ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
183,
480-489
(1977)
Enzyme Systems Involved in Phosphorylation and Dephosphorylation of Two Endogenous Phosphoproteins in Neuronal Membranes’ ISA0 Department
UNO,
TETSUFUMI
of Pharmacology, New
UEDA,”
AND
PAUL
Yale University School of Medicine, Haven, Connecticut O&51 0 Received
April
GREENGARD 333 Cedar
Street,
1, 1977
Adenosine 3’:5’-monophosphate-dependent protein kinase and phosphoprotein phosphatases were solubilized by Triton X-100, from a particulate fraction of bovine cerebral cortex enriched in synaptic membranes, and partially purified. The properties of these partially purified enzymes were studied using two substrates, Protein I and Protein II, prepared from the synaptic membrane fraction, as well as the substrates protamine and histone. The results suggest that the phosphorylation of Protein I and Protein II, as well as protamine and histone, are catalyzed by a single species of CAMP-dependent protein kinase. Thus, a single peak of protein kinase activity was observed, upon DEAEcellulose chromatography of the Triton X-100 extract of the synaptic membrane preparation, which catalyzed the phosphorylation of all four substrate proteins. Moreover, the activity of this partially purified protein kinase toward the various substrate proteins was altered in a parallel fashion, either when the protein kinase preparation was subjected to heat inactivation or pH inactivation, or when the enzyme was assayed in the presence of various concentrations of cyclic nucleotides or of a protein kinase modulator. The individual protein substrates acted as competitive inhibitors with respect to one another. Upon sucrose density gradient centrifugation, the protein kinase activity toward the various substrates sedimented as a single peak. Finally, the relative specific activities toward the various substrates did not change significantly during a ZOOO-fold purification of the enzyme. In contrast to these observations with protein kinase, two peaks of protein phosphatase activity, with markedly different specificities toward Protein I and Protein II, were found upon DEAE-cellulose and Bio-Gel P-200 column chromatography of the Triton X-100 extract of the synaptic membrane fractions. One peak catalyzed the dephosphorylation of Phosphoprotein I but not of Phosphoprotein II, whereas the other peak catalyzed the dephosphorylation of Phosphoprotein II but not of Phosphoprotein I. The dephosphorylation of Phosphoprotein I by Phosphoprotein I phosphatase was not affected by adenosine 3’:5’-monophosphate, whereas the dephosphorylation of Phosphoprotein II by Phosphoprotein II phosphatase required the presence of this nucleotide. Moreover, the two phosphatases differed from one another with respect to Stokes’ radius as well as sedimentation coefficient.
The adenosine 3’:5’-monophosphate3-dependent phosphorylation, by endogenous
protein kinase, of two endogenous substrate proteins, designated Protein I and Protein II, has been demonstrated in preparations of nervous tissue enriched in synaptic membranes (1, 2). Moreover, Protein I (3) and Protein II (41, as well as Protein I kinase (5), Protein II kinase (4), and Pro&in 11 phosphatase (4), activities have been solubilized from such membrane preparations. The studies reported in this paper were undertaken to determine whether Protein I kinase and Protein II kinase represent the same or different spe-
’ This study was supported by United States Publit Health Service Grants MH-17387 and NS-08440. 2 Recipient of a Postdoctoral Fellowship from the National Institute of Mental Health, Fellowship No. MH-00270. ” Abbreviations used: CAMP, adenosine 3’:5’monophosphate; cGMP, guanosine 3’:5’-monophosphate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid; PIPES, piperazine-NJ’-bis(2ethanesulfonic acid); 8-N,-CAMP, 8-azido-adenosine 3’:5’-monophosphate; DEAE, diethylaminoethyl; DTT, dithiothreitol; SDS, sodium dodecyl sulfate. 480 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9961
PROTEIN
KINASE
AND
PHOSPHATASE
ties of CAMP-dependent protein kinase, whether Protein I phosphatase activity could be demonstrated in extracts of synaptic membrane preparations and, if so, whether Protein I phosphatase and Protein II phosphatase represent the same or different species of phosphoprotein phosphatase. EXPERIMENTAL
PROCEDURES
Materials. Fresh calf brains were obtained from a local slaughterhouse and transported in ice to the laboratory. Cyclic nucleotides were purchased from Schwarz/Mann. DEAE-cellulose 52 and phosphocellulose paper (P81) were from Whatman. Histone mixture and protamine were from Sigma. Bio-Gel P200 was from Bio-Rad. Protein I, purified through step 7 to apparent homogeneity, was prepared as described by Ueda and Greengard (31. Protein I kinase, purified through step 9 to apparent homogeneity, was prepared as described by Uno et al. (5). [y:“PlATP was prepared by the method of Post and Sen (61. lR2P1cAMP was purchased from New England Nuclear. 1:‘YPl-8-N,-cAMP was prepared by a slight modification of the micromethod described by Haley (7). Preparation of fractions enriched in synaptic membranes; Triton X-100 extraction. Synaptic membrane fractions, designated M-l, M-l (0.91, and M-l (1.01, were prepared from bovine cerebral cortex, as described previously (31. In preliminary experiments, Triton X-100 extracts of the purified synaptic membrane preparations, M-l (0.9) and M-l (1.01, were shown to yield results similar to those obtained using Triton X-100 extracts of the crude synaptic membrane fraction, M-l, with regard to the chromatographic separation of Protein I kinase, Protein II kinase, Protein I phosphatase, and Protein II phosphatase. Therefore, the M-l fraction was used for Triton X-100 extraction in all experiments to be described. To prepare the Triton X-100 extract, the M-l fraction was incubated at 4°C for 30 min in the presence of 0.1% Triton X-100 and 10m4 M DTT and centrifuged at 150,OOOg for 30 min, as described previously (4). The resulting supernatant fluid was used as the Triton extract. Protein kinase assay. Protein I kinase activity, Protein II kinase activity, protamine kinase activity, and histone kinase activity were measured in the presence of CAMP by methods described elsewhere (5). Preparation of [“2P/Phosphoprotein I and [:“p/Phosphoprotein ZZ. To prepare substrate for use in the assay of Phosphoprotein I phosphatase and Phosphoprotein II phosphatase, Protein I and Protein II were phosphorylated by incubating with ly““PIATP in the presence of CAMP-dependent Protein I kinase. The incubation mixture contained, in a
FROM
NEURONAL
MEMBRANES
481
