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Autophosphorylation of adenosine 3′,5′-monophosphate-dependent protein kinase from bovine brain
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
164,
551-559
(1974)
Autophosphorylation
of Adenosine
3’, 5’-Monophosphate-Dependent Protein
Kinase from
BovineBrain
l
HIROO MAENO, PROCERFINA L. REYES, TETSUFUMI UEDA, STEPHEN A. RUDOLPH,2 AND PAUL GREENGARD Department
of Pharmacology,
Yale University
School of Medicine,
New Haven,
Connecticut
06510
Received April 8, 1974 A highly purified adenosine 3’) 5’-monophosphate-dependent protein kinase from bovine brain has been found to catalyze its own phosphorylation. The incorporated phosphate was shown to be associated with the cyclic AMP-binding subunit (R-protein) of the protein kinase. The catalytic subunit exhibited no detectable incorporation of phosphate into itself, but was required for the phosphorylation of R-protein. The molecular weight of R-protein was determined by polyacrylamide gel electrophoresis to be about 48,000 in the presence of sodium dodecyl sulfate. Cyclic AMP strikingly inhibited the rate of autophosphorylation observed in the presence of ZnCl,, CaCl,, NiCl,, or Fe&, but had no significant effect in the presence of MgCl, or CoCl,. The concentration of cyclic AMP required to give half-maximal inhibition of phosphorylation was 3 x lo-’ M in the presence of either CaCl, or ZnCl,. Guanosine 3’, 5’-monophosphate was far less effective under the same experimental conditions than cyclic AMP. R-protein appears to be similar to a phosphoprotein recently discovered in synaptic membrane fractions from rat and bovine cerebral cortex.
The possibility that the phosphorylation and dephosphorylation of certain synaptic membrane proteins plays an important role in neural function is supported by observations of the enrichment in synaptic membrane fractions of cyclic AMP3dependent protein phosphorylation and of protein dephosphorylation systems (l-3) and is compatible with other biochemical (4-8) and physiological (8,9) evidence. Most previous studies of cyclic AMP-dependent protein kinases have focused at1This work was supported by Grants NS-08440 and MH-17387 from the United States Public Health Service. 2Postdoctoral Fellow of the Damon Runyon Memorial Fund for Cancer Research. 3Abbreviations used: Cyclic AMP, adenosine 3’) 5’-monophosphate; cyclic IMP, inosine 3’, 5’-monophosphate; cyclic GMP, guanosine 3’) 5’-monophosphate; cyclic CMP, cytidine 3’, 5’-monophosphate; cyclic UMP, uridine 3’, 5’-monophosphate; DodSO,-, sodium dodecyl sulfate.
tention on the enzymological properties of these enzymes, and have been carried out with artificial substrates, such as histone or casein (e.g., 10-13). However, in order to elucidate the biochemical basis for the physiological effects of cyclic AMP, it is necessary to identify and isolate the endogenous substrate proteins of cyclic AMPdependent protein kinases. Recently, cyclic AMP was demonstrated to regulate the endogenous phosphorylation of two specific protein components of synaptic membrane fractions from rat, bovine, and porcine cerebral cortex (7,14). It was suggested that the state of phosphorylation of these protein constituents might control the permeability of some neuronal membranes to inorganic ions, thereby mediating postsynaptic potential changes of the membrane. One of the proteins, referred to as Protein II, was of particular interest, since the effect of cyclic AMP on its phosphorylation was either stimulatory or inhibitory
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MAENO
depending on the precise experimental conditions (14). In the present study, we have been able to demonstrate that a highly purified cyclic AMP-dependent protein kinase from bovine brain can catalyze its own phosphorylation and that the incorporated phosphate is associated with the cyclic AMP-binding subunit (R-protein) of the protein kinase. This subunit comigrates with Protein II electrophoretically on DodSO,--polyacrylamide gels. MATERIALS
AND
METHODS
Materials. Synaptic membrane fractions were prepared from fresh bovine cerebral cortex according to a method previously described for rat brain (1). Cyclic AMP-dependent protein kinase II was partially purified from a 13,000g supernatant of bovine brain homogenate by protamine sulfate treatment, ammonium sulfate precipitation, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography as described by Miyamoto et al. (15). Except where specified otherwise, this partially purified protein kinase was used in the experiments to be described. Further purification was achieved by Sepharose 6B gel filtration and preparative polyacrylamide gel electrophoresis (15). The specific activity of the enzyme at various stages of purification was similar to that described previously (15). Calf thymus histone, cyclic [G-SH]AMP (16.3 Ci/mole) and ATP were obtained from Schwarz-Mann. Cyclic AMP, cyclic IMP, cyclic GMP, cyclic UMP, and cyclic CMP were from Boehringer-Mannheim. Bovine serum albumin (fraction V) was purchased from Sigma. [Y-~~P]ATP was prepared by the method of Post and Sen (16). Assay for cyclic AMP-dependent histone kinase octiuity. Protein kinase activity was routinely assayed using histone as a substrate. The assay was done in a total volume of 0.2 ml at pH 6.2 in the presence of 50 mM sodium acetate, 10 mM magnesium acetate, 5 PM [y-32P]ATP (l-3 x lo6 cpm/nmole), 100 c(g of calf thymus histone, and an appropriate amount of protein kinase, in the presence or absence of 5 pi cyclic AMP. The reaction was initiated by the addition of the [-r-12P]ATP, carried out for 5 min at 30°C and terminated by the addition of 2.0 ml of 5% trichloroacetic acid containing 0.25% sodium tungstate. The procedure for washing and counting radioactive histone in the precipitate was as described previously (17). One unit of histone kinase activity was defined as the amount of enzyme which transferred 1 pmole of 2zP from [Y-~~P]ATP to histone under the standard assay conditions. Assay for endogenous phosphorylation of protein kinuse. The standard reaction mixture with a total
ET AL. volume of 0.1 ml at pH 6.2 contained 50 mM sodium acetate, 10 mM MgCl,, 0.1 mM EDTA, 5 PM [-rs2P]ATP (l-3 x 10’ cpm per nmole), and an appropriate amount of enzyme in the presence or absence of 5 PM cyclic AMP. The reaction was carried out for 15 set at 20°C and terminated by the addition of 0.05 ml of 9% DodSO,in 0.03 M Tris-HCl buffer, pH 8.0, containing 6% mercaptoethanol, 3 mM EDTA, and 15% glycerol. A O.l-ml aliquot of this reaction mixture was then subjected to flat plate polyacrylamide gel electrophoresis (7,14) in the presence of DodSO,- as described by Fairbanks et al. (18). The gel was stained for protein with 0.025% Coomassie blue in 25% isopropyl alcohol and 10% acetic acid and was destained with 10% isopropyl alcohol and 10% acetic acid containing 0.0025% Coomassie blue, followed by 10% acetic acid (18). The gel was dried on Whatman No. 50 filter paper under vacuum and heat, and an autoradiogram of the gel was made using Kodak X-Omat Royal X-ray film to visualize 3zP incorporation into specific proteins (7,14). The optical density of the bands on the autoradiogram was measured with a Joyce-Loebl microdensitometer. The optical density of the band on the autoradiogram corresponding to the protein showing cyclic AMP-dependent phosphorylation was taken to be proportional to the amount of 3T incorporated into this protein (14). When protein kinase (either partially purified through hydroxylapatite or purified to apparent homogeneity) was assayed for endogenous phosphorylation, the molecular weight of the phosphorylated subunit was determined to be 48,000 by electrophoresis on flat plate polyacrylamide gels in the presence of DodSO,-, using standard marker proteins of known molecular weights, as described previously (14). Using DodSO,gel electrophoresis, it was possible to study the total distribution of 32P incorporation into protein. Of the 3zP-labeled protein applied to the gels, approximately 90% was recovered after correction for quenching. Of the total radioactivity appearing on the gels, greater than 90% was in the 48,000-dalton band when the incubation was carried out in the presence of Mg*+, and virtually 100% in the presence of Zna+ (see Fig. 5). Therefore, whenever endogenous phosphorylation was studied, it was the phosphorylation of this particular protein that was measured. Assay for cyclic AMP-binding activity. Cyclic AMP-binding activity was determined by adsorption of cyclic AMP-binding protein onto Millipore filters according to the method of Gilman (19), except for omission of the heat-stable inhibitor. Other methods. In order to identify the site of endogenous phosphate incorporation, SIP-labeled protein was hydrolyzed in 6 N HCi at 106°C for 6 hr, dried in uacuo, and subjected to high-voltage paper electrophoresis for 3 hr, as described previously (2). After the paper was dried, phosphoserine and phosphothreonine were located by staining with ninhydrin. The
