Identification, purification, and partial characterization of plasma protein kinases

ARCHIVES

OF BIOCHEMISTRY

Vol. 243, No. 2, December,

AND

BIOPHYSICS

pp. 530-541,

Identification,

1985

Purification, and Partial Characterization of Plasma Protein Kinases

THOMAS

M. CHIANG’

AND

ANDREW

H. KANG

Veterans Administration Medical Center, Departments of Medicine and Biochemistry, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38104 Received

April

22, 1985, and in revised

form

August

20,1985

Two isomeric forms of protein kinases, FI and FII, were isolated from human plasma. These two isomeric enzymes were isolated to apparent homogeneity on NaDodS04PAGE by using (NH&SO4 fractionation, DEAE-cellulose, hydroxylapatite, Affi-Gel blue, and high-pressure liquid column chromatography. Polyclonal antibodies were obtained from immunized rabbits and both enzymes cross-reacted with each other. Furthermore, immunoaffinity-purified anti-F1 and anti-F11 antibodies inhibited the enzyme activity of both FI and FII. These enzymes are cyclic nucleotides, Ca’+, calmodulin and phosphatidylserine-independent enzymes which can phosphorylate exogenously added histone, casein, protamine, phosvitin, and platelet surface proteins. The phosphorylated proteins of intact platelets by these enzymes in the presence of exogenously added [y32P]ATP ranged in apparent molecular weights from 13.5K to 200K, as estimated by their mobility during NaDodSO,-PAGE. Trypsin removed the label from the platelet surface phosphoproteins without affecting the intracellularly located phosphoproteins labeled endogenously by 32P04-prelabeling of intact platelets. These observations raise the possibility that these enzymes could play a role in modulating the properties of platelets through phosphorylation of the platelet surface proteins. o 19% Academic PM. I~C.

Phosphorylation of enzymatic or nonenzymatic proteins by protein kinases has been shown to be an important means of regulating their functions (l-8). The activities of more than twenty enzymes have been shown to be modulated by phosphorylation [for review, see Ref. (l)]. At least four general types of protein kinase activities have previously been described: CAMP’-dependent, cGMP-dependent, cal-

cium-dependent, and independent enzyme activities. Recently, a calcium- and phospholipid-dependent kinase has also been reported (9,10). This enzyme is stimulated by the tumor promotor, phorbol ester (11). Two CAMP-dependent and one cGMP-dependent protein kinases have already been extensively characterized (12-17). Platelets have been shown to contain several protein kinase activities. Cyclic AMP-dependent and independent kinases have been found in both the 100,OOOg supernatant (18,19) as well as in the platelet membrane fraction (20). Recently, a cal-

i Author to whom correspondence should be addressed: Research Service (151), V.A. Medical Center, 1030 Jefferson Avenue, Memphis, Tennessee 38104. 2 Abbreviations used: CAMP, adenosine 3’,5’-cyclic monophosphate; cGMP, guanosine 3’,5’-cyclic monophosphate; BSA, bovine serum albumin; TCA, trichloroacetic acid; PRP, platelet-rich plasma; TrisEDTA buffer, 20 mM Tris HW130 ItIM NaWl mM ethylenediaminetetraacetate, pH 7.4; NaDodSO1PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Tris-glycerol buffer, 20 mM Tris HCl/ 0003-9861/&j Copyright All rights

$3.00

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

5 mM P-mercaptoethanol/l% glycerol, pH 7.4; glycylglycine-glycerol, 20 mM glycylglycine/5 mM 8-mercaptoethanol/O.l% glycerol, pH 7.0; ELISA, enzymelinked immunosorbent assay; saline-Tween, 150 mM NaClO.O5% Tween 20; PBS-Tween, 20 mM phosphate/ 150 mM NaCl-0.05% Tween-20, pH 7.4. 530

INDEPENDENT

cium-phospholipid and phorbol esterstimulatable protein-kinase C has been described in platelets prelabeled with 32Pi (11). In this paper, we demonstrate the existence of two protein kinases in normal human plasma. These two protein kinases are independent enzymes which can phosphorylate exogenously added histone, intact platelets, and other substrates. These two enzymes have been purified to apparent homogeneity by NaDodS04 polyacrylamide gel electrophoresis. Polyclonal antibodies raised against the two purified protein kinases are cross-reactive with each other. An immunoreactive band was detected in the 100,OOOg supernatant of broken platelets in transblot experiments suggesting that these two plasma protein kinases might originate from platelets. METHODS Sampks. Human blood was collected by venipuncture from healthy volunteers who were not on any drugs after an overnight fast. Blood was collected in polypropylene tubes containing either 3.8% sodium citrate (1:9), heparin (10 USP units/ml blood), or no anti-coagulant but incubated at 37°C for 30 min for clotting (21). After centrifuging at 250g for 10 min, the plasma or serum was collected and recentrifuged at 100,OOOg (Beckman L5-75, SW 41 Ti rotor, 33,000 rpm) for 2 h at 4°C. In some experiments, the plasma or serum was filtered through a Millipore filter (0.45 pm pores, Millipore Corp., Bedford, Mass.) prior to the ultracentrifugation. The supernatant was collected for immediate assay for protein kinase activity or kept in a -20°C freezer. Assay of plasma protein kinase. Protein kinase aetivity was measured in the following manner. In a final volume of 0.1 ml, an appropriate amount of sample was incubated in 30 mM Tris, pH 7.4,2 mM MgClz, 2 mM NaF, 25 pg of histone type II (Sigma Chemical Co., St. Louis, MO.) and 10 WM [T-“P]ATP (0.5 &i; ICN Pharmaceuticals, Inc., Irvine, Calif.) in the presence or absence of CAMP, (1 pM; Sigma Chemical Co.) at 30°C for 5 min. At the end of the incubation, 1 ml of 10% TCA was added to stop the reaction. The carrier protein (BSA, 200 pg) was added to the tubes and mixed. Another 1 ml of 10% TCA was then added, followed by mixing. The mixtures were centrifuged at 2OOOg for 20 min at 4’C. The supernatants were discarded. The precipitates were subjected to two washes with 2 ml of 10% TCA. The washed precipitates were dissolved in a total volume of 2 ml of Aquasol (1 ml at a time) and placed into individual vials each

