgC12, pH 8.3, at 37°C for 60 rain [20]. After these enzymatic treatments, ice-cold HC104 was added to 0.5 M (final) and the acid precipitates were collected by repeated centrifugation at 3000 rpm for 5 min. Amounts of protein were determined by the method of Lowry et al. [21] with bovine serum albumin as a standard. In order to analyze phosphomonoester linkages, the reaction product was subjected to partial hydrolysis with 6 M HC1 at l l 0 ° C for 4 h according to Kinzel et al. [22] with modifications. High-voltage electrophoresis of the hydrolysate was carried out to identify phosphoamino acids on a microfine cellulose-coated thin film (Tokyo Kasei, Tokyo), saturated with pyridine/acetic a c i d / H 2 0 (1:10:289, v/v), pH 3.5, and electrophoresed at 50 V/cm for 30 min, according to Witte et al. [23]. To test the possibility of the presence of phosphomannosyl residues, the reaction products were subjected to endoglycosidase H treatment according to Muramatsu et al. [24]. Acid precipitates were washed with ethanol/ether (3:1, v/v), airdried, and digested with 20 #g trypsin in 0.25 ml modified Hanks' balanced salt solution containing 10 mM NaF and 10 mM sodium phosphate buffer, pH 7.4, at 37°C for 15 min. After the tryptic digestion, 40 #g soybean trypsin inhibitor was added and the mixture was incubated with 0.1 U endoglycosidase H at 37°C for 24 h, and fractionated by Sephadex G-25 column chromatography (Sephadex G-25, fine, column size 0.9 × 27 cm). In another experiment, the trypsinates were mixed with Triton X-100 to a final concentration of 1% and fractionated by Con A-Sepharose column chromatography eluted with

a 0-0.5 M glucose linear gradient in 20 mM Tris-HC1 buffer, containing 0.15 M NaC1, 1 mM MnCI 2, 1 mM CaC12 and 1% Triton X-100, pH 7.4. SDS-polyacrylamide gel electrophoresis. Cells were phosphorylated in the presence or absence of exogenous proteins with [7-32P]ATP with a higher specific activity (1-2 Ci/mmol), precipitated with 0.5 M HC104 and washed with 0.5 ml ethanol/ether (3 : 1) as described. The final pellets were air-dried, solubilized with sample buffer according to Laemmli [25] containing 2% SDS, 10% glycerol, 5% fl-mercaptoethanol, 0.0015% bromphenol blue and 0.5 /~1 2 M Tris, and then boiled at 100°C for 5 min. Myosin (prepared from guinea pig skeletal muscle, according to Richards et al. [26], heavy chain, 200 K and light chain, 20 K), phosphorylase b from rabbit muscle (97 K., Sigma), albumin from bovine plasma (68 K, Armour Pharmaceutical Co., Chicago, IL, U.S.A.), actin from rabbit skeletal muscle (43 K, a generous gift from Dr. H. Shimizu, the University of Tokyo), and apoferritin from horse spleen (subunit 18 K, Miles Laboratories, Elkhart, IN, U.S.A.), were treated similarly and used as marker proteins. Slab gel electrophoresis was performed with 7.5% or 10% polyacrylamide gels containing 0.1% SDS according to Laemmli [25]. Samples were run first at 12 mA for 2 h, and then at 25 mA for another 2 h. After electrophoresis, the gels were stained with 0.25% Coomassie brilliant blue R-250 (Sigma) in methanol/acetic acid/H20 (5:1:4, v/v), destained in methanol/acetic acid/H20 (2:3:35, v/v), dried on Whatman 3 MM paper and placed in contact with Fuji RX film (Fuji Photo Film Co., Ashigara, Kanagawa, Japan) and an intensifying screen, Lightning-plus (Du Pont, Wilmington, DE) at - 7 0 ° C for 5-7 days for autoradiography. Turnover of phosphorylated protein. To test the turnover of the phosphorylated proteins, the phosphorylated cells were washed three times with ice-cold modified Hanks' balanced salt solution containing 0.1 mM ATP, resuspended in 0.5 ml modified Hanks' balanced salt solution containing 10 mM MgCI2, 0.1 mM ATP and 10 mM Hepes buffer, pH 7.4, and further incubated at 37°C for the indicated period. The reaction products were then precipitated with 0.5 M HC104 containing

166

unlabeled 1 mM ATP, and analyzed by SDS-polyacrylamide gel electrophoresis as described. Duplicate samples were processed as above and half of them was mildly digested with 10/~g trypsin at 37°C for 2 min before acid precipitation in order to test the trypsin-sensitivity of the phosphorylated proteins.