AUTOPHOSPHORYLATION
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OF BRAIN PROTEIN KINASE
paper was then cut into 0.5-cm strips, and the radioactivity of the strips was counted by liquid scintillation spectrometry. Protein concentrations were determined by the method of Lowry et al, (20) or by modification of a turbidimetric method (21) in which the turbidity of protein solutions in the presence of 5% trichloroacetic acid was determined spectrophotometrically. In either method, bovine serum albumin was used as the standard protein. RESULTS
Evidence for autophosphorylution of cyclic AMP-dependent protein kinase. Cyclic AMP-dependent protein kinase, purified through the step of hydroxylapatite chromatography, was subjected to preparative polyacrylamide disc gel electrophoresis and the gel was then assayed for histone kinase activity, endogenous phosphorylation, and cyclic AMP binding activity. As illustrated in Fig. 1, all three activities migrated on the gel at the same speed (R, 0.18 against bromphenol blue). (When the peak fractions were pooled, an aliquot subjected to reelectrophoresis as described in the legend to Fig. 1, and that gel stained for protein, a single major band, containing over 95% of the protein stain, was observed.) These results indicated that the cyclic AMP-dependent protein kinase might be catalyzing its own phosphorylation. In order to determine whether the phosphate was incorporated into the regulatory or catalytic subunit, the enzyme, obtained by polyacrylamide disc gel electrophoresis, was phosphorylated with [T-~~P]ATP and then incubated with cyclic [G-3H]AMP to dissociate the enzyme into its regulatory and catalytic subunits. When this preparation was subjected to electrophoresis on a cylindrical polyacrylamide gel, in the absence of DodSO,-, the 32Pand 3H elution profiles were found to be identical (Fig. 2). (The catalytic subunit, as judged by the ability to phosphorylate histone, was not detected in these fractions; presumably, the dissociated catalytic subunit, which has an isoelectric point of 7.8 (Vi), migrated into the upper buffer under the conditions of electropboresis used.) In experiments not shown here, when the holoenzyme was endogenously phosphorylated
OA 0
0.5 Rt
A 1.0
FIG. 1. Polyacrylamide gel electrophoresis of protein kinase. The enzyme preparation (550 ag) from a hydroxylapatite column was subjected to electrophoresis on a 7.5% polyacrylamide cylindrical gel (1.0 x 7 cm), pH 8.9, with concentrating gel (22,23). The upper and lower buffers were glycine-Tris (pH 8.3) and Tris-HCI (pH 8.1), respectively, both of which contained 1 mM dithiothreitol and 0.1 mM EDTA. After electrophoresis for 5 hr at 350 V, at 4”C, gel slices (2 mm) were homogenized in 10 mM Tris-HCI buffer, pH 7.5, containing 1 mM dithiothreitol and 0.1 mM EDTA, and then centrifuged at 100,OOOgfor 15 min. The clear supematants were used for the measurement of histone kinase activity in the presence (-C-) and absence (--O-) of cyclic AMP, endogenous phosphorylation in the absence of cyclic AMP, and cyclic AMP-binding activity, under standard assay conditions. In the experiment illustrated, the recoveries of cyclic AMP-dependent histone kinase activity, of endogenous phosphorylation, and of cyclic AMP-binding activity were 72, 70, and 61%, respectively.
with [T-~~P]ATP, dissociated in the presence of cyclic [G-3H]AMP and histone, and then applied to a Sephadex G-100 column, the catalytic subunit was found to contain no radioactive phosphate, and the majority of protein-bound 32Premained at the top of the column along with most of the proteinbound 3H. Experiments were also carried out to determine whether the isolated catalytic
554
MAENO ET AL.
5
10 Slier
15
20
$
Number
FIG. 2. Polyacrylamide gel electrophoresis of phosphorylated purified protein kinase. Cyclic AMPdependent protein kinase, which had been purified by polyacrylamide disc gel electrophoresis, as described in the legend to Fig. 1, was phosphorylated by incubation at 20°C for 2 min in a volume of 0.37 ml, in the presence of 50 mM sodium acetate, pH 6.2, 0.1 mM EDTA, 5 NM [Y-~?P]ATP (247 cpm/pmole), and 2.5 mM ZnCl,. EDTA, dithiothreitol, and cyclic [G*H]AMP (7000 cpm/pmole) were then added to final concentrations of 5 mM, 1.25 mM, and 0.6 PM, respectively. The incubation mixture was kept at 0°C for 10 min, and then cyclic AMP (final concentration, 6.25 PM) was added to achieve complete dissociation of the enzyme. The mixture was subjected to electrophoreis on a 7.5% cylindrical polyacrylamide gel (1.0 x 6.0 cm), without concentrating gel. The electrophoresis was carried out at 85 V at 4°C for 11 hr in the presence of 25 mM sodium acetate buffer, pH 6.0, containing 0.5 mM dithiothreitol and 0.05 mM EDTA. Gel slices (2.5 mm) were homogenized, to extract proteins, in 0.5 ml of 50 mM sodium acetate buffer (pH 6.0) containing 1 mM dithiothreitol and 0.1 mM EDTA, and the homogenates centrifuged at 100,OOOgfor 15 min. In order to obtain maximal binding of radioactive cyclic AMP, aliquots of the supernatant were again incubated with cyclic [G-*HIAMP, this time according to the conditions of Gilman et al. (19), and then filtered through Millipore filter discs (19). The radioactivity (s’P: O---O; SH: O--O) of the doubly labeled protein on the discs was then counted. Slices are numbered from the origin. In the experiment illustrated, the recoveries of protein-bound 3*P and cyclic [G-SH]AMP were 97 and 60%, respectively.
subunit, the isolated regulatory subunit, or the reconstituted holoenzyme could be selfphosphorylated. The results of these experiments are presented in Table I. The free catalytic subunit showed cyclic AMPindependent histone kinase activity, but no incorporation of phosphate into itself. The regulatory subunit, which was indicated (Fig. 2) to be the substrate in the autophosphorylation reaction, was not itself capable of autophosphorylation. How-
ever, when the isolated regulatory and catalytic subunits were incubated together, the reconstituted holoenzyme was capable of autophosphorylation (Table I). Moreover, the ratio of cyclic AMP-binding activity to autophosphorylation activity observed with the reconstituted holoenzyme was virtually the same as that observed with the intact holoenzyme from which the subunits were prepared (data not shown). Thus, using cyclic AMP-binding activity as the basis for comparison, the reconstituted holoenzyme was as effective as the parent holoenzyme in carrying out autophosphorylation. In order to determine the amount of phosphate incorporated per mole of Rprotein, the enzyme obtained after preparative polyacrylamide gel electrophoresis was incubated for 2 min, in a reaction volume of 1.0 ml, under standard conditions for endogenous phosphorylation in the absence of cyclic AMP. Cyclic AMP (final concentration 10e5 M) was then added to the reaction mixture and the incubation continued for a further 5 min at 30°C to dissociate the catalytic and regulatory subunits. The preparation was then subjected to preparative polyacrylamide gel electrophoresis and the gel then sliced, homogenized, and centrifuged, as described in the legend to Fig. 2. An aliquot of the clear supernatant from each slice was used for protein determination by the turbidimetric method. The remainder of the supernatant was applied to a Millipore filter disc, washed thoroughly with 0.02 M potassium phosphate buffer, pH 6.0, and then the radioactivity on the filter disc was measured. In two experiments, the amount of incorporated phosphate was calculated to be 0.63 and 0.46 mole per mole of the isolated R-protein. In another experiment, the phosphorylated regulatory subunit, purified by electrophoresis, as described in the legend to Fig. 2, was hydrolyzed in 6 N HCl and analyzed for phosphoserine and phosphothreonine by high-voltage paper electrophoresis as described in Methods. After correction for the hydrolysis of phosphoserine and phosphothreonine, 96% of the total activity was found to be in phosphoserine.