PROTEIN

KINASE

531

containing 5 ml of Aquasol. Radioactivity was determined using a Packard Tricarb scintillation counter. Kinase activity was calculated by subtracting the radioactivity of the background (sum of cpm of two sets of controls, one set consisting of plasma and [y=P]ATP without histone, and the other set consisting of histone and [y-32P]ATP without plasma) from that of the samples (histone, plasma, and [y-“PIATP). The incorporation of radioactivity into histone was linear in a time-dependent manner up to 10 min. Identificaticm of the phosphoamino acids. The phosphoamino acid content of histone was identified after acid hydrolysis. Samples were phosphorylated as described under assay of plasma kinases except [yazP]ATP was increased to 10 /ICUtest. Triehloroacetic acid (TCA) was added to 50%. The precipitate was then washed twice with TCA followed by washing with a mixture of ether and ethanol (1:l). After centrifugation, the pellet was recovered and dried. The pellets were hydrolyzed with 6 N HCl under Nz for 3 h at 110°C and the recovered supernatant was lyophilized. The phosphoamino acids and added standards (P-serine, P-threonine, and P-tyrosine) were separated by two-dimensional thin-layer electrophoresis (glacial acetic acid, 88% formic acid, and Hz0 78:25:887 by volume, pH 1.9, at 900 V for 45 min) and ascending chromatography (isobutyric acid and 0.5 M NH,OH, 5:3 v/ v) and the amino acids were detected by ninhydrin staining. The corresponding spots of P-Thr, P-Ser, and P-Tyr were scraped out from plates, decolored with 6% H202, and the radioactivity was measured by scintillation spectrometry (21). Washed platelets. Human blood was collected by venipuncture using a two-syringe technique from normal volunteers not ingesting any drugs after an overnight fast, and put into polypropylene tubes containing 0.1 vol of 3.8% sodium citrate. Platelet-rich plasma (PRP) was prepared by centrifuging the titrated blood at room temperature for 10 min at 226g (22). An equal volume of 20 mM Tris/l30 mM NaCl/l mM EDTA, pH 7.4 (Tris-EDTA buffer), was added to PRP. The suspension was spun at 1000 rpm for 5-10 min. The platelets were washed once more and finally suspended in Tris-EDTA buffer. Sodium dodecyl sulfate polyawylamide gel electre phoresis (NaDodSO,-PAGE) of phosphorylated platelets. All chemicals used in NaDodSO1-PAGE were purchased from Bio-Rad Laboratories (Rockville Center, N. Y.). Washed platelets (50 ~1) suspended in Tris-EDTA buffer (400,00O/jd) were incubated with and without purified plasma protein kinase (15 pg) in a reaction mixture containing 30 mM Tris, pH 7.4, 2 mM MgClz, 2 mM NaF, 100 mM NaCl, and 10 pM [y“P]ATP (0.5 &i/test) at 30°C for 5 min in a final volume of 0.1 ml. At the end of the incubation, equal volumes of concentrated NaDodSO1-PAGE sample buffer (23) were added to stop the reaction. Aliquots of 0.1 ml were withdrawn and boiled for 1 min. The

532

CHIANG

boiled samples were analyzed on a 10% NaDodSO,slab gel (23). After overnight electrophoresis at 20 V in Tris-glycine buffer, pH 8.3, or as otherwise modified as stated in the text or the legends, the gel was stained with Coomassie brilliant blue (Eastman Kodak Co., Rochester, N. Y.), destained with methanol-acetic acid (107%) solution, soaked in ATP solution, and dried under vacuum. The dried gel was radioautographed using a Kodak XAR-5 film (Picker International, Norcross, Ga.). Purification of plasma protein k&se. One liter of plasma obtained by Millipore filtration and/or centrifugation at 100,OOOg for 2 h was used for enzyme purification studies. All maneuvers were carried out at 4°C. The first step used was ammonium sulfate fractionation. The kinase activity was found in the 30-50% fraction. This fraction was dialyzed against 20 mM Tris/5 mM P-mercaptoethanol/l%glycerol, pH 7.4 (Tris glycerol buffer). The dialyzed fraction was applied to a DEAE-cellulose column (2.5 X 20 cm) and eluted with the same buffer. After the base line was established, a linear gradient of NaCl (O-300 mM) was superimposed for elution. Fractions of 6 ml were collected. The optical density at 280 nm was recorded by spectrophotometry. Aliquots of these fractions were assayed for kinase activity. Active fractions were pooled and dialyzed against phosphate-glycerol buffer (20 mM phosphate/5 mM @-mercaptoethanol/l% glycerol, pH 7.4). The dialyzed fractions were applied to hydroxylapatite columns (0.9 X 20 cm) and eluted with a linear gradient (125 ml each) of 20 to 320 mM phosphate/5 mM fl-mercaptoethanol/l% glycerol, pH 7.4. Aliquots of each fraction were assayed for kinase activity and active fractions were pooled, concentrated, and dialyzed against phosphate-glycerol buffer. The dialyzed fractions were applied to Affi-Gel blue (50100 mesh) columns (2.5 X 30 cm) and eluted with phosphate-glycerol buffer followed by 1.5 M NaCl in the same buffer to ensure that these kinases were not bound to the column. The active fractions (unbound) were pooled, dialyzed, and lyophilized. The lyophilized fraction was loaded on a TSK-125 column for highpressure liquid chromatography (HPLC; Waters Associates, Milford, Mass.). The column was equilibrated and the sample was eluted with glycylglycine-glycerol buffer, 20 mM glycylglycine/5 mM P-mercaptoethanol/ 0.1% glycerol, pH 7.0). Each fraction was assayed for kinase activity and active fractions were pooled, concentrated, and dialyzed against 20 mM glycylglycine buffer. These fractions were frozen at -80°C in small aliquots. Immunization of animals, Four New Zealand white female rabbits (two for each group; 1.5 kg) were each immunized with 100 fig of purified FI or FII in complete Freund’s adjuvant (GIBCO, Grand Island, N. Y.) intradermally. Each animal received booster injections of 100 Fg of the purified protein kinase in incomplete Freund’s adjuvant at 2 and 4 weeks after