12

a

10

S

,,, <

Results 0

Phosphorylation reaction of J774.1 cells with external A TP Intact J774.1 cells were incubated with [7,32p]ATP and the conditions for incorporation of the radioactivity into acid-insoluble materials were examined. The highest activity was obtained when the cells were incubated with [7-32p]ATP in the medium, pH 7.4, containing Mg 2÷ (Table I). Addition of NaF, a known inhibitor of phosphoprotein phosphatase [8,27], did not significantly increase the radioactivity. Cyclic AMP (cAMP) and dibutyryl cyclic AMP (DBcAMP) did not affect the activity, suggesting that this reaction is cAMP-in-

TABLE 1 C O N D I T I O N S F O R P H O S P H O R Y L A T I O N OF I N T A C T J774.1 CELLS W I T H E X T E R N A L [~,-32p]ATP 1-106 J774.1 cells were phosphorylated with [-/-32p]ATP (1 #Ci, 10 # M ) at 37°C for 15 min in 0.5 ml of modified Hanks' balanced salt solution containing 10 m M MgCI 2 and 10 m M N a F (complete system). Ca 2+ was omitted from the system unless otherwise noted. At the same time, incubations with the indicated omission from the mixture and addition of cyclic nucleotides were also performed. Values represent means _+S.E. for three different experiments. The value obtained without cells was used as a negative control in the following experiments, subtracting this value from the total incorporation. Factors

32p incorporated d p m / 1 0 6 cells per 15 rain

Expt. 1. Complete system 4 827 _+391 - cells 241 _+ 21 - M g 2+ 897_+ 54 - NaF 5113 _+191 Expt. 2. Complete system 3 071 _+393 + 100/~M c A M P 3231_+ 65 + 1 0 0 / x M D B c A M P 3446_+135 - cells 202 + 65

% 100+ 8 5+0 19+ 1 106_+ 4 100-[-13 105 _+ 2 112_+ 4 7_+ 2

I

0

6

0

5

10

~gC~ (rnM)

20

b

5

10 15 Incubotion time (min)

30

Fig. t. Characterization of the cell surface phosphorylation in J774.1 cells. J774.1 cells were phosphorylated at 37°C for 15 min as described in Table I except that the following reaction conditions were varied: (a) Mg 2+ (e) and EDTA ( O ) concentrations; (b) period of incubation at 37°C (e), 30°C (A), 18°C (11) and 0°C (O).

dependent. In addition, almost all the activity was associated with cell fractions, indicating that neither enzymes nor substrates were released from the ceils during the incubation (data not shown). Incorporation of [32P]radioactivity was proportional to the cell number up to 1 • 1 0 6 cells, indicating that cell-cell contact had no significant effect on this phosphorylation reaction. The requirement of Mg 2+ for the reaction is shown in Fig. la and the reaction was maximumal at more than 10 mM. Addition of 2-5 mM EDTA to the Mg2+-free medium completely abolished 32p incorporation, possibly due to removal of the endogenous Mg 2+ from the cells. The rate of the phosphorylation at 30-37°C showed a rapid initial phase for 5 min, followed by a slower rate of reaction for about 30 min (Fig. lb). At 0°C, the rate of reaction became much slower, showing the temperature dependence

167 of the reaction. A change of p H c h a n g e d the activity a n d the i n c o r p o r a t i o n was relatively lower at alkaline p H t h a n at neutral pH. The K m value for the external A T P in this p h o s p h o r y l a t i o n was d e t e r m i n e d from double-reciprocal plots a n d f o u n d to be 1.27 • 10 -4 M at 5 m i n of i n c u b a t i o n .

Characterization of phosphorylated products The p h o s p h o r y l a t e d products were treated with various chemicals a n d enzymes as shown in T a b l e I1 to investigate their nature. The radioactivity recovered in the acid-insoluble fractions decreased c o n s i d e r a b l y o n t r e a t m e n t with K O H , hot HC1 or trypsin, while it was n o t affected significantly by t r e a t m e n t with HC1 at 37°C, hydroxylamine, D N A a s e or R N A a s e . T r e a t m e n t with alkaline p h o s p h a t a s e (E. coli) released a b o u t 30% of the radioactivity. These results suggest that the phosp h o r y l a t e d p r o d u c t s were not polynucleotides b u t proteins which were devoid of acylphosphate-lin-