AUTOPHOSPHORYLATION
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OF BRAIN PROTEIN KINASE
TABLE I COMPARISON OF AIJTOPHOSPHORYLATION OF CATALYTICSUBUNIT,REGULATORY SUBUNIT,AND RECONSTITUTED HOLOENZYME~ Preparation
Cyclic [3H]AMP bound (cpm)
Histone fji;s;
-Cyclic AMP Regulatory subunit (20 ccl) Catalytic subunit (20 ~1) Regulatory subunit (20 @I)plus catalytic subunit (20 ~1)
1590 <30 1500
0 285 -
activity
+Cyclic AMP 0 287 280
Autophosphorylation activity ( +Mg2+) (arbitrary units) -%I%
+Cyclic AMP
DHoloenzyme with specific activity of 9.2 x 10’ units/mg protein, purified through the step of Sepharose 6B column chromatography according to the method of Miyamoto et al. (15), was used to prepare free catalytic and regulatory subunits. To prepare the catalytic subunit, the holoenzyme (0.47 mg protein) was dissociated by incubation in 0.4 ml of 50 mM sodium acetate buffer, pH 6.2, containing histone (1 mg/ml) and 5 PM cyclic AMP. The catalytic subunit was separated from the regulatory subunit by chromatography on Sephadex G-100 (0.9 x 57 cm). The column was preequilibrated with, and the protein eluted with, 5 mM potassium phosphate buffer (pH 7.0) containing 2 mM dithiothreitol, 0.1 mM EDTA, and histone (100 &ml). (The addition of histone to the holoenzyme, a procedure used to promote dissociation, resulted in precipitation of the R-protein, which remained at the top of the gel and was not eluted from the column.) The peak fractions from the column were combined and solid ammonium sulfate was added to 70% saturation. This procedure precipitated the catalytic subunit, but left the histone in the supernatant. The resulting precipitate was collected by centrifugation, dissolved in 0.2 ml of 5 mM potassium phosphate, pH 7.0, containing 0.1 mM EDTA, and dialyzed for 16 hr against 2 liters of solution of the same composition. To prepare the regulatory subunit, the holoenzyme (0.47 mg protein) was incubated at 20°C for 2 min in 50 mM sodium acetate, pH 6.2, containing 2.5 mM ZnCl, and 0.1 mM EDTA. EDTA, dithiothreitol, and cyclic AMP were then added in final concentrations of 5 mM, 1.25 mM, and 6.25 @M,respectively. The mixture was kept at 0°C for 10 min, and the dissociated regulatory subunit was prepared by means of gel electrophoresis, using the procedure described in the legend to Fig. 2. Cyclic AMP-binding activity, histone kinase activity, and autophosphorylation activity (endogenous phosphorylation) were determined on 20-~1 aliquots of the indicated preparations.
In order to determine whether phosphorylation of the regulatory subunit affected cyclic AMP-binding activity, the holoenzyme preparation was incubated in the presence and absence of nonradioactive ATP for 2 min under the standard assay conditions, in the absence of cyclic AMP. After the 2-min incubation, cyclic AMPbinding activity of the enzyme was measured according to the method of Gilman (19). The phosphorylated and nonphosphorylated enzymes had cyclic AMP-binding activities within 5% of each other. General characteristics of the autophosphorylation reaction. The rate of autophos-
phorylation of the brain protein kinase was quite rapid under the standard incubation conditions, as shown in the upper part of Fig. 3. Under the conditions used, phosphorylation reached a maximum in about 20 to 30 sec. Divalent metal ions were required for the autophosphorylation reac-
tion. In the presence of certain of these metal ions, cyclic AMP was found to have a marked effect on the autophosphorylation (Fig. 3, Table II). Thus, cyclic AMP caused a decrease in phosphate incorporation in the presence of MnCl,, CaCl,, or ZnCl,, but had no effect when MgCl, or CoCl, was used. When autophosphorylation was carried out in the presence of ZnCl, for 30 set, and cyclic AMP then added, no decrease in the level of phosphorylation was observed (data not shown). In other experiments, cyclic AMP was also found to be inhibitory in the presence of NiCl,, CuCl,, or FeCl,. Since detailed kinetic experiments were not carried out for each metal ion, we cannot state whether the effects of the metal ions and cyclic AMP were due to changes in the rate of autophosphorylation or to changes in the total amount of phosphate that could be incorporated. In order to determine whether the inhibi-
556
MAENO ET AL. lOmM
0
MqCI2
; 0
,
.
40
60
*
20 Incubation
Time
(ret)
FIG. 3. Autophosphorylation
of protein kinase as a function of incubation time. Autophosphorylation of 11 pg of protein kinase with [-y-82P]ATP was performed in the absence (O---O) or presence (W---O) of 5 pM cyclic AMP under standard assay conditions, except for the variation in incubation time, and, where indicated, the replacement of 10 mM MgCl, by 2.5 mM ZnCl,. TABLE II EFFECT OF VARIOUS DIVALENT METAL IONS ON THE ENINXENOUS PHOSPHORYLATIONOF R-PR~TEW
Met& ionMe at
Incorporation of phosphate (arbitrary units) -Cyclic AMP
+Cyclic AMP
ered by about 70% (Table III). However, when 4 mM EDTA was added between the preincubation and incubation periods in order to chelate the Zn2+, followed by the addition of 10 mM MgCl,, the inhibition by cyclic AMP of the rate of autophosphorylation was abolished, indicating that the effect of Zn2+ and cyclic AMP was readily reversible. Moreover, in another experiment, it was found that when the protein kinase was first incubated with [y32P]ATP in the presence of 2.5 mM ZnCl, plus 5 FM cyclic AMP for 15 set, negligible phosphorylation occurred, but that immediately upon the addition of 4 mM EDTA, and then 10 mM MgC12, to this reaction mixture, autophosphorylation began, indicating the reversibility of the inhibitory effect. The percentage of inhibition by 5 PM cyclic AMP in the presence of 2.5 mru ZnCl, or 10 mM CaCl, was not altered by varying the concentration of ATP from 1 to 8 FM. Over the same range of ATP concentrations, cyclic AMP had no effect on the reaction in the presence of 10 mru MgCl,. The effect of varying the concentration of cyclic AMP on the autophosphorylation reaction was studied in the presence of MgCl*, of CaCl,, and of ZnCl,. When the reaction mixture contained 10 mM MgCl,, TABLE III
None M&l, CoCl, MnCl, CaCl, ZnCl,
7 44 56 65 52 53
5 46 59 44 16 15
” Autophosphorylation of 11 pg of protein kinase with [-y-s2P]ATP was carried out under standard assay conditions, as described in the text, except for the variation in the kind and amount of each metal ion as indicated.
tion of enzyme activity by cyclic AMP in the presence of divalent ions was reversible, protein kinase was preincubated with 2.5 mM ZnCl,, in the absence or presence of 5 x lo6 M cyclic AMP at 20°C for 15 set and then incubated with [y-S2P]ATP for an additional 15 sec. In the presence of cyclic AMP, the autophosphorylation was low-
REVERSIBILITY OF THE INHIBITORY EFFECT OF CYCLIC AMP ON THE AUTOPHOSPHORYLATIONOF PROTEIN KINAW Tube
No.
1 2 3 4
Preincubation
-Cyclic + Cyclic -Cyclic + Cyclic
AMP AMP AMP AMP
Addition of EDTA
Incorporation of 3aP (arbitrary units)
+ +
100 29 133 130
a Protein kinase (11 pg) was preincubated with 2.5 mM ZnC1, in 50 mM sodium acetate buffer (pH 6.2) for 15 set at 20°C in the absence or presence of 5 NMcyclic AMP, as indicated. Tubes 1 and 2 were then directly incubated with 5 PM [Y-~~PJATP for 15 set at 20°C. After pmincubation, tubes 3 and 4 had 4 mru EDTA added and mixed, and immediately thereafter these tubes were incubated with 5 FM [y-32P]ATP and 10 mM MgCl, for 15 set at 20°C.
AUTOPHOSPHORYLATION
the rate of autophosphorylation of protein kinase was not affected by any concentration of cyclic AMP tested. When MgCl, was replaced by CaCl, or ZnCl,, more than 80% of the activity could be inhibited by cyclic AMP, and the concentration of cyclic AMP required to give half-maximal inhibition was approximately 3 x lo-’ M (Fig. 4). In other experiments the effect of various cyclic nucleotides on the autophosphorylation reaction was studied in the presence of 2.5 mM ZnCl,. Cyclic AMP was the most effective inhibitor. At a concentration of 10e5 M, cyclic AMP, cyclic IMP, cyclic GMP, cyclic UMP, and cyclic CMP caused an inhibition of 97, 70, 16, 9, and O%, respectively. The effect of pH on autophosphorylation of the protein kinase was also studied. In the presence of 2.5 mM ZnCl,, an inhibitory effect of cyclic AMP was observed over the whole pH range tested (4.0-9.0). In the presence of 10 mM MgC12, there was a broad optimum for the autophosphorylation reaction, from pH 5.5 to pH 8.5, with no cyclic AMP dependence. Comparison
of R-protein
and Protein II.