final volume of 1 ml: 50 kmol of HEPES buffer (pH 7.4); 10 units of Protein I kinase [purified through step 4 (511; 200 wg of Protein I [purified through step 4 (311 or 100 pg of a partially purified preparation of Protein II (described below); 1 nmol of l-y-“?PlATP (about 1 to 4 x 10’ cpm); 10 pmol of magnesium chloride; and 10 nmol of CAMP. The reaction was carried out at 3O”C, for 60 min in the case of Protein I and 20 min in the case of Protein II. The reaction was terminated by the addition of 100 nmol of unlabeled ATP and 20 pmol of EDTA. The reaction mixture was dialyzed at 4”C, first against 1.5 liter of 100 mM potassium phosphate buffer (pH 7.01, containing lo-’ M DTT, 5 x lOmy M EDTA, and lo-” M cold ATP for 4 h, and then against 1.5 liter of 100 mM potassium phosphate buffer (pH 7.0) containing 10 ’ M DTT twice for 8 h. Phosphoprotein I phosphatase assay. The standard reaction mixture for the Phosphoprotein I phosphatase assay contained, in a final volume of 100 ~1, 40 ng of [“‘PlPhosphoprotein I (about 6000 cpm); 5 pmol of PIPES buffer (pH 7.01; and 1 pmol of magnesium chloride. The reaction was carried out at 30°C for 20 or 60 min, and terminated by the addition of 50 ~1 of “SDS-stop solution” (31. Radioactivity remaining in the Protein I band was measured by sodium dodecyl sulfate-gel electrophoresis and autoradiography as described in the assay method for Protein I (3). One unit of enzyme activity was defined as the amount of enzyme which removed 1 pmol of [“‘PIphosphate from 1”2P1Phosphoprotein I in 20 min at 30°C. Enzyme activity was proportional to the amount of enzyme and time of incubation, under the experimental conditions used. Phosphoprotein II phosphatase assay. Phosphoprotein II phosphatase activity was measured by the method used to assay Phosphoprotein I phosphatase activity, except that 20 ng of [“‘PJPhosphoprotein II (about 3000 cpml was used as substrate, the incubation time was 20 min, and 10 p,M CAMP was present in the reaction mixture. One unit of enzyme activity was defined as the amount of enzyme which removed 1 pmol of [32Plphosphate from I:12P]Phosphoprotein II in 20 min at 30°C. Enzyme activity was proportional to amount of enzyme and time of incubation, under the experimental conditions used. Protumine phosphatase assay. Protamine phosphatase activity was assayed by measuring the release of radioactive orthophosphate from [“‘Plphosphoprotamine, as described elsewhere (8). Protein II assay. The amount of Protein II was measured by the method for assaying Protein I (31, except that 0.65 units of Protein I kinase (containing trace amounts of Protein II) was used, and the incubation time was 2 min. Photoaffinity labeling with 8-N,cAMP. Photoaffinity labeling experiments, in which [:j2P]-8-N,CAMP was incorporated covalently into protein kinase regulatory subunit, were carried out by the
482
UNO,
UEDA,
AND
method of Pomerantz et al. (91, except that sodium dodecyl sulfate-gel electrophoresis and autoradiography were carried out as described elsewhere (3). Preparation of a Protein II fraction free from Protern II kinase and Phosphoprotein II phosphataw. Ten milliliters (1.5 mg of protein/ml) of the 0.1% Triton extract of the M-l fraction, in 10 mM potassium phosphate buffer tpH 7.0) containing 10 ’ M DTT, were applied to a DEAE-cellulose column (0.9 x 30 cm), which had previously been equilibrated with the same buffer containing 10 ’ M DTT. Proteins were eluted from the column with a linear gradient of phosphate buffer (10 to 500 mM), pH 7.0. in 10 ’ M DTT, using a total volume of 200 ml. Fractions (2.5 ml each) were collected and assayed for Protein II, Protein II kinase activity, and Phosphoprotein II phosphatase activity, as described above. Using this procedure, Protein II was prepared essentially free from Protein II kinase and Phosphoprotein II phosphatase (Fig. 11. Those fractions containing the highest amounts of Protein II (Fractions 57 through 63) were combined and used for the studies of Protein II kinase and Protein II phosphatase activity described in this paper. The amount of Protein 11 present in this partially purified preparation was determined by measuring the maximal incorporation of radioactive phosphate. Evidence presented elsewhere (4, 10, 111 suggested that the Protein II band observed in synaptic membrane fractions represented the regulatory subunit of a CAMP-dependent protein kinase present in these membranes. In support of that suggestion, it was found in the present study that Protein II present in the partially purified preparation had the same electrophoretic mobility as the regulatory sub-
10
20
30 FRACTION
GREENGARD unit of purified membrane-derived CAMP-dependent protein kinase (Fig. 2) when the two proteins were subjected to sodium dodecyl sulfate gel ctlcctrophoresis, as determined both by phosphorylation and by incorporation of the photoaffinity label 8-N,,-cAMP Moreover, the ability of the regulatory subunit of the purified synaptic membrane-derived protein kinasc to inhibit the catalytic subunit of the synaptic membrane-derived protein kinase. but not the cata lytic subunit of brain cytosol or heart cytosol protein kinase (see Ref. 15), Table VIII, was mimicked by the partially purified preparation of Protein II (data not shown:. Sucrose dr~nsity gradient centrifugatiou. Sucrose density gradient centrifugation of protein kinasc was carried out as described by Martin and Ames 1121, except that 50 mM potassium phosphate buffer was used, 0.1 mM DTT was present, and centrifugation was carried out for 17 h. Prrparation of protc~in kinasc modulator. Protein kinase modulator was prepared from bovine brain by the method of Donnelly ct al. (13). Protein concentration was determined by the method of Lowry et nl. 114) with bovine serum albumin as standard.
Protein
RESULTS
AND
DISCUSSION
I Kinase
and Protein
II Kinase
DEAE-cellulose column chromatography. When a Triton extract of the M-l synaptic membrane fraction was subjected to DEAE-cellulose column chromatography, single peaks of Protein I kinase activ-
40
50
60
70
NUMBER
FIG. 1. Separation of Protein II, Protein II kinase, and Phosphoprotein II phosphatase by DEAE-cellulose column chromatography. A Triton extract of the M-l membrane fraction was subjected to DEAE-cellulose column chromatography, and aliquots of each fraction (2.5 ml) were analyzed for Protein II (x-x), Protein II kinase activity in the absence (CI--0) or presence (W-W) of CAMP, and Phosphoprotein II phosphatase activity in the absence (a-& or presence (A-A) of CAMP. All values are expressed per fraction
PROTEIN
AND
PHOSPHATASE
CAMPBindkng
Phosphorylatcon PK
KINASE
Pll
PK
PII Orlgln
t-
52 .OOO
FIG. 2. Autoradiograph of a sodium dodecyl sulfate-polyacrylamide gel showing the incorporation of a2P from ly-“‘PJATP (left) and of l:‘2P]-8-N,-cAMP (right) into the protein kinasc regulatory subunit (PK) and Protein II (PH. Membrane-derived protein kinase (2.0 units, 0.80 ng), purified through step 9 (51, was subjected to autophosphorylation under standard conditions (5). Protein II (5.2 ng) was phosphorylated for 1 min with 0.02 units of Protein II kinase in the presence of 10 pM CAMP under standard conditions (51. Other aliquots of protein kinase (5.2 ngl and Protein II (2.8 ng) were incubated with 0.2 FM l:“P]-8-N,-CAMP for photoaffinity labeling, as described under Experimental Procedures. These four preparations were then subjected to gel electrophoresis, with the results shown above.