kages as revealed b y the resistance to hydroxyla m i n e [28]. T r e a t m e n t with e t h a n o l / e t h e r ( 3 : 1 ) decreased the radioactivity by a b o u t 30%. However, this was n o t due to the c o n t r i b u t i o n of the labeled phospholipids b u t rather to the loss of the phosphorylated proteins during repeated washing with the organic solvent. I n fact, the specific radioactivity of the sample was n o t changed by these t r e a t m e n t s (untreated, 3.03 + 0.25; treated, 3.17 + 0.15 c p m / ~ g protein, m e a n s + S.E. for three different experiments), a n d no radioactivity was extracted in c h l o r o f o r m / m e t h a n o l (2: 1) b y the m e t h o d of Bligh a n d Dyer [29]. These n a t u r e s of the products are similar to those described previously for protein p h o s p h o r y l a t i o n s b y ecto-protein kinases [10,22]. F u r t h e r characterization of the phosphorylated proteins was d o n e by partial acid-hydrolysis a n d s u b s e q u e n t analysis of p h o s p h o a m i n o acids. As shown in Fig. 2, the radioactivity was m a i n l y

TABLE II CHARACTERISTICS OF THE PROTEIN KINASE REACTION PRODUCTS J774.1 cells were phosphorylated at 37°C for 15 rain and then precipitated with cold 0.5 M HCIO4 as shown in Fig. 1. The labeled pellets were washed three times with HCIO4 and then treated with chemicals and enzymes as indicated. After these treatments, the radioactivity recovered in the HC104-insoluble fractions was counted. In Expt. 1, the samples treated with HC! or KOH were neutralized before HClO4-precipitation. In Expt. 3, the reaction products were treated with ethanol/ether (3 : 1) at 37°C for 30 rain and the pellets were recovered by centrifugation (3000 rpm, 5 min) and then air-dried. This treatment removed about 30-45% of the labeled materials. The samples were then treated as indicated with enzymes in 0.5 ml solutions as follows: Trypsin in modified Hanks' balanced salt solution with 10 mM NaF and 10 mM sodium,phosphate buffer, pH 7.4; DNAase I in 20 mM sodium acetate buffer with 2 mM MgC!2, pH 5.0; RNAase A in 20 mM sodium acetate buffer, pH 5.0; alkaline phosphatase in 20 mM Hepes buffer with 1 mM MgCI2, pH 8.3. Values are means_+S.E. for three different experiments. Numbers in parenthesis show the significance of differences between control and experimental values, determined by Student's t-test Treatment of reaction products Expt. 1. None 1 M KOH, 37°C, 30 min 1 M HCI, 37°C, 30 min 1 M HCI, 100°C, 30 min Expt. 2. None 0.1 M Hydroxylamine, 37°C, 30 min 0.8 M Hydroxylamine, 37°C, 30 min Ethanol/ether (3 : 1, v/v), 37°C, 30 min Expt. 3 Ethanol/ether (3 : 1, v/v), 37°C, 30 min + 10 #g Trypsin, 37°C, 15 rain + 98 U DNAase I, 37°C, 60 min 4 U RNAase A, 37°C, 60 rain + 0.75 U Alkaline phosphatase, 37°C, 60 min

32p incorporated (pmol/106 ceils per 15 rain)

(%)

6.75 _+0.33 1.47 _+0.47 6.67 _+0.40 1.22_+0.35

100+ 22+ 99+ 18_+

5 7 (P < 0.01) 6 5 (P < 0.01)

7.42 _+0.60 6.54_+0.30 6.31 _+0.34 4.98 _+0.64

100_+ 88_+ 85_+ 67_+

8 4 5 9 (P < 0.05)

5.49 _+0.69 0.74 _+0.10 4.85_+0.88 4.94 _+0.70 3.98 + 0.57

100_+13 13_+ 2 (P < 0.01) 88_+16 90_+13 72 _+10

168

® pi

161

'~ i

0.

P-Ser

P- Thr ,.j

6

u12

,

i

',

x

P-Tyr O

.Q

I

0 10 x

20 30 Fraction number

40

50

origin

® Fig. 2. Analysis of phosphoamino acids in the reaction products. The acid precipitates of the phosphorylated products obtained as described in Table I were air-dried, solubilized with 0.1% SDS, and then passed through a Sephadex G-25 column. The fractions near the void volume were collected, and precipitated with 100% ethanol. The dried samples were hydrolyzed with 6 M HCI at l l 0 ° C for 4 h and then subjected to high-voltage electrophoresis on microfine cellulose film. The dotted circles marked P-Ser, P-Thr and P-Tyr indicate the positions of ninhydrin-stained phosphoserine, phosphothreonine and phosphotyrosine standards, respectively. Pi, inorganic phosphate.