In a previous paper (14) it was shown that
.: OP-----i
557
OF BRAIN PROTEIN KINASE
M - I (0.9)
Purified Protein Kinose
M -I (I.01
nr-7 + -
+
‘+
-
cyclic AMP origin
BPB front
FIG. 5. Comparison of the electrophoretic migration of R-protein with that of Protein II from synaptic membrane fractions M-l (0.9) and M-l (1.0) of bovine cerebrum. Twenty-two micrograms of cyclic AMPdependent protein kinase, 25Opg of M-l (0.9), and 113 pg of M-l (1.0) were subjected to autophosphorylation with [-r-*T]ATP in the absence or presence of 5 PM cyclic AMP under standard assay conditions, as described in the text, except for the presence of 2.5 mM ZnCl, instead of MgCl,. After termination of the reaction by the addition of DodSO,-, the mixtures were subject to slab gel electrophoresis and autoradiography. The photograph shows the autoradiograph of the dried gel on X-ray film. The minus and plus signs indicate the absence or presence of cyclic AMP in the incubation mixture. BPB, bromphenol blue.
the endogenous phosphorylation of a protein (Protein II), of molecular weight 48,000, from synaptic membrane fractions M-l (0.9) and M-l (1.0) was regulated by cyclic AMP. Since the molecular weight of R-protein appeared similar to that of Protein II, the 32P-phosphorylated proteins were subjected to electrophoresis on the Cancmption Of Cyclic AMP (-lop M) same polyacrylamide gel in the presence of FIG. 4. Effect of varying the concentration of cy- DodSO,- in order to compare directly the clic AMP on the autophosphorylation reaction. Autoelectrophoretic migration of these proteins. phosphorylation of 11 rg of protein kinase with The autoradiogram of this gel is shown in [y-s*P]ATP was performed under standard assay Fig. 5, where it can be seen that these conditions, as described in the text, except for the variation in the concentration of cyclic AMP and the proteins ran in identical positions. In addireplacement of 10 mM MgCl, by 2.5 mru CaCI, or tion, these proteins showed the same response to cyclic AMP in the presence of ZnCl, as indicated.
558
MAENO ET AL.
Zna+. However, unlike the autophosphorylation reaction studied in the present investigation, the phosphorylation of Protein II was greatly stimulated by cyclic AMP in the presence of Mg2+ or Mn2* (14). Also, the phosphorylation of Protein II exhibits a sharp pH maximum at pH 6.2 (14), unlike the autophosphorylation reaction of the purified protein kinase. DISCUSSION
The data presented in this paper suggest that a protein kinase from mammalian brain uses a subunit of itself as an endogenous substrate. On polyacrylamide disc gel electrophoresis, protein kinase activity using histone as substrate, endogenous phosphorylation, and cyclic AMP-binding activity were inseparable. In addition, the rate of endogenous phosphorylation varied linearly with enzyme concentration in the absence of cyclic AMP (data not shown), suggesting that the enzyme and substrate were part of a complex over the range of concentrations studied. It was also found that cyclic AMP bound to the phosphorylated substrate and dissociated it from the holoenzyme (Fig. 2). These observations lead us to conclude that the regulatory subunit of the brain protein kinase is an endogenous substrate of this enzyme. There are some similarities and some differences between the endogenous phosphorylation and the phosphorylation of exogenous substrates by the enzyme with regard to the effect of divalent metal ions. It was found by Miyamoto et al. (15) that cyclic AMP stimulated histone kinase activity of the enzyme in the presence of Mg2+, Co2+, or Mn2+, but inhibited histone kinase activity in the presence of certain concentrations of Zna+, Fea+, Ca2+, Cu+, or Cu2+. In contrast to the phosphorylation of exogenous substrates such as histone, the autophosphorylation was not significantly affected by cyclic AMP in the presence of Mg2+. In the presence of a number of other divalent metal ions, including Zn2+, Ca2+, Mna+, Ni2+, Cu2+, and Fez+, cyclic AMP inhibited the endogenous phosphorylation. The pH dependence of the autophosphorylation reaction contrasts with that observed for histone phosphorylation. The
rate of autophosphorylation in the presence of Mga+ is almost independent of pH in the pH range 5.5-8.5; in contrast, the enzyme exhibits a fairly sharp maximum at pH 6.2 with exogenous substrates (12). The extent to which the state of dissociation of the subunits of protein kinase may account for the autophosphorylation data observed in the present study needs to be investigated. For example, it seems possible that the inhibition by cyclic AMP of the autophosphorylation reaction, which occurs in the presence of certain divalent metal ions, results from a cyclic AMPinduced dissociation of the enzyme. However, it is difficult to test thispossibility, since the techniques necessary to ascertain the state of dissociation, such as sucrose density-gradient centrifugation, require much longer periods of interaction of the enzyme, cyclic nucleotide, and other reagents than do the enzymological measurements carried out in the present study. We have also considered the possibility that the inhibition of autophosphorylation by cyclic AMP is due to the activation of an endogenous protein phosphatase. However, Zn2+ is known to inhibit protein phosphatase (3,24), and the most striking effect of cyclic AMP on the autophosphorylation reaction is observed in the presence of Zn2+. Also, the purified protein kinase exhibited no detectable protein phosphatase activity, in the absence or presence of cyclic AMP, either using protamine phosphate as substrate according to the method of Maeno and Greengard (3), or using endogenously labeled R-protein as substrate, according to the method of DeLorenzo and Greengard (24). It has recently been reported that cyclic AMP-dependent protein kinases from human erythrocytes (25) and from bovine heart (26,27) can phosphorylate their own regulatory subunits. However, the effect of cyclic AMP on the autophosphorylation of those enzymes was apparently not investigated. It would be of interest to know if the erythrocyte and heart preparations show the same characteristics, with respect to cyclic AMP and divalent metal ions, as the brain enzyme. As mentioned previously, there are some
AUTOPHOSPHORYLATION
intriguing similarities between the autophosphorylation reaction and the endogenous phosphorylation of Protein II, a protein present in synaptic membrane fractions (14). The two proteins run with the same mobility on DodSO,--polyacrylamide gels and have the same response to cyclic AMP in the presence of Zn2+ and certain other metal ions. However, a critical evaluation of the possible identity of the two proteins must await solubilization and purification of the membrane-bound Protein II.
1. 2. 3. 4.
5. 6. 7.
8.
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10. 11.
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KINASE
559
12. MNAMOTO, E., Kuo, J. F., AND GREENCARD, P. (1969) J. Biol. Chem. 244, 6395-6402. 13. Kuo, J. F., AND GREENGARD, P. (1969) Proc. Nat. Acad. Sci. USA 64, 1349-1355. 14. UEDA, T., MAENO, H., AND GREENGARD, P. (1973) J. Biol. Chem. 248, 8295-8305. 15. MNAMOTO, E., PETZOLD, G. L., Kuo, J. F., AND GREENCARD, P. (1973) J. Biol. Chem. 248, 179-189. 16. POST, R. L., AND SEN, A. K. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 773-776, Academic Press, New York. 17. Kuo, J. F., AND GREENGARD, P. (1970) J. Biol. Chem. 245, 4067-4073. 18. FAIRBANKS, G., SRECK, T. L., AND WALLACH, D. F. REFERENCES H. (1971) Biochemistry 10, 2608-2617. 19. GILMAN, A. G. (1970) Proc. Nat. Acad. Sci. USA MAENO, H., JOHNSON, E. M., AND GREENGARD, P. 67,305-312. (1971) J. Biol. Chem. 246, 134-142. JOHNSON, E. M., MAENO, H., AND GREENGARD, P. 20. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., (1971) J. Biol. Chem. 246, 7731-7739. AND RANDALL, R. J. (1951) J. Biol. Chem. 193, MAENO, H., AND GRLZENGARD,P. (1972) J. Biol. 265275. 21. LAYNE, E. (1957) in Methods in Enzymology Chem. 247, 3269-3277. (Colowick, S. P., and Kaplan, N. O., eds.), Vol. DEROBERTIS, E., DELORES ARNAIZ, G., ALBERICI, 3, pp. 447-454, Academic Press, New York. M., BUTCHER, R. W., AND SUTHERLAND, E. W. 22. ORNSTEIN, L. (1964) Ann. N. Y. Acad. Sci. 121, (1967) J. Biol. Chem. 242, 3487-3493. 321-349. CHEIJNG, W. Y., AND SALGANICOFF,L. (1967) Na23. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, ture (London) 214.90-91. 404-427. FLORENDO, N., BARNETT, R. J., AND GREENCARD,P. 24. DELORENZO, R. J., AND GREENGARD, P. (1973) (1971) Science 173,745-748. JOHNSON, E. M., UEDA, T., MAENO, H., AND Proc. Nat. Acad. Sci. USA 70, 1831-1835. GREENGARD, P. (1972) J. Biol. Chem. 247, 25. GUTHROW, C. E., JR., BRUNSWICK, D. J., COOPER5650-5652. MAN, B. S., AND RASMUSSEN,H. (1973) Abstracts GREENGARD, P., MCAFEE, D. A., AND KEBABIAN, J. of the Fifth Annual Miami Winter Symposia, p. W. (1972) Advan. Cyclic Nucleotide Res. 1, 111. 337-355. 26 ROSEN, 0. M., RUBIN, C. S., AND ERLICHMAN, J. HOFFER, B. J., SIGGINS, G. R., OLIVER, A. P., AND (1973) in Protein Phosphorylation in Control BLOOM, F. E. (1972) Advan. Cyclic Nucleotide Mechanisms (Huijing, F., and Lees, E. Y. C., Res. 1, 411-423. eds.), Vol. 5, pp. 67-82, Academic Press, New York. WALSH, D. A., PERKINS, J. P., AND KREBS, E. G. (1968) J. Biol. Chem. 243, 3763-3765. 27. ERLICHMAN, J., RUBIN, C. S., AND ROSEN, 0. M. LANGAN, T. A. (1968) Science 162,579-580. (1973) Fed. Proc. 32, 643 Abstr.