ity, Protein II kinase activity, protamine kinase activity, and histone kinase activity were obtained, and all of these peaks were eluted in the same position, as shown in Fig. 3. The most active fractions (Fractions 40 through 46) were pooled. This partially purified enzyme preparation was used for all further studies of protein kinase activity, except where indicated. Sucrose density gradient centrifugation. When this partially purified preparation of protein kinase was subjected to sucrose density gradient centrifugation, Protein I kinase activity, Protein II kinase activity, and protamine kinase activity again appeared in the same position (data not shown), suggesting that the phosphorylation of Protein I, Protein II, and protamine might be catalyzed by a single species of protein kinase. Inactivation by pH and by heat. Pre-
FROM
NEURONAL
MEMBRANES
483
treatment of the partially purified preparation of protein kinase at various pH values for 10 min at 30°C led to similar losses of Protein I kinase, Protein II kinase, and protamine kinase activity (Fig. 4). Moreover, pretreatment of this protein kinase preparation for various periods of time at 45°C caused parallel losses of Protein I kinase, Protein II kinase, and protamine kinase activity (Fig. 51. In addition, pretreatment of this enzyme preparation at various temperatures from 35 to 50°C for 10 min also caused parallel losses of the three types of enzyme activity (data not shown). Inhibition by modulator. The effect of various concentrations of a protein kinase modulator from brain (13) on Protein I kinase, Protein II kinase, and protamine kinase activity was examined in the presence of 10 pM CAMP, using 0.25 units of Protein I kinase activity. All three types of enzyme activity were inhibited approximately in parallel by increasing concentrations of the modulator. Fifty percent inhibition was obtained with 0.35 to 0.50 mg of modulator/ml (data not shown). Effect of various concentrations of CAMP and cGMP. The effect of various concentrations of CAMP and cGMP on Protein I kinase, Protein II kinase, and protamine kinase activity is shown in Fig. 6. CAMP stimulated the three types of protein kinase activity in a similar fashion. At higher concentrations, cGMP also stimulated the three types of enzyme activity in a similar fashion. The concentrations of CAMP and cGMP required for half-maximal activation were approximately 1 x 10 ’ and 5 x lo-” M, respectively, for all three types of protein kinase substrate. Substrate specificity. The substrate specificity of the membrane-derived protein kinase, at various stages of a 2000-fold purification, is shown in Table I. The relative specific activities of the protein kinase toward Protein Ia, Protein Ib, Protein II, protamine, and histone did not change significantly throughout the entire purification procedure. Competitive inhibition by alternative substrate proteins. In experiments in which Protein I and Protein II were both
484
UNO,
UEDA,
AND
GREENGARD
FIG. 3. Elution profile of Protein I kinase, Protein II kinase, protamine kinase, and histone kinase activity upon DEAE-cellulose column chromatography. Ten milliliters (1.5 mg of protein/ml) of a Triton extract of the M-l fraction, in 100 rnM potassium phosphate buffer (pH 7.0)-IO-” M DTT, were applied to a DEAE-cellulose column (0.9 x 30 cm) previously equilibrated with the same buffer. Protein kinase activity was eluted with a linear gradient of phosphate buffer (100 to 400 mM), pH 7.0, in lo- ’ M DTT, using a total volume of 200 ml. Fractions (2.5 ml each) were collected and aliquots were assayed for protein kinase activity using four different substrates, in the absence (open symbols) or presence (filled symbols) of 10 WM CAMP. All values are expressed per fraction.
4
FIG. 4. pH inactivation of Protein I kinase, Protein II kinase, and protamine kinase activity. The partially purified preparation of protein kinase (2ml aliquot) was dialyzed overnight against 1 liter of 10 mM potassium phosphate buffer (pH 7.0) containing 10el M DTT. Aliquots (100 ~11, containing 5.1 units of Protein I kinase activity, were added to 100 ~1 of each of the following buffer solutions (50 mM): (1) sodium acetate buffer (pH 4.5, 5.0, and 5.5); (2) potassium phosphate buffer (pH 6.0, 6.5, and 7.0); and (3) Tris-HCl buffer (pH 7.5, 8.0, and 9.0). Each enzyme solution was incubated at 30°C for 10 min, cooled to o”C, and mixed with 1.8 ml of 400 rnM potassium phosphate buffer (pH 7.0). Aliquots (20 ~1) of each of the resulting enzyme solutions were then assayed for Protein I kinase (A), Protein II kinase (01 and protamine kinase (0) activity in the presence of 10 +M CAMP. Protein kinase activity is expressed as percentage of that found in the sample which had been preincubated at pH 7.0.
FIG. 5. Thermal inactivation of Protein I kinase, Protein II kinase, and protamine kinase activity. The partially purified preparation of protein kinase (l-ml aliquot containing 2.5 units of Protein I kinase and 150 pg of protein) was preincubated at 45°C for various periods of time. Aliquots (20 ~1) of each sample were then assayed for Protein I kinase (al, Protein II kinase (D), and protamine kinase (0) activity, in the presence of 10 FM CAMP. Protein kinase activity is expressed as log of the percentage of the activity found in samples which had not been preincubated.
present, they acted as alternative strates for the protein kinase. Thus, uring the rate of phosphorylation of of these proteins, the other acted apparent competitive inhibitor. In tion, protamine acted as an apparent
submeaseither es an ::ddicom-
PROTEIN
KINASE
AND
PHOSPHATASE
petitive inhibitor both of Protein I and Protein II phosphorylation. The Ki values obtained in these experiments (Table II) agreed closely with the K,,, values (see 5) for the same proteins acting as substrates for the purified protein kinase. These findings suggest that the same enzyme can catalyze the phosphorylation of all of these proteins. The data also indicate that Protein I and Protein II can interact with the
FROM
NEURONAL
same catalytic site on the protein kinase catalytic subunit. Since Protein II seems to be the regulatory subunit of this protein kinase (see Experimental Procedures), these results support the suggestion of Witt and Roskoski (16) that the regulatory subunit shields the active site of the catalytic subunit thereby inhibiting catalytic activity. In conclusion, the variety of experimental results presented above strongly suggest that the same species of protein kinase can catalyze the phosphorylation of Protein Ia, Protein Ib, Protein II, protamine, and histone. TABLE K, VALUES Protein
II
OF PROTEIN I KINASE FOR PROTEIN PROTEIN II, AND PROTAMINE”
used as inhibitor
Protein
CYCLIC
NUCLEOTIDES
(-log
(/AM)
SPECIFICITY
PROTEIN
KINASE
Substrate Protein
M-l fraction Triton extract Triton supernatant First DEAE-cellulose column eluate Ammonium sulfate precipitate Bio-Gel P-200 column eluate Second DEAE-cellulose column eluate Sepharose 4B column eluate Hydroxylapatite column eluate
II
0.88 6.5
I
OF MEMBRANE-DERIVED
Step
1. 2. 3. 4. 5. 6. 7. 8. 9.
0.92 6.2
Protein
” The K, values shown were calculated from Dixon plots (15) which were linear at various fixed substrate concentrations. Similar K, values were obtained from Lineweaver-Burk plots (15), which were linear at various fixed inhibitor concentrations. The range of concentrations of substrates and inhibitors varied from 4 x lo-!’ to 4 x 10 i M.
TABLE SUBSTRATE
I
-
Protein I Protein II Protamine
Ml
I,
used as substrate
Protein
FIG. 6. Stimulation by various concentrations of CAMP and cGMP of Protein I kinase, Protein II kinase, and protamine kinase activity. Protein I kinase (a, A), Protein II kinase (0, W, and protamine kinase (0, 0) activities were measured in the presence of various concentrations of CAMP (open symbols) or cGMP (closed symbols). Protein kinase activity is expressed as percentage of that found in the presence of 10 PM CAMP.