observed with phosphoserine and also with phosphothreonine, but no spot was seen with phosphotyrosine under the present conditions. This confirmed that external ATP and protein kinase made phosphomonoester linkages to serine and threonine residues of the proteins. It was of interest to examine whether 32p radioactivity was also incorporated in part into mannosyl residues of glycoproteins of the cell surface. At first, the reaction products were treated with trypsin (40 /~g/ml), followed by treatment with endoglycosidase H (0.4 U/ml) to liberate phosphomannosyl residues if present, and then analyzed by Sephadex G-25 column chromatography. The elution profile was not changed by the endoglycosidase H-treatment, while the trypsinized peptides were further digested by pronase into smaller sizes (Fig. 3). Secondly, the phosphorylated proteins or their trypsinized peptides were solubilized with 1%

Fig. 3. Sephadex G-25 column chromatography of the enzymatic hydrolysates of the reaction products. The phosphorylated products after incubation at 37°C for 15 min were precipitated with HCIO4 and then treated with ethanol/ether (3 : 1, v/v). The pellets were air-dried and then incubated with 20 ttg trypsin at 37°C for 15 min in 250/xl of modified Hanks' balanced salt solution containing 10 mM NaF and 10 mM sodium phosphate buffer, pH 7.4. The tryptic digestion was stopped by addition of 40 ~g soybean trypsin inhibitor and the sample was applied to the column (D). At the same time, separate samples digested with trypsin were further treated with either 0.1 U endoglycosidase H at 37°C for 24 h (=), or 930 U pronase at 37°C for 72 h (O) and were also applied to a Sephadex G-25 column. The samples were then fractionated by elution with 10 mM Tris-HCl buffer, containing 0.15 M NaCI, pH 7.4, and the radioactivity of each fraction was measured. Recovery of the radioactivity in each sample after the column chromatography was as follows: Trypsin alone, 55%; trypsin and endoglycosidase H, 71%; and trypsin and pronase, 85%. Arrows indicate the peak fractions of the following markers: a, blue dextran (M r >106); b, Suc-Gly-Pro-Leu-Gly-Pro-MCA (M r 696); c, p-nitrophenyl phosphate (M r 390).

Triton X-100 and subjected to Con A-Sepharose column chromatography with equilibration with the detergent. However, no radioactivity expected for mannosyl residues was retained in this affinity column (data not shown). These results taken together showed that the mannosyl residues of glycoproteins were not phosphorylated by external [732p]ATP.

Analysis by SDS-polyacrylamide gel electrophoresis of protein phosphorylations on the cell surface Possible substrates for the protein kinase and their localization was determined by SDS-poly-

169 b

200

c

d

e

f

a

200

k -

k -

97

k-

b

....

c

a'

b'

C~

....

~i

97k68k68k43 k

43k

-

9

n--

10 ~ S k

-

6 a i Fig. 4. Analysis of SDS-polyacrylamide gel electrophoresis of protein phosphorylation. The cells were phosphorylated as described in the text at 37°C for the following periods of time: 2 min (b); 5 rain (c); 10 min (d); 15 min (e); 30 min (f). The products were analyzed on SDS-polyacrylamide gels (7.5%) and subsequent autoradiography. The arrows with numbers indicate the bands with molecular weights of 215 K (1), 180 K (2), 170 K (3), 130 K (4), 110 K (5), 99 K (6), 67 K (7), 57 K (8), 39 K (9) and 34 K (10). In lane (a), a Coomassie brilliant blue-staining pattern of lane (e) is shown, since all the patterns from (b) to (f) were nearly identical.

a c r y | a m i d e gel electrophoresis a n d subsequent aut o r a d i o g r a p h y . M a j o r p h o s p h o r y l a t e d b a n d s were seen of p r o t e i n s with m o l e c u l a r weights of 130 K, 110 K, 67 K a n d 57 K (Fig. 4). These p h o s p h o r y l a t i o n s were o b s e r v e d as early as 2 min i n c u b a t i o n a n d their intensities increased with i n c u b a t i o n time. A s d e s c r i b e d previously, M g 2÷ was essential a n d all the p h o s p h o r y l a t e d b a n d s were d i m i n i s h e d w h e n M g 2+ was o m i t t e d or E D T A was a d d e d . M g 2÷ could be r e p l a c e d b y M n 2÷ b u t n o t b y C a 2÷ ( d a t a not shown). A d d i t i o n of C a 2÷ to the M g 2÷c o n t a i n i n g m e d i u m h a d little effect b u t Z n 2÷ (1 m M ) i n h i b i t e d m o s t of the p h o s p h o r y l a t i o n s a n d C u 2÷ (1 m M ) p r o m o t e d them ( d a t a not shown). T o d e t e r m i n e the localization of the p h o s p h o r y lated proteins, i n t a c t cells p h o s p h o r y l a t e d with [y-32p]ATP were i m m e d i a t e l y t r y p s i n i z e d for 2 min. By this treatment, all the p h o s p h o r y l a t e d b a n d s d i s a p p e a r e d c o m p l e t e l y from the gel, while