OF BIOCHEMISTRY
AND
BIOPHYSICS
164,
551-559
(1974)
Autophosphorylation
of Adenosine
3’, 5’-Monophosphate-Dependent Protein
Kinase from
BovineBrain
l
HIROO MAENO, PROCERFINA L. REYES, TETSUFUMI UEDA, STEPHEN A. RUDOLPH,2 AND PAUL GREENGARD Department
of Pharmacology,
Yale University
School of Medicine,
New Haven,
Connecticut
06510
Received April 8, 1974 A highly purified adenosine 3’) 5’-monophosphate-dependent protein kinase from bovine brain has been found to catalyze its own phosphorylation. The incorporated phosphate was shown to be associated with the cyclic AMP-binding subunit (R-protein) of the protein kinase. The catalytic subunit exhibited no detectable incorporation of phosphate into itself, but was required for the phosphorylation of R-protein. The molecular weight of R-protein was determined by polyacrylamide gel electrophoresis to be about 48,000 in the presence of sodium dodecyl sulfate. Cyclic AMP strikingly inhibited the rate of autophosphorylation observed in the presence of ZnCl,, CaCl,, NiCl,, or Fe&, but had no significant effect in the presence of MgCl, or CoCl,. The concentration of cyclic AMP required to give half-maximal inhibition of phosphorylation was 3 x lo-’ M in the presence of either CaCl, or ZnCl,. Guanosine 3’, 5’-monophosphate was far less effective under the same experimental conditions than cyclic AMP. R-protein appears to be similar to a phosphoprotein recently discovered in synaptic membrane fractions from rat and bovine cerebral cortex.
The possibility that the phosphorylation and dephosphorylation of certain synaptic membrane proteins plays an important role in neural function is supported by observations of the enrichment in synaptic membrane fractions of cyclic AMP3dependent protein phosphorylation and of protein dephosphorylation systems (l-3) and is compatible with other biochemical (4-8) and physiological (8,9) evidence. Most previous studies of cyclic AMP-dependent protein kinases have focused at1This work was supported by Grants NS-08440 and MH-17387 from the United States Public Health Service. 2Postdoctoral Fellow of the Damon Runyon Memorial Fund for Cancer Research. 3Abbreviations used: Cyclic AMP, adenosine 3’) 5’-monophosphate; cyclic IMP, inosine 3’, 5’-monophosphate; cyclic GMP, guanosine 3’) 5’-monophosphate; cyclic CMP, cytidine 3’, 5’-monophosphate; cyclic UMP, uridine 3’, 5’-monophosphate; DodSO,-, sodium dodecyl sulfate.
tention on the enzymological properties of these enzymes, and have been carried out with artificial substrates, such as histone or casein (e.g., 10-13). However, in order to elucidate the biochemical basis for the physiological effects of cyclic AMP, it is necessary to identify and isolate the endogenous substrate proteins of cyclic AMPdependent protein kinases. Recently, cyclic AMP was demonstrated to regulate the endogenous phosphorylation of two specific protein components of synaptic membrane fractions from rat, bovine, and porcine cerebral cortex (7,14). It was suggested that the state of phosphorylation of these protein constituents might control the permeability of some neuronal membranes to inorganic ions, thereby mediating postsynaptic potential changes of the membrane. One of the proteins, referred to as Protein II, was of particular interest, since the effect of cyclic AMP on its phosphorylation was either stimulatory or inhibitory
552
MAENO
depending on the precise experimental conditions (14). In the present study, we have been able to demonstrate that a highly purified cyclic AMP-dependent protein kinase from bovine brain can catalyze its own phosphorylation and that the incorporated phosphate is associated with the cyclic AMP-binding subunit (R-protein) of the protein kinase. This subunit comigrates with Protein II electrophoretically on DodSO,--polyacrylamide gels. MATERIALS
AND
METHODS
Materials. Synaptic membrane fractions were prepared from fresh bovine cerebral cortex according to a method previously described for rat brain (1). Cyclic AMP-dependent protein kinase II was partially purified from a 13,000g supernatant of bovine brain homogenate by protamine sulfate treatment, ammonium sulfate precipitation, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography as described by Miyamoto et al. (15). Except where specified otherwise, this partially purified protein kinase was used in the experiments to be described. Further purification was achieved by Sepharose 6B gel filtration and preparative polyacrylamide gel electrophoresis (15). The specific activity of the enzyme at various stages of purification was similar to that described previously (15). Calf thymus histone, cyclic [G-SH]AMP (16.3 Ci/mole) and ATP were obtained from Schwarz-Mann. Cyclic AMP, cyclic IMP, cyclic GMP, cyclic UMP, and cyclic CMP were from Boehringer-Mannheim. Bovine serum albumin (fraction V) was purchased from Sigma. [Y-~~P]ATP was prepared by the method of Post and Sen (16). Assay for cyclic AMP-dependent histone kinase octiuity. Protein kinase activity was routinely assayed using histone as a substrate. The assay was done in a total volume of 0.2 ml at pH 6.2 in the presence of 50 mM sodium acetate, 10 mM magnesium acetate, 5 PM [y-32P]ATP (l-3 x lo6 cpm/nmole), 100 c(g of calf thymus histone, and an appropriate amount of protein kinase, in the presence or absence of 5 pi cyclic AMP. The reaction was initiated by the addition of the [-r-12P]ATP, carried out for 5 min at 30°C and terminated by the addition of 2.0 ml of 5% trichloroacetic acid containing 0.25% sodium tungstate. The procedure for washing and counting radioactive histone in the precipitate was as described previously (17). One unit of histone kinase activity was defined as the amount of enzyme which transferred 1 pmole of 2zP from [Y-~~P]ATP to histone under the standard assay conditions. Assay for endogenous phosphorylation of protein kinuse. The standard reaction mixture with a total
ET AL. volume of 0.1 ml at pH 6.2 contained 50 mM sodium acetate, 10 mM MgCl,, 0.1 mM EDTA, 5 PM [-rs2P]ATP (l-3 x 10’ cpm per nmole), and an appropriate amount of enzyme in the presence or absence of 5 PM cyclic AMP. The reaction was carried out for 15 set at 20°C and terminated by the addition of 0.05 ml of 9% DodSO,in 0.03 M Tris-HCl buffer, pH 8.0, containing 6% mercaptoethanol, 3 mM EDTA, and 15% glycerol. A O.l-ml aliquot of this reaction mixture was then subjected to flat plate polyacrylamide gel electrophoresis (7,14) in the presence of DodSO,- as described by Fairbanks et al. (18). The gel was stained for protein with 0.025% Coomassie blue in 25% isopropyl alcohol and 10% acetic acid and was destained with 10% isopropyl alcohol and 10% acetic acid containing 0.0025% Coomassie blue, followed by 10% acetic acid (18). The gel was dried on Whatman No. 50 filter paper under vacuum and heat, and an autoradiogram of the gel was made using Kodak X-Omat Royal X-ray film to visualize 3zP incorporation into specific proteins (7,14). The optical density of the bands on the autoradiogram was measured with a Joyce-Loebl microdensitometer. The optical density of the band on the autoradiogram corresponding to