AND

KANG

the primary immunization. Sera were obtained after immunization and at e-week intervals thereafter. All sera were heat inactivated at 56°C for 30 min and then stored at 4°C. Puti&catian of IgG fractions from immune sera IgG was partially purified from immune sera by using affinity column chromatography (protein kinase Sepharose 4B). The affinity column (1.6 X 20 cm) was prepared according to the method of Wilchek et al. (24) using cyanogen bromide-activated Sepharose 4B and the purified plasma protein kinases. Four milliliters of sera were loaded on a column (1.6 X 20 cm) which was equilibrated with 20 mM phosphate buffer, pH 7.2. The column was eluted with the same buffer until the baseline was established. The bound IgG fraction was then eluted from these columns with 100 ml of 0.2 M glycine, pH 3.8. The peak fractions were pooled and extensively dialyzed, lyophilized, and reconstituted in an appropriate buffer before being used. Enzyme-linked immurw.wrbent assay (ELISA). Antiprotein kinase antibodies were detected by ELISA as described (25, 26). Briefly, assay wells (96-well plate) were coated with 0.2 ml of 50 mM NazC03, pH 9.6, containing 10 pg/ml of purified protein kinases. The plates were incubated at 37°C for 3 h and then washed extensively with 150 mM NaCl with 0.05% Tween 20 (Bio-Rad Lab, Rockville Center, N. Y.) (saline Tween). Various dilutions of antisera in 20 mM phosphate/l50 mM NaCI (pH 1.4; PBS) containing 0.05% Tween (PBSTween) were then added to the wells and incubated at 37°C for 3 h. The wells were again washed with saline-Tween and peroxidase-conjugated goat antirabbit IgG (l/2000, Cappel Lab., Westchester, Pa.) was added to each well. The plates were then incubated at 37’C for 2 h, washed with saline-Tween and enzyme substrate (5-aminosalicylic acid, Aldrich Chemical Co., Milwaukee, Wis. and HzOz, Fisher Scientific, St. Louis, MO.) was added. The absorbance was recorded with a Minireader II (Dynatech Lab, Inc., Alexandria, Va.) at 450 nm. The titers are expressed as the reciprocal for the highest dilution of serum yielding an absorbance of 0.100. Immunoblot assay. Samples were analyzed by NaDodSOI-PAGE (7.5%)and then transblotted onto nitrocellulose papers (Bio-Rad Lab.) with 25 m&i TrisHCl, 192 mM glycine/20% methanol, pH 8.3, for 5 h with a constant current of 200 mA. The nitrocellulose paper was immersed in the blocking buffer 20 mM Tris, 500 mM NaCl (pH 7.4, TBS), containing 3% horse serum (v/v) for 3 h. After five washes with TBS, the nitrocellulose paper was treated with the first antibody solution (l/1500 dilution of the antisera to plasma protein kinase in TBS containing 1% horse serum (KC Biological, Lenexa, Kans.) overnight in the cold room (4’C). At the end of the incubation, the nitrocellulose paper was washed 5 times with TBS and transferred to the second antibody solution (goat anti-rabbit IgG conjugated with horseradish peroxidase (l/3000 di-

INDEPENDENT

PROTEIN

lution in TBS) and incubated for 2 h at room temperature. The nitrocellulose paper was washed 5 times with TBS and then transferred to HRP color development (Bio-Rad Lab.) solution (120 ml) containing 60 mg HRP color development reagent in 20 ml of cold methanol and 60 ~1 of ice-cold HzOz (30% ,Fisher Scientific) (27, 28). Protein concentration determinaticm. Protein concentrations were determined by the method of Lowry

et al. (29). RESULTS

Incubation of plasma with [T-~‘P]ATP, in the presence of exogenously added histone, resulted in a net incorporation of =P04 into plasma proteins and histone. The incorporation of 32P04 into histone can be demonstrated by autoradiography (data not shown). Two plasma proteins were phosphorylated in addition to added histone, these two proteins have apparent molecular weights of 54K and 45K. The radioactivity incorporated into histone or plasma proteins cannot be replaced by unlabeled ATP in the destain solution suggesting that the radioactivity was covalently linked to histone and proteins. The identity of the two phosphorylated plasma