485
MEMBRANES
100 85 91 96 98 92 70 85 87
Ia Protein
DURING
(relative Ib
Protein
ITS PURIFICATION”
specific
activity)
II Protamine
Histone
100 163 17 11 100 150 20 15 100 162 18 12 100 150 17 12 100 155 17 13 100 150 16 11 100 167 18 13 100 160 18 11 100 ___. 163 17 12 (1 Bovine brain membrane-derived protein kinase was solubilized and purified to homogeneity, as described elsewhere (5). At each step of the purification procedure, substrate specificity of the enzyme was determined, using 5 pg of Protein I (1.6 pg of Protein Ia and 3.4 pg of Protein Ib) purified through step 7 (3), 7.0 pg of Protein II, 5.0 fig of protamine, or 5.0 pg of histone, as substrate. The phosphorylation of each substrate was carried out in the presence of 10 ~.LM CAMP, and the phosphorylated protein analyzed by sodium dodecyl sulfate gel electrophoresis and liquid scintillation spectrometry, as in the standard Protein I kinase assay method. At each step of enzyme purification, the moles of phosphate incorporated into each substrate is expressed as a percentage of that incorporated into Protein Ib.
486
UNO,
Phosphoprotein I Phosphatase phoprotein II Phosphatase
UEDA,
AND
and Phos-
DEAE-cellulose column chromatography. When a Triton extract of the M-l membrane fraction was subjected to column chromatography on DEAE-cellulose, two peaks of protein phosphatase activity were observed (Fig. 7). The first peak catalyzed the dephosphorylation of phosphoprotamine and Phosphoprotein II, but not of Phosphoprotein I. The second peak catalyzed the dephosphorylation of phosphoprotamine and Phosphoprotein I, but not of Phosphoprotein II. The rate of dephosphorylation of Phosphoprotein II by the first peak was stimulated by CAMP (Fig. 7); in contrast, the rate of dephosphorylation of Phosphoprotein I by the second peak, and the dephosphorylation of phosphoprotamine by either peak of activity, was unaffected by CAMP (data not shown). The most active fractions of Phosphoprotein I phosphatase (Fractions 22 through 28) and Phosphoprotein II phosphatase (Fractions 6 through 12) were separately pooled and concentrated to 1.0 ml by means of ultrafiltration on a Diaflo mem-
GREENGARD
brane (PM101 under pressure of nitrogen gas. Bio-Gel P-200 gel filtration. When the partially purified preparations of phosphoprotein phosphatase, obtained by DEAEcellulose column chromatography, were separately subjected to Bio-Gel P-200 column chromatography, Phosphoprotein I phosphatase activity, and Phosphoprotein II phosphatase activity were eluted in different positions (Fig. 8). The Stokes’ radii for Phosphoprotein I phosphatase and Phosphoprotein II phosphatase, calculated from the elution position by the method of Siegel and Monty (17), using appropriate marker proteins (3), were 55 and 44 A, respectively. The most active fractions of Phosphoprotein I phosphatase (Fractions 18 through 22) and of Phosphoprotein II phosphatase (Fractions 21 through 25) were separately pooled and concentrated to 1.0 ml by means of ultrafiltration on a Diaflo membrane (PM101 under pressure of nitrogen gas. These partially purified enzyme preparations were used for all further studies of protein phosphatase activity. Effect of CAMP on Phosphoprotein II
FIG. 7. Separation of Phosphoprotein I phosphatase from Phosphoprotein II phosphatase activity by DEAE-cellulose column chromatography. Ten milliliters (1.5 mg of protein/ml) of a Triton extract of the M-l fraction, in 100 mM potassium phosphate buffer (pH 7.0)-lOma M DTT, were applied to a DEAE-cellulose column (0.9 x 30 cm) previously equilibrated with the same buffer. Phosphoprotein phosphatase activity was eluted with a linear gradient of phosphate buffer (100 to 400 mM), pH 7.0, in 10. ’ M DTT, using a total volume of 200 ml. Fractions (2.5 ml each) were collected and aliquots were assayed for phosphoprotamine phosphatase activity (0- - -0) and Phosphoprotein I phosphatase activity (A- - -A1 in the absence of CAMP, and for Phosphoprotein II phosphatase activity in the absence (0 - -0) or presence (m-m) of 10 pM CAMP. All values are expressed per fraction.
PROTEIN
KINASE
AND
PHOSPHATASE
FROM
FIG. 8. Bio-Gel P-200 gel filtration of Phosphoprotein I phosphatase and Phosphoprotein II phosphatase activity. (A) Phosphoprotein I phosphatase (1.0 ml containing 21 units) and (B) Phosphoprotein II phosphatase (1.0 ml containing 72 units) obtained by DEAE-cellulose column chromatography were separately applied to Bio-Gel P-200 columns (0.9 60 cm), which had been equilibrated with 10 mM Tris-HCl buffer (pH 7.4). The columns were eluted with the same buffer, and l.O-ml fractions were collected. Aliquots were assayed for Phosphoprotein I phosphatase (a) and phosphoprotamine phosphatase (0) activity in the absence of CAMP, and for Phosphoprotein II phosphatase (W) activity in the presence of 10 CAMP. All values are expressed per fraction. x
FM
TABLE OF CAMP
Phosphatase
peak
PHOSPHATASE
-
ACTIVITY”
Substrate
-CAMP (units I phosphatase II phosphatase
III
ON PHOSPHOPROTEIN
Phosphoprotein
Phosphoprotein Phosphoprotein
487
MEMBRANES
on CAMP (Table III). In contrast, phosphoprotamine phosphatase activity, which cochromatographed with CAMP-dependent Phosphoprotein II phosphatase activity during the Bio-Gel P-200 gel filtration step (Fig. 8), was not affected by CAMP (Table III). Moreover, no effect of CAMP was observed on the dephosphorylation of either Phosphoprotein I or phosphoprotamine by the Phosphoprotein I phosphatase peak. [ :‘H]cAMP-binding activity could not be detected in the Bio-Gel P-200 preparation of Phosphoprotein II phosphatase nor was photoaffinity labeling of this preparation by B-N,,-CAMP observed (data not shown); in contrast, Phosphoprotein II itself is a CAMP-binding protein, and the regulatory subunit of membrane-bound protein kinase, as described under Experimental Procedures. These results support the suggestion (11, 18, 19) that CAMP causes an increased accessibility of Phosphoprotein II to Phosphoprotein II phosphatase, by acting directly on Phosphoprotein II, with a resultant stimulation of the Phosphoprotein II phosphatase reaction. Sucrose density gradient centrifugation. When the preparations of phosphoprotein phosphatase purified through the Bio-Gel P-200 gel filtration step were separately subjected to sucrose density gradient centrifugation, Phosphoprotein I phosphatase activity and Phosphoprotein II phosphatase activity appeared in different positions (Fig. 9). The positions of the two activities corresponded to .s~,,,~ values of 7.1 and 5.0 S, respectively. The molecular weights of Phosphoprotein I phosphatase and Phosphoprotein II phosphatase, calculated from their Stokes’
phosphatase activity. As was observed with the Phosphoprotein II phosphatase activity peak from the DEAE-cellulose column (Fig. 71, the dephosphorylation of Phosphoprotein II by Phosphoprotein II phosphatase purified through the Bio-Gel P-200 step showed an absolute dependence
EFFECT
NEURONAL
24
OF
BIOCHEMISTRY
AND
BIOPHYSICS
183,
480-489
(1977)