the protein showing cyclic AMP-dependent phosphorylation was taken to be proportional to the amount of 3T incorporated into this protein (14). When protein kinase (either partially purified through hydroxylapatite or purified to apparent homogeneity) was assayed for endogenous phosphorylation, the molecular weight of the phosphorylated subunit was determined to be 48,000 by electrophoresis on flat plate polyacrylamide gels in the presence of DodSO,-, using standard marker proteins of known molecular weights, as described previously (14). Using DodSO,gel electrophoresis, it was possible to study the total distribution of 32P incorporation into protein. Of the 3zP-labeled protein applied to the gels, approximately 90% was recovered after correction for quenching. Of the total radioactivity appearing on the gels, greater than 90% was in the 48,000-dalton band when the incubation was carried out in the presence of Mg*+, and virtually 100% in the presence of Zna+ (see Fig. 5). Therefore, whenever endogenous phosphorylation was studied, it was the phosphorylation of this particular protein that was measured. Assay for cyclic AMP-binding activity. Cyclic AMP-binding activity was determined by adsorption of cyclic AMP-binding protein onto Millipore filters according to the method of Gilman (19), except for omission of the heat-stable inhibitor. Other methods. In order to identify the site of endogenous phosphate incorporation, SIP-labeled protein was hydrolyzed in 6 N HCi at 106°C for 6 hr, dried in uacuo, and subjected to high-voltage paper electrophoresis for 3 hr, as described previously (2). After the paper was dried, phosphoserine and phosphothreonine were located by staining with ninhydrin. The
AUTOPHOSPHORYLATION
553
OF BRAIN PROTEIN KINASE
paper was then cut into 0.5-cm strips, and the radioactivity of the strips was counted by liquid scintillation spectrometry. Protein concentrations were determined by the method of Lowry et al, (20) or by modification of a turbidimetric method (21) in which the turbidity of protein solutions in the presence of 5% trichloroacetic acid was determined spectrophotometrically. In either method, bovine serum albumin was used as the standard protein. RESULTS
Evidence for autophosphorylution of cyclic AMP-dependent protein kinase. Cyclic AMP-dependent protein kinase, purified through the step of hydroxylapatite chromatography, was subjected to preparative polyacrylamide disc gel electrophoresis and the gel was then assayed for histone kinase activity, endogenous phosphorylation, and cyclic AMP binding activity. As illustrated in Fig. 1, all three activities migrated on the gel at the same speed (R, 0.18 against bromphenol blue). (When the peak fractions were pooled, an aliquot subjected to reelectrophoresis as described in the legend to Fig. 1, and that gel stained for protein, a single major band, containing over 95% of the protein stain, was observed.) These results indicated that the cyclic AMP-dependent protein kinase might be catalyzing its own phosphorylation. In order to determine whether the phosphate was incorporated into the regulatory or catalytic subunit, the enzyme, obtained by polyacrylamide disc gel electrophoresis, was phosphorylated with [T-~~P]ATP and then incubated with cyclic [G-3H]AMP to dissociate the enzyme into its regulatory and catalytic subunits. When this preparation was subjected to electrophoresis on a cylindrical polyacrylamide gel, in the absence of DodSO,-, the 32Pand 3H elution profiles were found to be identical (Fig. 2). (The catalytic subunit, as judged by the ability to phosphorylate histone, was not detected in these fractions; presumably, the dissociated catalytic subunit, which has an isoelectric point of 7.8 (Vi), migrated into the upper buffer under the conditions of electropboresis used.) In experiments not shown here, when the holoenzyme was endogenously phosphorylated
OA 0
0.5 Rt
A 1.0
FIG. 1. Polyacrylamide gel electrophoresis of protein kinase. The enzyme preparation (550 ag) from a hydroxylapatite column was subjected to electrophoresis on a 7.5% polyacrylamide cylindrical gel (1.0 x 7 cm), pH 8.9, with concentrating gel (22,23). The upper and lower buffers were glycine-Tris (pH 8.3) and Tris-HCI (pH 8.1), respectively, both of which contained 1 mM dithiothreitol and 0.1 mM EDTA. After electrophoresis for 5 hr at 350 V, at 4”C, gel slices (2 mm) were homogenized in 10 mM Tris-HCI buffer, pH 7.5, containing 1 mM dithiothreitol and 0.1 mM EDTA, and then centrifuged at 100,OOOgfor 15 min. The clear supematants were used for the measurement of histone kinase activity in the presence (-C-) and absence (--O-) of cyclic AMP, endogenous phosphorylation in the absence of cyclic AMP, and cyclic AMP-binding activity, under standard assay conditions. In the experiment illustrated, the recoveries of cyclic AMP-dependent histone kinase activity, of endogenous phosphorylation, and of cyclic AMP-binding activity were 72, 70, and 61%, respectively.
with [T-~~P]ATP, dissociated in the presence of cyclic [G-3H]AMP and histone, and then applied to a Sephadex G-100 column, the catalytic subunit was found to contain no radioactive phosphate, and the majority of protein-bound 32Premained at the top of the column along with most of the proteinbound 3H. Experiments were also carried out to determine whether the isolated catalytic
554
MAENO ET AL.
5
10 Slier
15
20
$
Number
FIG. 2. Polyacrylamide gel electrophoresis of phosphorylated purified protein kinase. Cyclic AMPdependent protein kinase, which had been purified by polyacrylamide disc gel electrophoresis, as described in the legend to Fig. 1, was phosphorylated by incubation at 20°C for 2 min in a volume of 0.37 ml, in the presence of 50 mM sodium acetate, pH 6.2, 0.1 mM EDTA, 5 NM [Y-~?P]ATP (247 cpm/pmole), and 2.5 mM ZnCl,. EDTA, dithiothreitol, and cyclic [G*H]AMP (7000 cpm/pmole) were then added to final concentrations of 5 mM, 1.25 mM, and 0.6 PM, respectively. The incubation mixture was kept at 0°C for 10 min, and then cyclic AMP (final concentration, 6.25 PM) was added to achieve complete dissociation of the enzyme. The mixture was subjected to electrophoreis on a 7.5% cylindrical polyacrylamide gel (1.0 x 6.0 cm), without concentrating gel. The electrophoresis was carried out at 85 V at 4°C for 11 hr in the presence of 25 mM sodium acetate buffer, pH 6.0, containing 0.5 mM dithiothreitol and 0.05 mM EDTA. Gel slices (2.5 mm) were homogenized, to extract proteins, in 0.5 ml of 50 mM sodium acetate buffer (pH 6.0) containing 1 mM dithiothreitol and 0.1 mM EDTA, and the homogenates centrifuged at 100,OOOgfor 15 min. In order to obtain maximal binding of radioactive cyclic AMP, aliquots of the supernatant were again incubated with cyclic [G-*HIAMP, this time according to the conditions of Gilman et al. (19), and then filtered through Millipore filter discs (19). The radioactivity (s’P: O---O; SH: O--O) of the doubly labeled protein on the discs was then counted. Slices are numbered from the origin. In the experiment illustrated, the recoveries of protein-bound 3*P and cyclic [G-SH]AMP were 97 and 60%, respectively.