Fig. 5. Tryptic digestion of the phosphorylated cell surface proteins. Coomassie blue-staining patterns (a-c) and the corresponding patterns on autoradiography (a'-c') are shown with the molecular weight standards indicated on the left. The ceils were phosphorylated at 37°C for 15 min as shown in Table I and the products precipitated with HCIO4 are shown in lanes a and a'. Other tubes were further incubated at 37°C for 2 min with 1 mM ATP in the presence (b, b') or absence (c, c') of 10 ~g trypsin, followed by HCIO4 precipitation, SDS-polyacrylamide gel electrophoresis and autoradiography. This trypsin treatment did not change viability of the cells. Longer exposure of the films was carried out in order to detect any band on the autoradiographs after trypsinization (b').

the trypsinized cells were shown to be in an intact state a n d profiles of the p r o t e i n s stained with C o o m a s s i e blue d i d not change (Fig. 5). This suggests that the p h o s p h o r y l a t e d p r o t e i n s were present on the cell surface a n d could be r e m o v e d b y m i l d trypsinization.

Phosphorylation of exogenous proteins T o further p r o v e p r o t e i n p h o s p h o r y l a t i o n on the cell surface, the effect of exogenous p r o t e i n s o n the p h o s p h o r y l a t i o n was d e t e r m i n e d . A d d i t i o n o f histone, casein o r p h o s v i t i n to the r e a c t i o n m i x t u r e increased 32p i n c o r p o r a t i o n into the acidi n s o l u b l e fractions in a d o s e - d e p e n d e n t m a n n e r , while a l b u m i n showed no effect (Fig. 6). The exogenously a d d e d p h o s v i t i n was a p p a r e n t l y phosp h o r y l a t e d as shown in Fig. 7b. As expected, the

170

a

40

b

c

d

\

f

i~ ¸

200 k---- !i!i!! ! ~ii I E

e

~ii!?ii~ !i~~i! 68 k--,- iiiiii!i i!i i

3o u

43 k-~

~O

O

x

\ o

E

2O

18 k-o

6

....... w

.=

10

Q.

0 0

L

i

i

100

200

300

Proteins

added

L 400

i 500

(pg)

Fig. 6. Phosphorylation of exogenous proteins by ecto-protein kinase in J774.1 cells. The phosphorylation was carried out at 37°C for 15 rain in the complete medium shown in Table I in the presence of various amounts of histone (O), casein (A), phosvitin (11) and albumin (v), and the radioactivity incorporated into HClO4-insolublefractions was measured. The value of the control without exogenous proteins is shown at the original point (O) and the values in the figure represent the means of two different experiments.

p h o s p h o r y l a t e d phosvitin was separated from the cells b y low-speed centrifugation (Fig. 7d). These results strongly suggest that the exogenous proteins were p h o s p h o r y l a t e d by p r o t e i n kinase(s) o n the cell surface. A similar result was also o b t a i n e d with casein b u t the p h o s p h o r y l a t e d histone was tightly associated with the cells (data n o t shown). This was p r o b a b l y due to the positive charge of this protein, since the cells were aggregated in the presence of histone (data n o t shown). T h e possibility that histone could penetrate partly into the cells b y endocytosis [30] c a n n o t be completely ruled out at present. N e i t h e r c A M P n o r D B c A M P at 100 # M m o d u l a t e d the p h o s p h o r y l a t i o n s of these exogenous substrates ( d a t a n o t shown).

Fig. 7. Distribution of exogenous proteins phosphorylated by ecto-protein kinase. Two sets of protein phosphorylation by intact J774.1 cells were carried out in the absence or presence of 100 #g phosvitin as described in Fig. 6 and analyzed by SDS-polyacrylamide gel electrophoresis with 10% polyacrylamide gels and autoradiography. After phosphorylation, a set of the reaction mixture was directly precipitated with HCIO4 (a, b) and the other set was rapidly centrifuged at 3000 rpm for 2 rain after addition of cold modified Hanks' balanced salt solution containing 10 mM NaF and 0.1 mM ATP. Then supernatants and pellets were carefully separated from the latter set of samples, and the supernatants were further filtrated through a membrane filter (pore size, 0.45 #m; Sartorius, G6ttingen, F.R.G.). The proteins in each fraction were precipitated and washed with cold HCIO4 repeatedly in the presence of 50 #g bovine serum albumin as a carrier. The supernatant fractions (c, d) and the corresponding pellet fractions (e. f) are shown. Exposure of the autoradiograms in (c)-(f) was shorter than that in (a) and (b), so that the phosphorylated bands of the cell pellets became less evident (e, f).