Enzyme Systems Involved in Phosphorylation and Dephosphorylation of Two Endogenous Phosphoproteins in Neuronal Membranes’ ISA0 Department
UNO,
TETSUFUMI
of Pharmacology, New
UEDA,”
AND
PAUL
Yale University School of Medicine, Haven, Connecticut O&51 0 Received
April
GREENGARD 333 Cedar
Street,
1, 1977
Adenosine 3’:5’-monophosphate-dependent protein kinase and phosphoprotein phosphatases were solubilized by Triton X-100, from a particulate fraction of bovine cerebral cortex enriched in synaptic membranes, and partially purified. The properties of these partially purified enzymes were studied using two substrates, Protein I and Protein II, prepared from the synaptic membrane fraction, as well as the substrates protamine and histone. The results suggest that the phosphorylation of Protein I and Protein II, as well as protamine and histone, are catalyzed by a single species of CAMP-dependent protein kinase. Thus, a single peak of protein kinase activity was observed, upon DEAEcellulose chromatography of the Triton X-100 extract of the synaptic membrane preparation, which catalyzed the phosphorylation of all four substrate proteins. Moreover, the activity of this partially purified protein kinase toward the various substrate proteins was altered in a parallel fashion, either when the protein kinase preparation was subjected to heat inactivation or pH inactivation, or when the enzyme was assayed in the presence of various concentrations of cyclic nucleotides or of a protein kinase modulator. The individual protein substrates acted as competitive inhibitors with respect to one another. Upon sucrose density gradient centrifugation, the protein kinase activity toward the various substrates sedimented as a single peak. Finally, the relative specific activities toward the various substrates did not change significantly during a ZOOO-fold purification of the enzyme. In contrast to these observations with protein kinase, two peaks of protein phosphatase activity, with markedly different specificities toward Protein I and Protein II, were found upon DEAE-cellulose and Bio-Gel P-200 column chromatography of the Triton X-100 extract of the synaptic membrane fractions. One peak catalyzed the dephosphorylation of Phosphoprotein I but not of Phosphoprotein II, whereas the other peak catalyzed the dephosphorylation of Phosphoprotein II but not of Phosphoprotein I. The dephosphorylation of Phosphoprotein I by Phosphoprotein I phosphatase was not affected by adenosine 3’:5’-monophosphate, whereas the dephosphorylation of Phosphoprotein II by Phosphoprotein II phosphatase required the presence of this nucleotide. Moreover, the two phosphatases differed from one another with respect to Stokes’ radius as well as sedimentation coefficient.
The adenosine 3’:5’-monophosphate3-dependent phosphorylation, by endogenous
protein kinase, of two endogenous substrate proteins, designated Protein I and Protein II, has been demonstrated in preparations of nervous tissue enriched in synaptic membranes (1, 2). Moreover, Protein I (3) and Protein II (41, as well as Protein I kinase (5), Protein II kinase (4), and Pro&in 11 phosphatase (4), activities have been solubilized from such membrane preparations. The studies reported in this paper were undertaken to determine whether Protein I kinase and Protein II kinase represent the same or different spe-
’ This study was supported by United States Publit Health Service Grants MH-17387 and NS-08440. 2 Recipient of a Postdoctoral Fellowship from the National Institute of Mental Health, Fellowship No. MH-00270. ” Abbreviations used: CAMP, adenosine 3’:5’monophosphate; cGMP, guanosine 3’:5’-monophosphate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid; PIPES, piperazine-NJ’-bis(2ethanesulfonic acid); 8-N,-CAMP, 8-azido-adenosine 3’:5’-monophosphate; DEAE, diethylaminoethyl; DTT, dithiothreitol; SDS, sodium dodecyl sulfate. 480 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9961
PROTEIN
KINASE
AND
PHOSPHATASE
ties of CAMP-dependent protein kinase, whether Protein I phosphatase activity could be demonstrated in extracts of synaptic membrane preparations and, if so, whether Protein I phosphatase and Protein II phosphatase represent the same or different species of phosphoprotein phosphatase. EXPERIMENTAL
PROCEDURES
Materials. Fresh calf brains were obtained from a local slaughterhouse and transported in ice to the laboratory. Cyclic nucleotides were purchased from Schwarz/Mann. DEAE-cellulose 52 and phosphocellulose paper (P81) were from Whatman. Histone mixture and protamine were from Sigma. Bio-Gel P200 was from Bio-Rad. Protein I, purified through step 7 to apparent homogeneity, was prepared as described by Ueda and Greengard (31. Protein I kinase, purified through step 9 to apparent homogeneity, was prepared as described by Uno et al. (5). [y:“PlATP was prepared by the method of Post and Sen (61. lR2P1cAMP was purchased from New England Nuclear. 1:‘YPl-8-N,-cAMP was prepared by a slight modification of the micromethod described by Haley (7). Preparation of fractions enriched in synaptic membranes; Triton X-100 extraction. Synaptic membrane fractions, designated M-l, M-l (0.91, and M-l (1.01, were prepared from bovine cerebral cortex, as described previously (31. In preliminary experiments, Triton X-100 extracts of the purified synaptic membrane preparations, M-l (0.9) and M-l (1.01, were shown to yield results similar to those obtained using Triton X-100 extracts of the crude synaptic membrane fraction, M-l, with regard to the chromatographic separation of Protein I kinase, Protein II kinase, Protein I phosphatase, and Protein II phosphatase. Therefore, the M-l fraction was used for Triton X-100 extraction in all experiments to be described. To prepare the Triton X-100 extract, the M-l fraction was incubated at 4°C for 30 min in the presence of 0.1% Triton X-100 and 10m4 M DTT and centrifuged at 150,OOOg for 30 min, as described previously (4). The resulting supernatant fluid was used as the Triton extract. Protein kinase assay. Protein I kinase activity, Protein II kinase activity, protamine kinase activity, and histone kinase activity were measured in the presence of CAMP by methods described elsewhere (5). Preparation of [“2P/Phosphoprotein I and [:“p/Phosphoprotein ZZ. To prepare substrate for use in the assay of Phosphoprotein I phosphatase and Phosphoprotein II phosphatase, Protein I and Protein II were phosphorylated by incubating with ly““PIATP in the presence of CAMP-dependent Protein I kinase. The incubation mixture contained, in a
FROM
NEURONAL
MEMBRANES
481