subunit, the isolated regulatory subunit, or the reconstituted holoenzyme could be selfphosphorylated. The results of these experiments are presented in Table I. The free catalytic subunit showed cyclic AMPindependent histone kinase activity, but no incorporation of phosphate into itself. The regulatory subunit, which was indicated (Fig. 2) to be the substrate in the autophosphorylation reaction, was not itself capable of autophosphorylation. How-
ever, when the isolated regulatory and catalytic subunits were incubated together, the reconstituted holoenzyme was capable of autophosphorylation (Table I). Moreover, the ratio of cyclic AMP-binding activity to autophosphorylation activity observed with the reconstituted holoenzyme was virtually the same as that observed with the intact holoenzyme from which the subunits were prepared (data not shown). Thus, using cyclic AMP-binding activity as the basis for comparison, the reconstituted holoenzyme was as effective as the parent holoenzyme in carrying out autophosphorylation. In order to determine the amount of phosphate incorporated per mole of Rprotein, the enzyme obtained after preparative polyacrylamide gel electrophoresis was incubated for 2 min, in a reaction volume of 1.0 ml, under standard conditions for endogenous phosphorylation in the absence of cyclic AMP. Cyclic AMP (final concentration 10e5 M) was then added to the reaction mixture and the incubation continued for a further 5 min at 30°C to dissociate the catalytic and regulatory subunits. The preparation was then subjected to preparative polyacrylamide gel electrophoresis and the gel then sliced, homogenized, and centrifuged, as described in the legend to Fig. 2. An aliquot of the clear supernatant from each slice was used for protein determination by the turbidimetric method. The remainder of the supernatant was applied to a Millipore filter disc, washed thoroughly with 0.02 M potassium phosphate buffer, pH 6.0, and then the radioactivity on the filter disc was measured. In two experiments, the amount of incorporated phosphate was calculated to be 0.63 and 0.46 mole per mole of the isolated R-protein. In another experiment, the phosphorylated regulatory subunit, purified by electrophoresis, as described in the legend to Fig. 2, was hydrolyzed in 6 N HCl and analyzed for phosphoserine and phosphothreonine by high-voltage paper electrophoresis as described in Methods. After correction for the hydrolysis of phosphoserine and phosphothreonine, 96% of the total activity was found to be in phosphoserine.
AUTOPHOSPHORYLATION
555
OF BRAIN PROTEIN KINASE
TABLE I COMPARISON OF AIJTOPHOSPHORYLATION OF CATALYTICSUBUNIT,REGULATORY SUBUNIT,AND RECONSTITUTED HOLOENZYME~ Preparation
Cyclic [3H]AMP bound (cpm)
Histone fji;s;
-Cyclic AMP Regulatory subunit (20 ccl) Catalytic subunit (20 ~1) Regulatory subunit (20 @I)plus catalytic subunit (20 ~1)
1590 <30 1500
0 285 -
activity
+Cyclic AMP 0 287 280
Autophosphorylation activity ( +Mg2+) (arbitrary units) -%I%
+Cyclic AMP
DHoloenzyme with specific activity of 9.2 x 10’ units/mg protein, purified through the step of Sepharose 6B column chromatography according to the method of Miyamoto et al. (15), was used to prepare free catalytic and regulatory subunits. To prepare the catalytic subunit, the holoenzyme (0.47 mg protein) was dissociated by incubation in 0.4 ml of 50 mM sodium acetate buffer, pH 6.2, containing histone (1 mg/ml) and 5 PM cyclic AMP. The catalytic subunit was separated from the regulatory subunit by chromatography on Sephadex G-100 (0.9 x 57 cm). The column was preequilibrated with, and the protein eluted with, 5 mM potassium phosphate buffer (pH 7.0) containing 2 mM dithiothreitol, 0.1 mM EDTA, and histone (100 &ml). (The addition of histone to the holoenzyme, a procedure used to promote dissociation, resulted in precipitation of the R-protein, which remained at the top of the gel and was not eluted from the column.) The peak fractions from the column were combined and solid ammonium sulfate was added to 70% saturation. This procedure precipitated the catalytic subunit, but left the histone in the supernatant. The resulting precipitate was collected by centrifugation, dissolved in 0.2 ml of 5 mM potassium phosphate, pH 7.0, containing 0.1 mM EDTA, and dialyzed for 16 hr against 2 liters of solution of the same composition. To prepare the regulatory subunit, the holoenzyme (0.47 mg protein) was incubated at 20°C for 2 min in 50 mM sodium acetate, pH 6.2, containing 2.5 mM ZnCl, and 0.1 mM EDTA. EDTA, dithiothreitol, and cyclic AMP were then added in final concentrations of 5 mM, 1.25 mM, and 6.25 @M,respectively. The mixture was kept at 0°C for 10 min, and the dissociated regulatory subunit was prepared by means of gel electrophoresis, using the procedure described in the legend to Fig. 2. Cyclic AMP-binding activity, histone kinase activity, and autophosphorylation activity (endogenous phosphorylation) were determined on 20-~1 aliquots of the indicated preparations.
In order to determine whether phosphorylation of the regulatory subunit affected cyclic AMP-binding activity, the holoenzyme preparation was incubated in the presence and absence of nonradioactive ATP for 2 min under the standard assay conditions, in the absence of cyclic AMP. After the 2-min incubation, cyclic AMPbinding activity of the enzyme was measured according to the method of Gilman (19). The phosphorylated and nonphosphorylated enzymes had cyclic AMP-binding activities within 5% of each other. General characteristics of the autophosphorylation reaction. The rate of autophos-
phorylation of the brain protein kinase was quite rapid under the standard incubation conditions, as shown in the upper part of Fig. 3. Under the conditions used, phosphorylation reached a maximum in about 20 to 30 sec. Divalent metal ions were required for the autophosphorylation reac-
tion. In the presence of certain of these metal ions, cyclic AMP was found to have a marked effect on the autophosphorylation (Fig. 3, Table II). Thus, cyclic AMP caused a decrease in phosphate incorporation in the presence of MnCl,, CaCl,, or ZnCl,, but had no effect when MgCl, or CoCl, was used. When autophosphorylation was carried out in the presence of ZnCl, for 30 set, and cyclic AMP then added, no decrease in the level of phosphorylation was observed (data not shown). In other experiments, cyclic AMP was also found to be inhibitory in the presence of NiCl,, CuCl,, or FeCl,. Since detailed kinetic experiments were not carried out for each metal ion, we cannot state whether the effects of the metal ions and cyclic AMP were due to changes in the rate of autophosphorylation or to changes in the total amount of phosphate that could be incorporated. In order to determine whether the inhibi-
556
MAENO ET AL. lOmM
0
MqCI2
; 0
,
.
40
60
*
20 Incubation
Time
(ret)
FIG. 3. Autophosphorylation
of protein kinase as a function of incubation time. Autophosphorylation of 11 pg of protein kinase with [-y-82P]ATP was performed in the absence (O---O) or presence (W---O) of 5 pM cyclic AMP under standard assay conditions, except for the variation in incubation time, and, where indicated, the replacement of 10 mM MgCl, by 2.5 mM ZnCl,. TABLE II EFFECT OF VARIOUS DIVALENT METAL IONS ON THE ENINXENOUS PHOSPHORYLATIONOF R-PR~TEW
Met& ionMe at
Incorporation of phosphate (arbitrary units) -Cyclic AMP
+Cyclic AMP
ered by about 70% (Table III). However, when 4 mM EDTA was added between the preincubation and incubation periods in order to chelate the Zn2+, followed by the addition of 10 mM MgCl,, the inhibition by cyclic AMP of the rate of autophosphorylation was abolished, indicating that the effect of Zn2+ and cyclic AMP was readily reversible. Moreover, in another experiment, it was found that when the protein kinase was first incubated with [y32P]ATP in the presence of 2.5 mM ZnCl, plus 5 FM cyclic AMP for 15 set, negligible phosphorylation occurred, but that immediately upon the addition of 4 mM EDTA, and then 10 mM MgC12, to this reaction mixture, autophosphorylation began, indicating the reversibility of the inhibitory effect. The percentage of inhibition by 5 PM cyclic AMP in the presence of 2.5 mru ZnCl, or 10 mM CaCl, was not altered by varying the concentration of ATP from 1 to 8 FM. Over the same range of ATP concentrations, cyclic AMP had no effect on the reaction in the presence of 10 mru MgCl,. The effect of varying the concentration of cyclic AMP on the autophosphorylation reaction was studied in the presence of MgCl*, of CaCl,, and of ZnCl,. When the reaction mixture contained 10 mM MgCl,, TABLE III
None M&l, CoCl, MnCl, CaCl, ZnCl,
7 44 56 65 52 53
5 46 59 44 16 15
” Autophosphorylation of 11 pg of protein kinase with [-y-s2P]ATP was carried out under standard assay conditions, as described in the text, except for the variation in the kind and amount of each metal ion as indicated.
tion of enzyme activity by cyclic AMP in the presence of divalent ions was reversible, protein kinase was preincubated with 2.5 mM ZnCl,, in the absence or presence of 5 x lo6 M cyclic AMP at 20°C for 15 set and then incubated with [y-S2P]ATP for an additional 15 sec. In the presence of cyclic AMP, the autophosphorylation was low-
REVERSIBILITY OF THE INHIBITORY EFFECT OF CYCLIC AMP ON THE AUTOPHOSPHORYLATIONOF PROTEIN KINAW Tube
No.