Trypsin-sensitivity of the ecto-protein kinase activity T o test the effect of t r y p s i n - t r e a t m e n t o n the ecto-protein kinase activity of intact J774.1 cells, cells were treated with 10 btg trypsin at 37°C for 2 min, followed b y i n c u b a t i o n with 10 /~M [3'32P]ATP in the presence of 100 /~g exogenous p r o t e i n (histone, casein or phosvitin), a n d inc u b a t e d at 37°C for a further 15 min. However, S D S - p o l y a c r y l a m i d e gel electrophoresis profiles of these reaction products of e n d o g e n o u s a n d exogen o u s proteins revealed little change in the phosp h o r y l a t i o n (data not shown). These results suggest that ecto-protein kinase was insensitive to trypsin treatment.

171 a

b

c

d

e

f

g

h

200

9"/ 68

43

Fig. 8. Chase of the phosphorylated proteins. Autoradiographs (a-h) with the positions of the marker proteins are shown. The reaction mixtures of the complete system were incubated at 37°C for 15 min, chilled on ice by adding ice-cold modified Hanks' balanced salt solution with 0.1 mM ATP to stop the phosphorylation reaction, and then centrifuged to precipitate the cells. The pellets were resuspended in 0.5 ml of prewarmed modified Hanks' balanced salt solution containing 10 mM Mg 2+ and 0.1 mM ATP, and further incubated at 37°C for 0 rain (a, e), 30 min (b, f), 60 min (c, g) or 120 rain (d, h), and then the incubation was either stopped by addition of ice-cold 0.5 M HCIO4 containing 0.1 mM ATP (a-d) or followed by treatment with 10/~g trypsin at 37°C for 2 min before addition of HC104 with ATP (e-h). The precipitates were solubilized with SDS and then applied to 7.5% polyacrylamidegels. Longer exposure of the autoradiogram was carried out than usual io detect any band in the trypsinized samples (e-h), but in vain. Similar results were obtained when 10 mM NaF was present in the chasing medium above (data not shown).

Turnover of the phosphorylated proteins on the cell surface In order to investigate metabolism of the phosphorylated proteins on the cell surface, phosphorylated cells by external [y-a2P]ATP were further incubated for up to 2 h in the presence of 0.1 m M unlabeled A T P and 10 m M Mg 2+. However, no apparent decrease in the phosphorylation of the surface proteins was detected by SDS-polyacrylamide gel electrophoresis, showing that these phosphorylated proteins were metabolically very stable (Fig. 8). Besides, the phosphorylated proteins were shown to be sensitive to mild trypsinization throughout chasing with cold A T P for up to

2 h, thus suggesting they remained on the cell surface. Discussion In this study, we demonstrated the presence of protein kinase(s) and the phosphorylated products on the outer surface of the plasma membrane of a macrophage-like cell line, J774.1 cells. This conclusion is based on the following results. (1) The phosphorylation was carried out by incubation of intact cells with external [y-32 P]ATP which cannot enter into the cells normally. (2) There was no evidence that either the enzyme or substrates were released from the cells during the incubation. (3) The phosphorylated products were proteins with phosphomonoester linkages to serine and threonine residues, but with no linkages of acyl-phosphate nor phosphomannose. (4) These protein phosphorylations occurred at the surface of cell membranes, since the phosphorylated cellular proteins were completely digested on mild trypsinization of the cells without drastic cell damage. Moreover, exogenous proteins, such as casein and phosvitin, could also be phosphorylated by the cells and these phosphorylated proteins were separated easily from the cell fractions by low-speed centrifigation. (5) It was unlikely that extracellularly generated [32p]H3PO 4 from the labeled ATP was taken up by the cells and was the source for intracellular protein phosphorylation, since the same concentration (10/~M) of [32p]H3PO 4 did not induce protein phosphorylations under the same conditions and the profiles of phosphorylated peptides with [y-32p]ATP on the gels were different from those labeled with a 10-fold higher concentration of [32p]H3PO 4 in the specific radioactivity (data not shown). (6) An absolute requirement of extracellular Mg 2÷ for the phosphorylations supports an extracellular reaction, since one would expect that intracellular phosphorylation does not require external Mg 2÷. These characteristics were in line with those of other reported ecto-protein phosphorylations [10,15]. Many endogenous proteins with molecular weights from 215 K to 35 K were shown to be phosphorylated. Among them, 130 K, 110 K, 67 K and 57 K proteins were prominently phosphorylated as early as 2 rain incubation. These proteins,