final volume of 1 ml: 50 kmol of HEPES buffer (pH 7.4); 10 units of Protein I kinase [purified through step 4 (511; 200 wg of Protein I [purified through step 4 (311 or 100 pg of a partially purified preparation of Protein II (described below); 1 nmol of l-y-“?PlATP (about 1 to 4 x 10’ cpm); 10 pmol of magnesium chloride; and 10 nmol of CAMP. The reaction was carried out at 3O”C, for 60 min in the case of Protein I and 20 min in the case of Protein II. The reaction was terminated by the addition of 100 nmol of unlabeled ATP and 20 pmol of EDTA. The reaction mixture was dialyzed at 4”C, first against 1.5 liter of 100 mM potassium phosphate buffer (pH 7.01, containing lo-’ M DTT, 5 x lOmy M EDTA, and lo-” M cold ATP for 4 h, and then against 1.5 liter of 100 mM potassium phosphate buffer (pH 7.0) containing 10 ’ M DTT twice for 8 h. Phosphoprotein I phosphatase assay. The standard reaction mixture for the Phosphoprotein I phosphatase assay contained, in a final volume of 100 ~1, 40 ng of [“‘PlPhosphoprotein I (about 6000 cpm); 5 pmol of PIPES buffer (pH 7.01; and 1 pmol of magnesium chloride. The reaction was carried out at 30°C for 20 or 60 min, and terminated by the addition of 50 ~1 of “SDS-stop solution” (31. Radioactivity remaining in the Protein I band was measured by sodium dodecyl sulfate-gel electrophoresis and autoradiography as described in the assay method for Protein I (3). One unit of enzyme activity was defined as the amount of enzyme which removed 1 pmol of [“‘PIphosphate from 1”2P1Phosphoprotein I in 20 min at 30°C. Enzyme activity was proportional to the amount of enzyme and time of incubation, under the experimental conditions used. Phosphoprotein II phosphatase assay. Phosphoprotein II phosphatase activity was measured by the method used to assay Phosphoprotein I phosphatase activity, except that 20 ng of [“‘PJPhosphoprotein II (about 3000 cpml was used as substrate, the incubation time was 20 min, and 10 p,M CAMP was present in the reaction mixture. One unit of enzyme activity was defined as the amount of enzyme which removed 1 pmol of [32Plphosphate from I:12P]Phosphoprotein II in 20 min at 30°C. Enzyme activity was proportional to amount of enzyme and time of incubation, under the experimental conditions used. Protumine phosphatase assay. Protamine phosphatase activity was assayed by measuring the release of radioactive orthophosphate from [“‘Plphosphoprotamine, as described elsewhere (8). Protein II assay. The amount of Protein II was measured by the method for assaying Protein I (31, except that 0.65 units of Protein I kinase (containing trace amounts of Protein II) was used, and the incubation time was 2 min. Photoaffinity labeling with 8-N,cAMP. Photoaffinity labeling experiments, in which [:j2P]-8-N,CAMP was incorporated covalently into protein kinase regulatory subunit, were carried out by the
482
UNO,
UEDA,
AND
method of Pomerantz et al. (91, except that sodium dodecyl sulfate-gel electrophoresis and autoradiography were carried out as described elsewhere (3). Preparation of a Protein II fraction free from Protern II kinase and Phosphoprotein II phosphataw. Ten milliliters (1.5 mg of protein/ml) of the 0.1% Triton extract of the M-l fraction, in 10 mM potassium phosphate buffer tpH 7.0) containing 10 ’ M DTT, were applied to a DEAE-cellulose column (0.9 x 30 cm), which had previously been equilibrated with the same buffer containing 10 ’ M DTT. Proteins were eluted from the column with a linear gradient of phosphate buffer (10 to 500 mM), pH 7.0. in 10 ’ M DTT, using a total volume of 200 ml. Fractions (2.5 ml each) were collected and assayed for Protein II, Protein II kinase activity, and Phosphoprotein II phosphatase activity, as described above. Using this procedure, Protein II was prepared essentially free from Protein II kinase and Phosphoprotein II phosphatase (Fig. 11. Those fractions containing the highest amounts of Protein II (Fractions 57 through 63) were combined and used for the studies of Protein II kinase and Protein II phosphatase activity described in this paper. The amount of Protein 11 present in this partially purified preparation was determined by measuring the maximal incorporation of radioactive phosphate. Evidence presented elsewhere (4, 10, 111 suggested that the Protein II band observed in synaptic membrane fractions represented the regulatory subunit of a CAMP-dependent protein kinase present in these membranes. In support of that suggestion, it was found in the present study that Protein II present in the partially purified preparation had the same electrophoretic mobility as the regulatory sub-
10
20
30 FRACTION
GREENGARD unit of purified membrane-derived CAMP-dependent protein kinase (Fig. 2) when the two proteins were subjected to sodium dodecyl sulfate gel ctlcctrophoresis, as determined both by phosphorylation and by incorporation of the photoaffinity label 8-N,,-cAMP Moreover, the ability of the regulatory subunit of the purified synaptic membrane-derived protein kinasc to inhibit the catalytic subunit of the synaptic membrane-derived protein kinase. but not the cata lytic subunit of brain cytosol or heart cytosol protein kinase (see Ref. 15), Table VIII, was mimicked by the partially purified preparation of Protein II (data not shown:. Sucrose dr~nsity gradient centrifugatiou. Sucrose density gradient centrifugation of protein kinasc was carried out as described by Martin and Ames 1121, except that 50 mM potassium phosphate buffer was used, 0.1 mM DTT was present, and centrifugation was carried out for 17 h. Prrparation of protc~in kinasc modulator. Protein kinase modulator was prepared from bovine brain by the method of Donnelly ct al. (13). Protein concentration was determined by the method of Lowry et nl. 114) with bovine serum albumin as standard.
Protein
RESULTS
AND
DISCUSSION
I Kinase
and Protein
II Kinase
DEAE-cellulose column chromatography. When a Triton extract of the M-l synaptic membrane fraction was subjected to DEAE-cellulose column chromatography, single peaks of Protein I kinase activ-
40
50
60
70
NUMBER
FIG. 1. Separation of Protein II, Protein II kinase, and Phosphoprotein II phosphatase by DEAE-cellulose column chromatography. A Triton extract of the M-l membrane fraction was subjected to DEAE-cellulose column chromatography, and aliquots of each fraction (2.5 ml) were analyzed for Protein II (x-x), Protein II kinase activity in the absence (CI--0) or presence (W-W) of CAMP, and Phosphoprotein II phosphatase activity in the absence (a-& or presence (A-A) of CAMP. All values are expressed per fraction
PROTEIN
AND
PHOSPHATASE
CAMPBindkng
Phosphorylatcon PK
KINASE
Pll
PK
PII Orlgln
t-
52 .OOO
FIG. 2. Autoradiograph of a sodium dodecyl sulfate-polyacrylamide gel showing the incorporation of a2P from ly-“‘PJATP (left) and of l:‘2P]-8-N,-cAMP (right) into the protein kinasc regulatory subunit (PK) and Protein II (PH. Membrane-derived protein kinase (2.0 units, 0.80 ng), purified through step 9 (51, was subjected to autophosphorylation under standard conditions (5). Protein II (5.2 ng) was phosphorylated for 1 min with 0.02 units of Protein II kinase in the presence of 10 pM CAMP under standard conditions (51. Other aliquots of protein kinase (5.2 ngl and Protein II (2.8 ng) were incubated with 0.2 FM l:“P]-8-N,-CAMP for photoaffinity labeling, as described under Experimental Procedures. These four preparations were then subjected to gel electrophoresis, with the results shown above.