533

KINASE

proteins is not known. The activity of the kinase, 9 pmol/min/mg, was essentially the same in plasma collected with different kinds of anti-coagulants (heparin and Na citrate) as well as in serum. The activity was apparently unaffected by the use of the one or two syringe technique to obtain blood, or the superimposition of Millipore filtration prior to ultracentrifugation of the plasma or serum. The enzyme activity was purified by ammonium sulfate precipitation and chromatography on DEAE cellulose, hydroxylapatite, Affi-Gel blue, and HPLC successively. Kinase activity was found in the 3050% ammonium sulfate precipitate. The precipitate was solubilized and dialyzed against Tris-glycerol buffer, applied to a DEAE cellulose column and eluted with a linear gradient of O-O.3 M NaCl. Three peaks of kinase activity were found when assay was conducted in the absence of CAMP or cGMP (Fig. 1). These peaks eluted at NaCl concentrations of 0.08 M, 0.13 M, and 0.20 M, respectively. Each of the peaks was pooled separately, dialyzed, and chromatographed on hydroxylapatite columns. -c

-0

-0

L 0

5

IO

I5

20

25

?I0

Jb

40

45

50

55

Fmctions FIG. 1. Purification of plasma protein kinase on DEAE-cellulose. The dialyzed solution of a 3050% (NH&SO1 precipitate was loaded on a DEAE-cellulose column (2.5 X 20 cm). The column was equilibrated and eluted with 20 mM Tris/5 mM @mercaptoethanol/l% glycerol, pH 7.4. A linear gradient (500 ml each) of 0 to 0.3 M NaCl was started at the 15th tube, and fractions of 6.0 ml were collected. The optical density of the effluent was monitored at 280 nm using a Gilford spectrophotometer (-). An aliquot of 50 ~1 of each fraction was assayed for protein kinase activity as described under Methods (O---O).

534

CHIANG

AND

A single peak of activity was found with each of the DEAE fractions I and II (FI and FII) (Fig. 2). Fraction III (from DEAEcellulose, Fig. 1) lost its enzyme activity during hydroxylapatite chromatography and was not investigated further. Fractions I and II were loaded on Affi-Gel blue to remove serum albumin. The enzyme activity was recovered from the unbound fraction (figure not shown). This fraction was concentrated, dialyzed, and loaded on an HPLC column. Only one peak of activity was found in each run as shown in Fig. 3. The recovery and special activities of the

-20 0.2

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I .-s 5 0.3 % 4 3 a2

7 $ 8 >

5 -

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-0

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- 25 .g a :: - 20 .e Y -15 .i: h

-5

0

-0 IO

20

30

40

50

Fractions FIG. 2. Purification of plasma protein kinase on hydroxylapatite. The dialyzed fractions of DEAE-cellulose (FI and FII, Fig. 2) were loaded on hydroxylapatite columns (0.9 X 20 cm) separately. Each column was equilibrated and eluted with 20 mM phosphate/ 5 mM /3-mercaptoethanol/l% glycerol, pH 7.4. A linear gradient (125 ml each) of 20 to 320 mM phosphate/5 mM @-mereaptoethanol/l % glycerol was started at the 11th tube, and fractions of 5 ml were collected. The optical density of the effluent was monitored at 230 nm using a Gilford spectrophotometer (0). An aliquot of 50 ~1 of each fraction was assayed for protein kinase activity as described under Methods (0). (A) FI (Fig. 1); (B) FII (Fig. 1).

KANG

enzyme preparations during the purification procedure are shown in Table I. FI and FII were purified approximately 600- and 2000-fold with recovery of 3 and 19%, respectively, by the purification procedures employed. The final purification step was monitored by NaDodSO1-slab gel electrophoresis. A single band was obtained for each preparation with an apparent molecular weight of 80,000 (Fig. 4, inset). Protein kinase activity was measured in each of the gel slices obtained after separation of the purified enzyme by 10% polyacrylamide electrophoresis without NaDodSOl. Results are shown in Fig. 4. Enzyme activity was found with the Coomassie stained band of each fraction. These findings reflect the specificity of kinase activity which is coincident with the major protein band of each fraction. The migration of these enzymes was the same in both reduced and nonreduced samples suggesting these enzymes are single polypeptides. Furthermore, we have run these enzymes on a calibrated G-200 column and assayed enzyme activity of each run. The molecular weight of these kinases were calculated to be 90 f 0.5K (data not shown). The substrate specificity of the plasma protein kinases was examined. These enzymes phosphorylate histone and protamine equally. Casein and phosvitin were also phosphorylated, but to a lesser degree (approximately 50% as compared with histone or protamine; data not shown). The effect of divalent ions on the activity of plasma protein kinase was studied. Maximal activity of plasma protein kinase was obtained at 2 mM MgC& (49 pmol/min/ mg). Without MgC12, the activity of plasma protein kinase was 2.7 pmol/min/mg. At the same concentration of various divalent ions, the activity of plasma protein kinase was 37.3 pmol/min/mg for Mg(OAc)B; 35.3 pmol/min/mg for MnClz; 41 pmol/min/mg for BaCl,; 2.9 pmol/min/mg for ZnCls and not detectable for CaC&. The activity of plasma protein kinase in the presence of EDTA without adding extra divalent ions in the reaction mixture was 2.7 pmol/ min/mg. The effect of CAMP and cGMP on the activity of plasma protein kinases was ex-

INDEPENDENT

PROTEIN

535

KINASE

0ij 62q5 7:

; 0.00 is $0.06 0.04

4

0.02

2

I!