172 once phosphorylated, were metabolically stable as to be found on the cell surface even at 2 h after the phosphorylation. The present phosphorylation of endogenous and exogenous proteins was independent of cyclic nucleotides as reported by others [9,10,15,16], while many of the ecto-protein kinases were shown to be cAMP-dependent [10-14]. As mentioned, the phosphorylated moieties of these proteins were easily cleaved by mild treatment with trypsin. However, similar trypsinization prior to the reaction did not reduce the activity of ecto-protein kinases (data not shown). These facts may indicate that the acceptor proteins in the plasma membrane could change in localization after the phosphorylations probably due to the conformational changes. It was also noted that ecto-protein kinase(s) of J774.1 cells is present on the outer surface but insensitive to trypsin, although ecto-protein kinase activity of human skin fibroblasts was lost with trypsin [11]. Although the roles of ecto-protein kinase and its acceptor proteins in mammalian cells are largely unknown, the present results make it possible to study the roles in macrophage functions and macrophage activation with J774.1 cells. Schlaeger and KOhler suggested the relation of cAMP-dependent ecto-protein kinase with release of cAMP from rat C-6 glioma cells [14]. A close relationship between protein phosphorylations of the cell surface and insulin-mediated glucose transport was shown in rat fat cells [12]. Mastro showed that ecto-protein kinase was activated by growth-stimulating hormones in mouse fibroblasts [31]. There are also several reports concerning the effect of external ATP on permeability [32-39], histamine release [34,40] and morphological changes [40,41], although the direct involvement of ecto-protein kinase in these changes has not been proved yet. These facts suggest a role of ecto-protein kinase in some specific stimulus-dependent processes in macrophages. In fact, we have observed preliminary results that treatment of J774.1 cells with bacterial lipopolysaccharide, which is known to 'activate' macrophage functions, significantly increased ecto-protein kinase activity (unpublished data). Although the present and previous studies demonstrated the activity of ecto-protein kinases in a variety of mammalian cells, it is obscure whether

ATP, a substrate for the enzymes, is present on the outside of the cells in vivo; in serum and body fluids. It is, however, reported that chromaffin granules [42] and platelets [43] secrete ATP together with catecholamine and serotonin, respectively, when the cells are stimulated. It should be noted that such reactions on the cell surface could occur in the local sites of macrophage-dependent cell injury, bacterial and viral infection, and also killing of tumor cells. Therefore, it is possible to speculate that ATP is present in serum or body fluids at micromolar levels in these local sites. If present, macrophages could consistently phosphorylate the proteins on the cell surface in vivo, since their turnover could be slow as shown in this study. It was recently observed that ATP was present in fresh serum of humans and mouse at 2 - 4 /LM by determination with high performance liquid column chromatography (personal communication from Dr. M. Yoshioka, University of Tokyo). Further biochemical and genetic studies with J774.1 cells are in progress to elucidate biological roles of ecto-protein kinase(s) in macrophage functions. Acknowledgements

The authors thank Drs. K. Suzuki and T. Terao of the National Institute of Hygienic Sciences and Dr. M. Yoshioka of the University of Tokyo for technical suggestions as to analysis of phosphoamino acids, and Dr. A. Kobata of the Medical Institute of the University of Tokyo for useful comments on detection of phosphomannosylated glycopeptides. This work was partially supported by Grants-in-Aid for Scientific Research, Nos. 56771113, 56480339, 57780175 and 57570821, from the Ministry of Education, Science and Culture of Japan, and funds from the Science and Technology Agency of Japan and the Research Foundation for Pharmaceutical Sciences. References

1 Van Furth, R., Martina, M.C., Diesselhoff-den Dulk, Raeburn, J.A., Van Zwet, T.L., Crofton, R. and Van Oud Alblas, A.B. (1980)in MononuclearPhagocytes(Van Furth, R., ed.), pp. 279-298, Martinus Nijhoff. The Hague 2 Krahenbuhl, J.L., Remington, J.S. and McLeod, R. (1980) in Mononuclear Phagocytes (Van Furth, R., ed.), pp. 1631-1650, Martinus Nijhoff, The Hague