ity, Protein II kinase activity, protamine kinase activity, and histone kinase activity were obtained, and all of these peaks were eluted in the same position, as shown in Fig. 3. The most active fractions (Fractions 40 through 46) were pooled. This partially purified enzyme preparation was used for all further studies of protein kinase activity, except where indicated. Sucrose density gradient centrifugation. When this partially purified preparation of protein kinase was subjected to sucrose density gradient centrifugation, Protein I kinase activity, Protein II kinase activity, and protamine kinase activity again appeared in the same position (data not shown), suggesting that the phosphorylation of Protein I, Protein II, and protamine might be catalyzed by a single species of protein kinase. Inactivation by pH and by heat. Pre-
FROM
NEURONAL
MEMBRANES
483
treatment of the partially purified preparation of protein kinase at various pH values for 10 min at 30°C led to similar losses of Protein I kinase, Protein II kinase, and protamine kinase activity (Fig. 4). Moreover, pretreatment of this protein kinase preparation for various periods of time at 45°C caused parallel losses of Protein I kinase, Protein II kinase, and protamine kinase activity (Fig. 51. In addition, pretreatment of this enzyme preparation at various temperatures from 35 to 50°C for 10 min also caused parallel losses of the three types of enzyme activity (data not shown). Inhibition by modulator. The effect of various concentrations of a protein kinase modulator from brain (13) on Protein I kinase, Protein II kinase, and protamine kinase activity was examined in the presence of 10 pM CAMP, using 0.25 units of Protein I kinase activity. All three types of enzyme activity were inhibited approximately in parallel by increasing concentrations of the modulator. Fifty percent inhibition was obtained with 0.35 to 0.50 mg of modulator/ml (data not shown). Effect of various concentrations of CAMP and cGMP. The effect of various concentrations of CAMP and cGMP on Protein I kinase, Protein II kinase, and protamine kinase activity is shown in Fig. 6. CAMP stimulated the three types of protein kinase activity in a similar fashion. At higher concentrations, cGMP also stimulated the three types of enzyme activity in a similar fashion. The concentrations of CAMP and cGMP required for half-maximal activation were approximately 1 x 10 ’ and 5 x lo-” M, respectively, for all three types of protein kinase substrate. Substrate specificity. The substrate specificity of the membrane-derived protein kinase, at various stages of a 2000-fold purification, is shown in Table I. The relative specific activities of the protein kinase toward Protein Ia, Protein Ib, Protein II, protamine, and histone did not change significantly throughout the entire purification procedure. Competitive inhibition by alternative substrate proteins. In experiments in which Protein I and Protein II were both
484
UNO,
UEDA,
AND
GREENGARD
FIG. 3. Elution profile of Protein I kinase, Protein II kinase, protamine kinase, and histone kinase activity upon DEAE-cellulose column chromatography. Ten milliliters (1.5 mg of protein/ml) of a Triton extract of the M-l fraction, in 100 rnM potassium phosphate buffer (pH 7.0)-IO-” M DTT, were applied to a DEAE-cellulose column (0.9 x 30 cm) previously equilibrated with the same buffer. Protein kinase activity was eluted with a linear gradient of phosphate buffer (100 to 400 mM), pH 7.0, in lo- ’ M DTT, using a total volume of 200 ml. Fractions (2.5 ml each) were collected and aliquots were assayed for protein kinase activity using four different substrates, in the absence (open symbols) or presence (filled symbols) of 10 WM CAMP. All values are expressed per fraction.
4
FIG. 4. pH inactivation of Protein I kinase, Protein II kinase, and protamine kinase activity. The partially purified preparation of protein kinase (2ml aliquot) was dialyzed overnight against 1 liter of 10 mM potassium phosphate buffer (pH 7.0) containing 10el M DTT. Aliquots (100 ~11, containing 5.1 units of Protein I kinase activity, were added to 100 ~1 of each of the following buffer solutions (50 mM): (1) sodium acetate buffer (pH 4.5, 5.0, and 5.5); (2) potassium phosphate buffer (pH 6.0, 6.5, and 7.0); and (3) Tris-HCl buffer (pH 7.5, 8.0, and 9.0). Each enzyme solution was incubated at 30°C for 10 min, cooled to o”C, and mixed with 1.8 ml of 400 rnM potassium phosphate buffer (pH 7.0). Aliquots (20 ~1) of each of the resulting enzyme solutions were then assayed for Protein I kinase (A), Protein II kinase (01 and protamine kinase (0) activity in the presence of 10 +M CAMP. Protein kinase activity is expressed as percentage of that found in the sample which had been preincubated at pH 7.0.
FIG. 5. Thermal inactivation of Protein I kinase, Protein II kinase, and protamine kinase activity. The partially purified preparation of protein kinase (l-ml aliquot containing 2.5 units of Protein I kinase and 150 pg of protein) was preincubated at 45°C for various periods of time. Aliquots (20 ~1) of each sample were then assayed for Protein I kinase (al, Protein II kinase (D), and protamine kinase (0) activity, in the presence of 10 FM CAMP. Protein kinase activity is expressed as log of the percentage of the activity found in samples which had not been preincubated.
present, they acted as alternative strates for the protein kinase. Thus, uring the rate of phosphorylation of of these proteins, the other acted apparent competitive inhibitor. In tion, protamine acted as an apparent
submeaseither es an ::ddicom-
PROTEIN
KINASE
AND
PHOSPHATASE
petitive inhibitor both of Protein I and Protein II phosphorylation. The Ki values obtained in these experiments (Table II) agreed closely with the K,,, values (see 5) for the same proteins acting as substrates for the purified protein kinase. These findings suggest that the same enzyme can catalyze the phosphorylation of all of these proteins. The data also indicate that Protein I and Protein II can interact with the
FROM
NEURONAL
same catalytic site on the protein kinase catalytic subunit. Since Protein II seems to be the regulatory subunit of this protein kinase (see Experimental Procedures), these results support the suggestion of Witt and Roskoski (16) that the regulatory subunit shields the active site of the catalytic subunit thereby inhibiting catalytic activity. In conclusion, the variety of experimental results presented above strongly suggest that the same species of protein kinase can catalyze the phosphorylation of Protein Ia, Protein Ib, Protein II, protamine, and histone. TABLE K, VALUES Protein
II
OF PROTEIN I KINASE FOR PROTEIN PROTEIN II, AND PROTAMINE”
used as inhibitor
Protein
CYCLIC
NUCLEOTIDES
(-log
(/AM)
SPECIFICITY
PROTEIN
KINASE
Substrate Protein
M-l fraction Triton extract Triton supernatant First DEAE-cellulose column eluate Ammonium sulfate precipitate Bio-Gel P-200 column eluate Second DEAE-cellulose column eluate Sepharose 4B column eluate Hydroxylapatite column eluate
II
0.88 6.5
I
OF MEMBRANE-DERIVED
Step
1. 2. 3. 4. 5. 6. 7. 8. 9.
0.92 6.2
Protein
” The K, values shown were calculated from Dixon plots (15) which were linear at various fixed substrate concentrations. Similar K, values were obtained from Lineweaver-Burk plots (15), which were linear at various fixed inhibitor concentrations. The range of concentrations of substrates and inhibitors varied from 4 x lo-!’ to 4 x 10 i M.
TABLE SUBSTRATE
I
-
Protein I Protein II Protamine
Ml
I,
used as substrate
Protein
FIG. 6. Stimulation by various concentrations of CAMP and cGMP of Protein I kinase, Protein II kinase, and protamine kinase activity. Protein I kinase (a, A), Protein II kinase (0, W, and protamine kinase (0, 0) activities were measured in the presence of various concentrations of CAMP (open symbols) or cGMP (closed symbols). Protein kinase activity is expressed as percentage of that found in the presence of 10 PM CAMP.