1 2 3 4
Preincubation
-Cyclic + Cyclic -Cyclic + Cyclic
AMP AMP AMP AMP
Addition of EDTA
Incorporation of 3aP (arbitrary units)
+ +
100 29 133 130
a Protein kinase (11 pg) was preincubated with 2.5 mM ZnC1, in 50 mM sodium acetate buffer (pH 6.2) for 15 set at 20°C in the absence or presence of 5 NMcyclic AMP, as indicated. Tubes 1 and 2 were then directly incubated with 5 PM [Y-~~PJATP for 15 set at 20°C. After pmincubation, tubes 3 and 4 had 4 mru EDTA added and mixed, and immediately thereafter these tubes were incubated with 5 FM [y-32P]ATP and 10 mM MgCl, for 15 set at 20°C.
AUTOPHOSPHORYLATION
the rate of autophosphorylation of protein kinase was not affected by any concentration of cyclic AMP tested. When MgCl, was replaced by CaCl, or ZnCl,, more than 80% of the activity could be inhibited by cyclic AMP, and the concentration of cyclic AMP required to give half-maximal inhibition was approximately 3 x lo-’ M (Fig. 4). In other experiments the effect of various cyclic nucleotides on the autophosphorylation reaction was studied in the presence of 2.5 mM ZnCl,. Cyclic AMP was the most effective inhibitor. At a concentration of 10e5 M, cyclic AMP, cyclic IMP, cyclic GMP, cyclic UMP, and cyclic CMP caused an inhibition of 97, 70, 16, 9, and O%, respectively. The effect of pH on autophosphorylation of the protein kinase was also studied. In the presence of 2.5 mM ZnCl,, an inhibitory effect of cyclic AMP was observed over the whole pH range tested (4.0-9.0). In the presence of 10 mM MgC12, there was a broad optimum for the autophosphorylation reaction, from pH 5.5 to pH 8.5, with no cyclic AMP dependence. Comparison
of R-protein
and Protein II.
In a previous paper (14) it was shown that
.: OP-----i
557
OF BRAIN PROTEIN KINASE
M - I (0.9)
Purified Protein Kinose
M -I (I.01
nr-7 + -
+
‘+
-
cyclic AMP origin
BPB front
FIG. 5. Comparison of the electrophoretic migration of R-protein with that of Protein II from synaptic membrane fractions M-l (0.9) and M-l (1.0) of bovine cerebrum. Twenty-two micrograms of cyclic AMPdependent protein kinase, 25Opg of M-l (0.9), and 113 pg of M-l (1.0) were subjected to autophosphorylation with [-r-*T]ATP in the absence or presence of 5 PM cyclic AMP under standard assay conditions, as described in the text, except for the presence of 2.5 mM ZnCl, instead of MgCl,. After termination of the reaction by the addition of DodSO,-, the mixtures were subject to slab gel electrophoresis and autoradiography. The photograph shows the autoradiograph of the dried gel on X-ray film. The minus and plus signs indicate the absence or presence of cyclic AMP in the incubation mixture. BPB, bromphenol blue.
the endogenous phosphorylation of a protein (Protein II), of molecular weight 48,000, from synaptic membrane fractions M-l (0.9) and M-l (1.0) was regulated by cyclic AMP. Since the molecular weight of R-protein appeared similar to that of Protein II, the 32P-phosphorylated proteins were subjected to electrophoresis on the Cancmption Of Cyclic AMP (-lop M) same polyacrylamide gel in the presence of FIG. 4. Effect of varying the concentration of cy- DodSO,- in order to compare directly the clic AMP on the autophosphorylation reaction. Autoelectrophoretic migration of these proteins. phosphorylation of 11 rg of protein kinase with The autoradiogram of this gel is shown in [y-s*P]ATP was performed under standard assay Fig. 5, where it can be seen that these conditions, as described in the text, except for the variation in the concentration of cyclic AMP and the proteins ran in identical positions. In addireplacement of 10 mM MgCl, by 2.5 mru CaCI, or tion, these proteins showed the same response to cyclic AMP in the presence of ZnCl, as indicated.
558
MAENO ET AL.
Zna+. However, unlike the autophosphorylation reaction studied in the present investigation, the phosphorylation of Protein II was greatly stimulated by cyclic AMP in the presence of Mg2+ or Mn2* (14). Also, the phosphorylation of Protein II exhibits a sharp pH maximum at pH 6.2 (14), unlike the autophosphorylation reaction of the purified protein kinase. DISCUSSION
The data presented in this paper suggest that a protein kinase from mammalian brain uses a subunit of itself as an endogenous substrate. On polyacrylamide disc gel electrophoresis, protein kinase activity using histone as substrate, endogenous phosphorylation, and cyclic AMP-binding activity were inseparable. In addition, the rate of endogenous phosphorylation varied linearly with enzyme concentration in the absence of cyclic AMP (data not shown), suggesting that the enzyme and substrate were part of a complex over the range of concentrations studied. It was also found that cyclic AMP bound to the phosphorylated substrate and dissociated it from the holoenzyme (Fig. 2). These observations lead us to conclude that the regulatory subunit of the brain protein kinase is an endogenous substrate of this enzyme. There are some similarities and some differences between the endogenous phosphorylation and the phosphorylation of exogenous substrates by the enzyme with regard to the effect of divalent metal ions. It was found by Miyamoto et al. (15) that cyclic AMP stimulated histone kinase activity of the enzyme in the presence of Mg2+, Co2+, or Mn2+, but inhibited histone kinase activity in the presence of certain concentrations of Zna+, Fea+, Ca2+, Cu+, or Cu2+. In contrast to the phosphorylation of exogenous substrates such as histone, the autophosphorylation was not significantly affected by cyclic AMP in the presence of Mg2+. In the presence of a number of other divalent metal ions, including Zn2+, Ca2+, Mna+, Ni2+, Cu2+, and Fez+, cyclic AMP inhibited the endogenous phosphorylation. The pH dependence of the autophosphorylation reaction contrasts with that observed for histone phosphorylation. The
rate of autophosphorylation in the presence of Mga+ is almost independent of pH in the pH range 5.5-8.5; in contrast, the enzyme exhibits a fairly sharp maximum at pH 6.2 with exogenous substrates (12). The extent to which the state of dissociation of the subunits of protein kinase may account for the autophosphorylation data observed in the present study needs to be investigated. For example, it seems possible that the inhibition by cyclic AMP of the autophosphorylation reaction, which occurs in the presence of certain divalent metal ions, results from a cyclic AMPinduced dissociation of the enzyme. However, it is difficult to test thispossibility, since the techniques necessary to ascertain the state of dissociation, such as sucrose density-gradient centrifugation, require much longer periods of interaction of the enzyme, cyclic nucleotide, and other reagents than do the enzymological measurements carried out in the present study. We have also considered the possibility that the inhibition of autophosphorylation by cyclic AMP is due to the activation of an endogenous protein phosphatase. However, Zn2+ is known to inhibit protein phosphatase (3,24), and the most striking effect of cyclic AMP on the autophosphorylation reaction is observed in the presence of Zn2+. Also, the purified protein kinase exhibited no detectable protein phosphatase activity, in the absence or presence of cyclic AMP, either using protamine phosphate as substrate according to the method of Maeno and Greengard (3), or using endogenously labeled R-protein as substrate, according to the method of DeLorenzo and Greengard (24). It has recently been reported that cyclic AMP-dependent protein kinases from human erythrocytes (25) and from bovine heart (26,27) can phosphorylate their own regulatory subunits. However, the effect of cyclic AMP on the autophosphorylation of those enzymes was apparently not investigated. It would be of interest to know if the erythrocyte and heart preparations show the same characteristics, with respect to cyclic AMP and divalent metal ions, as the brain enzyme. As mentioned previously, there are some
AUTOPHOSPHORYLATION
intriguing similarities between the autophosphorylation reaction and the endogenous phosphorylation of Protein II, a protein present in synaptic membrane fractions (14). The two proteins run with the same mobility on DodSO,--polyacrylamide gels and have the same response to cyclic AMP in the presence of Zn2+ and certain other metal ions. However, a critical evaluation of the possible identity of the two proteins must await solubilization and purification of the membrane-bound Protein II.
1. 2. 3. 4.
5. 6. 7.
8.
9.
10. 11.
OF BRAIN
PROTEIN
KINASE
559
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