173 3 Kaplan, G. and M~rland, B. (1978) Exp. Cell Res. 115, 53-61 4 Ralph, P., Pilchard, J. and Cohn, M. (1975) J. Immunol. 114, 898-905 5 Muschel, R.J., Rosen, N. and Bloom, B.R. (1977) J. Exp. Med. 145, 175-186 6 Rosen, N., Piscitello, J., Schneck, J., Muschel, R.J., Bloom, B.R. and Rosen, O.M. (1979) J. Cell Physiol. 98, 125-136 7 Greengard, P. (1978) Science 199, 146-152 8 Rubin, C.S. and Rosen, O.M. (1975) Annu. Rev. Biochem. 44, 831-887 9 Ronguist, G. and Agren, G. (1974) Acta Chem. Scand. B. 28, 1169-1174 10 Mastro, A.M. and Rozengurt, E. (1976) J. Biol. Chem. 251, 7899-7906 11 Chiang, T.M., Kang, E.S. and Kang, A.H. (1979) Arch. Biochem. Biophys. 195, 518-525 12 Chang, K.-J. and Cuatrecasas, P. (1974) J. Biol. Chem. 249, 3170-3180 13 Kang, E.S., Gates, R.E. and Farmer, D.M. (1978) Biochem. Biophys. Res. Commun. 83, 1561-1569 14 Schlaeger, E.-J. and K6hler, G. (1976) Nature 260, 705-707 15 Kiibler, D., Pyerin, W. and Kinzel, V. (1982) J. Biol. Chem. 257, 322-329 16 Remold-O'Donnell, E. (1978) J. Exp. Med. 148, 1099-1104 17 Akagawa, K.S. and Tokunaga, T. (1980) Microbiol. Immunol. 24, 1005-1011. 18 Kunitz, M. (1950) J. Gen. Physiol. 33, 349-362 19 Kunitz, M. (1946) J. Biol. Chem. 164, 563-568 20 Bessey, O.A., Lowry, O.H. and Brock, M.J. (1946) J. Biol. Chem. 164, 321-329 21 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 22 Kinzel, V., Kiibler, D., Mastro, A.M. and Rosengurt, E. (1976) Biochim. Biophys. Acta 434, 281-285 23 Witte, O.N., Dasgupta, A. and Baltimore, D. (1980) Nature 283, 826-831

24 Muramatsu, T., Koide, N., Ceccarini, C. and Atkinson, P.H. (1976) J. Biol. Chem. 251. 4673-4679 25 Laemmli, U.K. (1970) Nature 227, 6~0-685 26 Richards, E.G., Chung, C.-S., Menzel, D.B. and Olcott, H.S. (1967) Biochemistry 6, 528-540 27 Shacter-Noiman, E. and Chock, P.B. (1983) J. Biol. Chem. 258, 4214-4219 28 Hokin, L.E., Sastry, P.S., Galsworthy, P.R. and Yoda, A. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 177-184 29 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917 30 Murphy, R.F., Jorgensen, E.D. and Cantor, C.R. (1982) J. Biol. Chem. 257, 1695-1701 31 Mastro, A.M. (1979) J. Cell Physiol. 99, 349-358 32 Hempling, H.G., Stewart, C.C. and Gasic, G. (1969) J. Cell Physiol. 73, 133-140 33 Trams, E.G. (1974) Nature 252, 480-482 34 Cockcroft, S. and Gomperts, B.D. (1979) Nature 279, 541-542 35 Rozengurt, E., Heppel, L.A. and Friedberg, I. (1977) J. Biol. Chem. 252, 4584-4590 36 Kitagawa, T. (1980) Biochem. Biophys. Res. Commun. 94, 167-173 37 Kitagawa, T. and Akamatsu, Y. (1981) Biochim. Biophys. Acta 649, 76-82 38 Kitagawa, T. and Akamatsu, Y. (1982) Jap. J. Med. Sci. Biol. 35, 213-219 39 Dahlquist, R., Diamant, B. and K.riiger, P.G. (1974) Int. Arch. Allergy 46, 655-675 40 Kriiger, P.G., Diamant, B. and Dahlquist, R. (1974) Int. Arch. Allergy 46, 676-688 41 Stewart, C.C., Gasic, G. and Hempling, H.G. (1969) J. Cell Physiol. 73, 125-132 42 Poisner, A.M. and Trifaro, J.M. (1967) Mol. Pharmacol. 3, 561-571 43 Da Prada, M. and Pletscher, A. (1968) Br. J. Pharmacol. 34, 591-597