0 %

0

10

20

FRACTION

30

40

50

60

NUMBER

FIG. 3. Purification of plasma protein kinases by high-pressure liquid chromatography. The dialyzed unbound fractions from Affi-Gel blue column (FI and FII, Fig. 3) were loaded on a TSK-125 column (300 X 75 mm, Bio-Rad Laboratories), separately. Each column was equilibrated with 20 mM glycylglycine/5 mu @-mercaptoethanol/O.l% glycerol, pH 7.0. Fractions of 0.5 ml were collected. The optical density of the effluent was monitored at 280 nm (-). Alternate effluent fractions were used to assay for the enzyme activity (O---O). (A) FI (Figs. 1 and 2); (B) FII (Figs. 1 and 2).

amined in the 100,000g supernatant and the partially purified fractions (FI and FII) from the DEAE column. The enzyme activity was not affected by additions of various concentrations of CAMP (0.5 to 2 PM), or cGMP (0.05 to 1 PM). The activity was not affected by the protein inhibitor for CAMP-dependent protein kinase (Sigma Chemical Co., St. Louis, MO.) or stelazine (Smith Kline Co., Carolina, P. R. ), an agent which can inhibit calcium-dependent protein kinases. The addition of Ca2+ (1 mM) and calmodulin (15 pg; Sigma) or of Ca2+ (1 mM) and phosphatidylserine (5 pg) (Sigma) had no effect. These results indicate that the plasma protein kinases are independent forms of protein kinase. These

protein kinases have the same property as platelet originated protein kinases which are not dependent on CAMP for activity (30, 31). The phosphorylated amino acids were identified. The major phosphorylated amino acid was serine followed by threonine. Tyrosine was not phosphorylated by these plasma protein kinases suggesting that they are not tyrosine kinases (Table II). Next, we investigated the immunologic properties of FI and FII. Polyclonal antibodies raised against purified FI were analyzed by ELISA for reactivity with FI and FII. Polyclonal antibodies raised against purified FII were similarly examined for

536

CHIANG

AND TABLE

KANG I

THEPURIFICATIONOFPLASMAPROTEIN

Fraction

Protein concentration (m/ml)

Ultracentrifuged plasma” 30-50% Ammonium sulfate DEAE-cellulose column FI FII

9 7.6 0.76 0.96

Hydroxylapatite FI FII

0.19 0.24

High-pressure liquid chromatography FI FII

0.3 0.29

Total protein (mg) 9000 1824

KINASE

Specific activity (pmol/min/mg) 10.2 (1) 49.6 (4.9)

38.7 46.1

Total activity 91,800 90,470

100 98

1,016 1,306

(99.6) (128)

39,325 60,206

43 65

0.66 1.2

6,395 21,562

(626) (2,113)

4,252 25,874

5 28

0.52 0.93

6,125 18,976

3,185 17,647

3 19

(600) (1,860)

Note. The protein kinse activity was assayed as described under Methods. a The starting plasma was filtered through a Millipore filter (0.4 grn pores) and/or centrifuged for 2 h at 4°C. The cumulative degree of purification for each step is shown in parentheses.

reactivity with FI and FII. Each of the antibodies recognized both FI and FII, indicating that FI and FII shared common antigenic site(s) (Table III). Moreover, the kinase activity of FI and FII could be completely inhibited by immunoaffinity purified anti-F1 and anti-F11 antibody (Table IV). Loss of kinase activity was recovered in the precipitates (data not shown) indicating that the antigenicity of protein kinase was not located at the active sites of the enzymes. Utilizing these antibodies, the possible source of the plasma kinases was examined, using immunoblot assay (Fig. 5). The anti-F1 antibodies reacted with purified FI (lane 5) and FII (lane 6). The 100,OOOg supernatant of broken platelets contained a band which reacted with the polyclonal antibodies as shown in lane 2. The 100,000~ supernatant of broken cell preparations of other cells, such as erythrocytes (lane l), T-cells (lane 3), and monocytes (lane 4), did not react with the antibodies. An identical set of results was obtained using polyclonal antibodies raised against purified FII (data not shown). These results suggest that the plasma protein kinases may originate from platelets.

% Recovery

at 100,OOOg

The physiologic role of the plasma kinases is not known at the present time. We consider the possibility that the kinases might be involved in modulating the function of cells such as platelets by altering the state of phosphorylation of the cell membrane proteins. Thus, we first investigated whether the plasma kinases could phosphorylate the outer surface proteins of platelets. Because the major activity existed in FII after purification, we used the FII enzyme to examine this possibility. When washed platelets were incubated with purified plasma kinase (FII) in the presence of [T-~~P]ATP, the radioactivity was incorporated into several protein bands as analyzed by NaDodSO,-PAGE and radioautography (lane 1, Fig. 6A). Ten prominently labeled components had apparent molecular weights of 13.5K, 23K, 31K, 46K, 57K, 62K, 75K, 94K, 155K, and 200K. The phosphorylation of the surface proteins of platelets was not enhanced by the addition of 1 PM CAMP or 0.1 cGMP (data not shown). In order to topographically localize the phosphorylated platelet proteins as intracellular or exposed to the cell surface, washed platelets were phosphorylated ei-

INDEPENDENT

Dye

15

-

PROTEIN

Front

1

537

KINASE

gel electrophoresis and radioautography. Trypsin treatment destroyed the radiolabeled bands of platelets phosphorylated using [T-~~P]ATP and plasma kinase (A), but not those endogenously phosphorylated using 32Pi (B, Fig. 6). These results indicate that platelet proteins which can be labeled by added plasma kinase are susceptible to trypsin digestion whereas proteins which are located intracellularly are resistant to trypsin treatment. These findings support the likelihood that platelet surface proteins could be substrates for plasma kinases whereas intracellularly located phosphoproteins are not. The phosphorylation of platelets in platelet-rich plasma by the addition of [y=P]ATP occurs to a lesser extent compared

10 -

TABLE IDENTIFICATION

5-

I 10

I 20

30

40

4 50

!