485
MEMBRANES
100 85 91 96 98 92 70 85 87
Ia Protein
DURING
(relative Ib
Protein
ITS PURIFICATION”
specific
activity)
II Protamine
Histone
100 163 17 11 100 150 20 15 100 162 18 12 100 150 17 12 100 155 17 13 100 150 16 11 100 167 18 13 100 160 18 11 100 ___. 163 17 12 (1 Bovine brain membrane-derived protein kinase was solubilized and purified to homogeneity, as described elsewhere (5). At each step of the purification procedure, substrate specificity of the enzyme was determined, using 5 pg of Protein I (1.6 pg of Protein Ia and 3.4 pg of Protein Ib) purified through step 7 (3), 7.0 pg of Protein II, 5.0 fig of protamine, or 5.0 pg of histone, as substrate. The phosphorylation of each substrate was carried out in the presence of 10 ~.LM CAMP, and the phosphorylated protein analyzed by sodium dodecyl sulfate gel electrophoresis and liquid scintillation spectrometry, as in the standard Protein I kinase assay method. At each step of enzyme purification, the moles of phosphate incorporated into each substrate is expressed as a percentage of that incorporated into Protein Ib.
486
UNO,
Phosphoprotein I Phosphatase phoprotein II Phosphatase
UEDA,
AND
and Phos-
DEAE-cellulose column chromatography. When a Triton extract of the M-l membrane fraction was subjected to column chromatography on DEAE-cellulose, two peaks of protein phosphatase activity were observed (Fig. 7). The first peak catalyzed the dephosphorylation of phosphoprotamine and Phosphoprotein II, but not of Phosphoprotein I. The second peak catalyzed the dephosphorylation of phosphoprotamine and Phosphoprotein I, but not of Phosphoprotein II. The rate of dephosphorylation of Phosphoprotein II by the first peak was stimulated by CAMP (Fig. 7); in contrast, the rate of dephosphorylation of Phosphoprotein I by the second peak, and the dephosphorylation of phosphoprotamine by either peak of activity, was unaffected by CAMP (data not shown). The most active fractions of Phosphoprotein I phosphatase (Fractions 22 through 28) and Phosphoprotein II phosphatase (Fractions 6 through 12) were separately pooled and concentrated to 1.0 ml by means of ultrafiltration on a Diaflo mem-
GREENGARD
brane (PM101 under pressure of nitrogen gas. Bio-Gel P-200 gel filtration. When the partially purified preparations of phosphoprotein phosphatase, obtained by DEAEcellulose column chromatography, were separately subjected to Bio-Gel P-200 column chromatography, Phosphoprotein I phosphatase activity, and Phosphoprotein II phosphatase activity were eluted in different positions (Fig. 8). The Stokes’ radii for Phosphoprotein I phosphatase and Phosphoprotein II phosphatase, calculated from the elution position by the method of Siegel and Monty (17), using appropriate marker proteins (3), were 55 and 44 A, respectively. The most active fractions of Phosphoprotein I phosphatase (Fractions 18 through 22) and of Phosphoprotein II phosphatase (Fractions 21 through 25) were separately pooled and concentrated to 1.0 ml by means of ultrafiltration on a Diaflo membrane (PM101 under pressure of nitrogen gas. These partially purified enzyme preparations were used for all further studies of protein phosphatase activity. Effect of CAMP on Phosphoprotein II
FIG. 7. Separation of Phosphoprotein I phosphatase from Phosphoprotein II phosphatase activity by DEAE-cellulose column chromatography. Ten milliliters (1.5 mg of protein/ml) of a Triton extract of the M-l fraction, in 100 mM potassium phosphate buffer (pH 7.0)-lOma M DTT, were applied to a DEAE-cellulose column (0.9 x 30 cm) previously equilibrated with the same buffer. Phosphoprotein phosphatase activity was eluted with a linear gradient of phosphate buffer (100 to 400 mM), pH 7.0, in 10. ’ M DTT, using a total volume of 200 ml. Fractions (2.5 ml each) were collected and aliquots were assayed for phosphoprotamine phosphatase activity (0- - -0) and Phosphoprotein I phosphatase activity (A- - -A1 in the absence of CAMP, and for Phosphoprotein II phosphatase activity in the absence (0 - -0) or presence (m-m) of 10 pM CAMP. All values are expressed per fraction.
PROTEIN
KINASE
AND
PHOSPHATASE
FROM
FIG. 8. Bio-Gel P-200 gel filtration of Phosphoprotein I phosphatase and Phosphoprotein II phosphatase activity. (A) Phosphoprotein I phosphatase (1.0 ml containing 21 units) and (B) Phosphoprotein II phosphatase (1.0 ml containing 72 units) obtained by DEAE-cellulose column chromatography were separately applied to Bio-Gel P-200 columns (0.9 60 cm), which had been equilibrated with 10 mM Tris-HCl buffer (pH 7.4). The columns were eluted with the same buffer, and l.O-ml fractions were collected. Aliquots were assayed for Phosphoprotein I phosphatase (a) and phosphoprotamine phosphatase (0) activity in the absence of CAMP, and for Phosphoprotein II phosphatase (W) activity in the presence of 10 CAMP. All values are expressed per fraction. x
FM
TABLE OF CAMP
Phosphatase
peak
PHOSPHATASE
-
ACTIVITY”
Substrate
-CAMP (units I phosphatase II phosphatase
III
ON PHOSPHOPROTEIN
Phosphoprotein
Phosphoprotein Phosphoprotein
487
MEMBRANES
on CAMP (Table III). In contrast, phosphoprotamine phosphatase activity, which cochromatographed with CAMP-dependent Phosphoprotein II phosphatase activity during the Bio-Gel P-200 gel filtration step (Fig. 8), was not affected by CAMP (Table III). Moreover, no effect of CAMP was observed on the dephosphorylation of either Phosphoprotein I or phosphoprotamine by the Phosphoprotein I phosphatase peak. [ :‘H]cAMP-binding activity could not be detected in the Bio-Gel P-200 preparation of Phosphoprotein II phosphatase nor was photoaffinity labeling of this preparation by B-N,,-CAMP observed (data not shown); in contrast, Phosphoprotein II itself is a CAMP-binding protein, and the regulatory subunit of membrane-bound protein kinase, as described under Experimental Procedures. These results support the suggestion (11, 18, 19) that CAMP causes an increased accessibility of Phosphoprotein II to Phosphoprotein II phosphatase, by acting directly on Phosphoprotein II, with a resultant stimulation of the Phosphoprotein II phosphatase reaction. Sucrose density gradient centrifugation. When the preparations of phosphoprotein phosphatase purified through the Bio-Gel P-200 gel filtration step were separately subjected to sucrose density gradient centrifugation, Phosphoprotein I phosphatase activity and Phosphoprotein II phosphatase activity appeared in different positions (Fig. 9). The positions of the two activities corresponded to .s~,,,~ values of 7.1 and 5.0 S, respectively. The molecular weights of Phosphoprotein I phosphatase and Phosphoprotein II phosphatase, calculated from their Stokes’
phosphatase activity. As was observed with the Phosphoprotein II phosphatase activity peak from the DEAE-cellulose column (Fig. 71, the dephosphorylation of Phosphoprotein II by Phosphoprotein II phosphatase purified through the Bio-Gel P-200 step showed an absolute dependence
EFFECT
NEURONAL
24