Phosphoamino

acids

(cpm)

S’O

Samples SLICE

ACIDS

Front

Dye 0

II

OF PHOSPHOAMINO

P-Ser

P-Thr

P-Tyr

NUMBER

FIG. 4. Correlation between protein kinase activity and protein stain. The purified enzyme fractions (I and II) were analyzed by 10% gel under bath reduced and nonreduced conditions without added NaDodSOI (22). The gel was prerun for 6 h in the cold room. Samples in different conditioned buffers (with and without fl-mercaptoethanol) were loaded and run overnight at a constant voltage of 35 V. At the end of electrophoresis, one of each sample was stained with Coomassie brilliant blue and the other sample was sliced into 2-mm slices. These slices were further broken into smaller pieces and were assayed for protein kinase activity. Identical results were obtained in the reduced and nonreduced gels. Results shown in this figure were under nonreduced conditions. (A) fraction I; (B) fraction II.

ther externally with [-y-32P]ATP and plasma protein kinase or internally with inorganic phosphate (““Pi). The platelets were then suspended in 20 mM Tris/l30 mM NaCVliter mM EDTA, pH 7.4, and subjected to trypsin (0.5 pg) digestion for different time intervals. At the end of the incubation, an equal volume of sample buffer of NaDodSO,-PAGE was added to stop the reaction before proteins were analyzed by

FI phosphorylated histone FI alone FII phosphorylated histone FII alone

930 0

470 0

6 0

650 0

80 0

0 0

Note. Histone (300 pg) was phosphorylated separately by the purified plasma kinase (I and II, 30 pg) in a reaction mixture which contained 30 mM TrisHCI, pH 7.4, 2 mM MgClz, 2 PM NaF, and 10 mM [y=P]ATP (10 &i/test) at 37°C for 10 min. Proteins were precipitated and washed with 50% TCA twice then once with ether and ethanol (1:l). Precipitates were then hydrolyzed with 6 N HCI, 3 h at 110°C under saturated Nz. Samples were diluted with 5 ml water and lyophilized. The material was reconstituted in water with standards (P-serine, P-threonine, and Ptyrosine), then analyzed by high voltage electrophoresis (solvent:glacial acetic acid/W% formic acid/HzO, 78/25/887, pH 1.9) at 900 V for 45 min and ascending chromatography (isobutyric acid/O.5 M NH,OH, pH 5.3). The phosphoamino acids were detected with ninhydrin. The corresponding spots of P-Ser, P-Thr, and P-Tyr were removed from the plates, decolored with 6% HzOz, and counted in a scintillation counter. The numbers listed in the table are the averages of duplicate samples. Histone and [-y-“P]ATP without enzyme served as a background control.

538

CHIANG TABLE OF ANTISERA

REACTIVITY

AND

KANG

III

WITH FI AND FII

BY ELISA -2OOK

Wells coated with 10 pg of Antibodies raised against

Dilution

FI

FII

FI

1:800

1.44

1:1600 1:3200

1.44 1.36

0.52 0.28 0.12

1:800

0.74 0.47 0.28

FII

1:1600 1:3200

-ISOK

1.38 1.33

1.07 -

Note. The details of this in the text. The absorbance

experiment was read

are described at 450 nm.

to the phosphorylation of washed platelets exposed to purified plasma kinases (data not shown). The majority of the radioactive phosphate was incorporated into plasma proteins suggesting that the plasma proTABLE

IV

THE INHIBITORY EFFECT OF IMMUNOAFFINITYPURIFIED ANTIBODIES ON THE PROTEIN KINASE ACTIVITY OF FI AND FII Enzyme activity (% of control) Additions Anti-F1 Anti-F11 Anti-F1 Anti-F11 Anti-F1 Anti-F11 Anti-F1 Anti-F11

IgG, IgG, IgG, IgG, IgG, IgG, IgG, IgG,

FI 6 pg 6 pg 3 Gg 3 pg 1.5 pg 1.5 wg 0.75 pg 0.75 pg

0 0 22

14 19 25 33 64

4516

i

FII 0 0

123

4

56

FIG. 5. Immunoblot of 100,OOOg supernatants of various broken cells. Samples (100 gg each) from red cells (lane l), platelets (lane 2), T-cells (lane 3), monocytes (lane 4), purified plasma protein kinases FI (lane 5) and FII (lane 6), and Bio-Rad markers were analyzed on NaDodSO1-PAGE (7.5%).At the end of electrophoresis, the gel was transblotted onto nitrocellulose paper with 25 mM Tris-HCVl92 mM glycine/ 20% (v/v) methanol, pH 8.3, for 5 h. The nitrocellulose paper was blocked with 3% horse serum in TBS. After washing the TBS, the first antibody l/1500 (anti-protein kinase antibody raised against FI) was added and incubated overnight at 4°C. The paper was washed and incubated with goat anti-rabbit IgG horseradish peroxidase conjugated (l/3000). Finally, the paper was incubated with HRP color developer. Identical results were obtained using anti-protein kinase antibody raised against FII.

23

19 34 30 35 50

Note. Solutions of FI and FII obtained from AffiGel blue chromatography were incubated with the indicated amounts of immunoaffinity-purified IgG at 4°C for 3 h. At the end of the incubation, 5 ~1 of a second antibody (goat anti-rabbit antibody, Cappel Lab., Westchester, Pa.) was added and the mixture was further incubated at 4°C overnight. The mixture was then centrifuged at 30,000~ for 20 min and the supernatant was assayed for protein kinase activity. The results are expressed as the percentage of control which was incubated with preimmune serum.

teins are preferred substrates for the plasma kinases. Washed erythrocytes can be phosphorylated with these purified plasma kinases. The molecular weights of the phosphorylated proteins were 13K, 35K, 65K, 94K, and 250K, (data not shown). Thus, there are several competing substrates for the plasma protein kinases in plasma. DISCUSSION

This paper demonstrates the existence of protein kinase in human plasma and serum. We have purified the enzyme by a

INDEPENDENT

1

2

3

4

5

6

1

2

3

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PROTEIN

5

FIG. 6. Radioautograms of phosphorylated platelets and the effects of trypsin treatment. (A) Platelets were labeled externally using [T-~P]ATP and plasma kinase. An aliquot (0.1 ml) of washed platelets was incubated with [y-aaP]ATP and plasma protein kinase FII (15 pg) for 4 min. At the end of this labeling period, the effect of trypsin was examined as follows: Lane 1, labeled platelets (positive control); Lane 2, labeled platelets + soybean trypsin inhibitor (20 pg) + trypsin (0.5 pg) for 1 min; Lane 3, labeled platelets + trypsin (0.5 rg) for 1 min; Lane 4, labeled platelets + soybean trypsin inhibitor (20 pg) + trypsin (0.5 pg) for 2 min; Lane 5, labeled platelets + trypsin (0.5 rg) for 2 min; Lane 6, unlabeled platelets + [T-~P]ATP (negative control). At the end of the incubation, the reaction was terminated by adding an equal volume of NaDodSO,-PAGE sample buffer. Aliquots of 0.1 ml of each sample were boiled and loaded on 10% gel. The proteins were eleetrophoresed, stained with Coomassie brilliant blue, and destained with acetic aeid/methanol (7/10%).The destained gel was dried and a radioautograph was obtained (A). In panel 5 are identical sets of experiments (lanes l-5) using platelets which were labeled endogenously with inorganic phosphate (aaPO,).

combination of (NH&SO4 fractionation, DEAE-cellulose, hydroxylapatite, Affi-Gel blue chromatography and HPLC. Although these three separate fractions of the enzyme activity were obtained from DEAEcellulose chromatography (Fig. 2), we were successful in further purifying only two of the fractions, FI and FII, approximately 600- and 2000-fold, respectively. These two fractions are probably isoenzymes with similar molecular weights but with separate elution positions on DEAE-cellulose column. The third fraction (FIII) could not be recovered from the subsequent chro-

KINASE

539

matography column. The reason for this is unclear. The plasma enzymes are independent kinases in that their activity is not affected appreciably by the addition of CAMP, cGMP, Ca2+, or phospholipids. The enzymes are capable of phosphorylating exogenously added histone, platelet surface proteins, and other substrates. The source of these enzymes is not certain. While immunoblot results indicate that circulating platelets contain an immunocross-reactive material suggesting that these kinases could be derived from platelets, levels of the enzyme are not different in serum or plasma collected under a variety of different conditions. This suggests that these kinases are components of plasma proteins. In addition, the activity of protein kinase in the 100,OOOg supernatant of plasma samples was not stimulated by the addition of CAMP or cGMP in the assay mixture. If the plasma kinases originated from broken platelets, we should see stimulation by cyclic nucleotides. The reported presence of a tyrosine kinases in serum (32) suggests that kinases are true plasma proteins. A CAMP-dependent protein kinase has been partially purified from the 100,OOOg supernatant of platelet homogenates and can be dissociated into catalytic and regulatory subunits by the addition of CAMP (18). In addition, a partially CAMP-dependent and another independent protein kinase have been demonstrated in platelet membrane preparations (19). The membrane-bound enzymes can transfer 32P of [T-~‘P]ATP to endogenous platelet substrates with apparent molecular weights of 52K, 31K, and 20K. The phosphorylation of the 52K molecular weight protein was CAMP dependent (20). If the kinase activities described in this paper arose from broken platelets during our preparation, one would have expected to see stimulatory effects of cyclic nucleotides on the 100,OOOg supernatant fraction. The activity of protein kinase in the 100,OOOg supernatant fraction was not stimulated by the addition of CAMP (0.5 to 2 PM) and cGMP (0.05 to 0.5 PM; data not shown). Recently, isolation of a cyclic nucleotide-independent, calcium-

540

CHIANG

activated, phospholipid-dependent protein kinase from platelet homogenates has been reported (33). This enzyme is dissimilar to the plasma kinases described in this paper in that there are differences in molecular weight (60,000 vs 80,000) of the substrates, as well as differences in the effect of calcium and phospholipid on the activity of the enzyme. The apparent molecular weight of the plasma protein kinase is 80,000 as established by NaDodSO,-PAGE, which is similar to that of the protease-activated kinase II proenzyme (34). The proenzyme can be activated with trypsin to a lower molecular weight active kinase (Mr 45K). The plasma enzymes cannot be further activated with trypsin. Instead, plasma kinase activity is destroyed by exposure to trypsin (data not shown). Irrespective of the source, the kinases appear to be valid components of plasma. Further, they appear to interact with at least three other components, plasma proteins, platelet membranes, and erythrocyte membranes (unpublished results). The only rate limiting factor to the phosphorylation of plasma proteins, platelets, and erythrocytes would appear to be the availability of ATP. Plasma proteins themselves appear to be preferred as substrates for the plasma kinases which would be a mechanism limiting the phosphorylations of platelets and erythrocyte membranes. Under normal conditions, the availability of ATP in plasma is limited. The function of these plasma protein kinases is not known. However, under certain conditions during which cells or tissues may be damaged or during platelet aggregation, ATP may become available locally, intravascularly. The phosphorylation of plasma proteins, platelets, and erythrocytes may occur under these circumstances in view of the presence of these plasma kinases. ACKNOWLEDGMENTS We thank Ms. D. Devall, Mr. S. Wathen, and Ms. V. Woo for their expert technical assistance. Sincere thanks is given to Ms. J. Rogers for typing the manuscript. This research was supported by the Veterans

AND

KANG

Administration and in part by U. S. Public Health Service Grants AM-16506, HL-20114, a grant in aid from the American Heart Association (84-799), and an investigatorship award from American Heart Association Tennessee Affiliate. REFERENCES 1. KREBS, E. G., AND BEAVO, J. A. (1979) Annzc, Rev. Biochem 48,923-959. 2. LAGAN, T. A. (1969) Proc. NatL Acad Sci. USA 64, 12’76-1283. 3. NISHIMURA, J., AND DEUEL, T. F. (1983) FEBS I&. 156,130-134. 4. TENG, C. S., TENG, C. T., AND ALLTREY, V. G. (1972) J. BioL Chem. 246,3597-3609. 5. DELORENZO, R. J., WALTON, K. G., CURRAN, P. F., AND GREENGARD, P. (1973) Proc Nat1 Acad Sci. USA 70,880-884. 6. Fox, J. E. B., AND PHILLIPS, D. R. (1982) J. BioL Chem. 257,4120-4126. 7. DAUGHERTY, J. J., PURI, R. K., AND TOFT, D. 0. (1982) J,BioL Chem 257,14,226-14230. 8. CHANG, K. H., AND CUATRECASAS, P. (1974) J. BioL Chem. 249,3170-3180. 9. Kuo, J. F., AND GREENGARD, P. (1970) J. Bid Chem. 245,2493-2498. 10. SCHATZMAN, R. C., WISE, B. C., AND Kuo, J. F. (1981) Biucti Biophys. Res. Commun. 98,669676. 11. CASTAGNA, M., TAKAI, Y., KAIBUCKI, K., SANO, K., KIKKAWA, U., AND NISHIZUKA, Y. (1982) J. BioL Chem. 257,7847-7851. 12. PACKHAM, M. A., WARRIOR, E. S., GLYNN, M. F., SERY, A. S., AND MUSTARD, J. F. (1967) J. Exp. Med 126.171-188. 13. SODERLING, T. R., CORBIN, J. D., AND PARK, C. R. (1973) J. BioL Chem 248,1822-1829. 14. CORBIN, J. D., KEELEY, S. L., AND PARK, C. R. (1975) J. BioL Chem 250,218-225. 15. GILL, C. N., HOLDY, K. E., WALTON, G. M., AND KANSTEN, C. B. (1976) Proe. N&L AuxL Sci USA 73,3918-3922. 16. HOFMANN, F., BEAVO, J. A., BECHTEL, P. J., AND KREBS, E. G. (1977) J. BioL Chem 252, 14411447. 17. LINCOLN, T. M., DILLS, W. L., JR., AND CORBIN, J. D. (1977) J. Bid Ch.em. 252.4269-4275. 18. BOOYSE, F: M., MARR, J., YANK, D. C., GUILIANI, D., AND RAFELSON, M. D., JR. (1976) Biocd Biaphys. Acta 422,60-72. 19. CARLSON, C. A., AND KIM, K. H. (1973) J. Bid Chem 248,378-380. 20. STEINER, M. (1975) Arch Biochem Biophys. 171, 245-254. 21. RUBIN, R. A., AND EARP, H. S. (1985) science (Washington, D. C.) 219,60-63.

INDEPENDENT 22. CHAING, T. M., BEACHEY, E. H., AND KANG, (1975) J. Bid Chm. 250,6916-6922. 23. LAMMELI, 285.

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26. VOLLER, A., BIRDWELL, D. E., AND BARTLEIT, (1976) in Immunoenzymatic techniques Feldman, ed.), pp. 167-173, North-Holland, Amersterdam.

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27. FLOCKERZI, V., SPEICHERMANN, N., AND HOFMANN, F. (1978) J. Bid Chxm. 253,3395-3399.

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28. WISE, B. C., RAYNOR, R. C., AND Kuo, J. F. (1982) J. Biol Chem 257,8481+X488. 29. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Bid C&m. 193, 265-275. 30. GALEBRU, J., KRUST, B., AND HOVANESSIAN, A. (1983) Biochxm. Biophys. Res. Ccnnmun 113, 370-376. 31. IKEDA, Y., AND STEINER, M. (1979) J. Biol. Chem. 254,66-74. 32. LIN, M. F., LEE, P. L., AND CLINTON, G. M. (1985) J. Bid Ch.em 260,1582-1587. 33. YAMAKI, T., NISHIKAWA, M., AND HIDAKA, H. (1982) B&hem Bicrphys. Acta 714,257-264. 34. LUBBEN, T. H., AND TRAUGH, J. A. (1983) J. Biol. Chem. 258,13,992-13,997.