THE JOURNAL OF BIOLOGICAL C~m.mwnv Vol. 252, No 11, Issue of June 10, pp. 3848-3853, 1977

Printed ,n U.S.A.

Endogenous Protein Kinase Inhibitors PURIFICATION, CHARACTERIZATION, AND DISTRIBUTION IN DIFFERENT TISSUES*

(Received for publication, November 18, 1976)

ANDRZEJ SZMIGIELSKI, ALESSANDRO GUIDOTTI, AND ERMINIO COSTA

From the Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D. C. 20032

A thermostable inhibition of ATP:protein phosphotrans- ferase (EC 2.7.1.37) (protein kinase) which is present in crude tissue extracts has been resolved by gel chromatogra- phy (Sephadex G-100) into two molecular forms. These two forms will be referred to as type I and type II inhibitor. The type I inhibitor (M, = 24,000) is specific for CAMP-dependent protein kinase and corresponds to the inhibitor described earlier (Walsh, D. A., Ashby, C. D.; Gonzalez, C., Calkins, D., Fisher, E. H., and Krebs, E. G. (1971j.J. Biol. Chem. 246, 1977-1985). The type II inhibitor (3Zr = 15,000) competes for the enzyme with various substrate proteins (histone, a-ca- sein, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). The type II inhibitor blocks protein phosphorylation catalyzed by several types of protein kinases (CAMP- and cGMP-de- pendent or cyclic nucleotide-independent protein kinases). The type II inhibitor from rat brain has been purified 1500- fold; this protein is thermostable, has acidic characteristics, and does not require Ca’+ ions for its activity. Different ratios and concentrations of type I and type II inhibitors of protein kinase are found in rat skeletal muscle, pancreas, cerebellum and corpus striatum, and in lobster tail muscle.

ATP:protein phosphotransferases (EC 2.7.1.37) (protein ki- nases) control a variety of cellular processes in nuclei, ribo- somes, and plasma membranes (for a review, see Ref. 1). This regulatory activity depends on the phosphorylation of specific proteins. However, at the present time, our knowledge of the kinetics and intracellular distribution of protein kinases is not sufficient to explain the selectivity of irz viva protein phospho- rylation. As suggested by Beavo et al. (2), other regulatory factors, such as small molecular weight proteins, may contrib- ute to the specificity of protein phosphorylation. A thermosta- ble protein inhibitor which binds to the catalytic subunits of CAMP-dependent protein kinase has been described in several tissues (3-5). A protein with similar biological and physico- chemical properties has also been found in lobster tail muscle and was termed “modulator” of protein kinase (6, 7). This modulator can either stimulate or inhibit the activity of cyclic nucleotide-dependent protein kinase according to the type and concentration of substrate protein, metal ion, and cyclic nu-

* This investigation was supported in part by Public Health Ser- vice International Research Fellowship F05TW02326 awarded to A. S.

cleotide (7). The modulator has been resolved into two compo- nents: one that is similar to the inhibitor described by Walsh et al. (3) and the other which stimulates cGMP-dependent pro- tein kinase (8, 9).

We now report on yet another heat-stable, low molecular weight protein, which is able to inhibit the activity of protein kinases. This protein may have profound influence on the mechanisms of phosphorylation in various tissues. In fact, this protein competitively inhibits the phosphorylation of acidic and basic proteins that are used as substrates for cyclic nucleo- tide-dependent and -independent protein kinases.

EXPERIMENTAL PROCEDURES

Methods

Assay for Protein Kinase Inhibitor Content in Tissue Prepara- tions-This assay measures the ability of tissue preparations to inhibit phosphate transfer from ATP to various proteins in a reaction catalyzed by partially purified protein kinase from various sources. Unless otherwise indicated, the standard assay system contained in a final volume of 120 ~1: 100 mM sodium acetate buffer (pH 6.01, 80 mM magnesium acetate, 40 pg of arginine-rich histone, 20 pmol of cGMP or CAMP, 0.1 mM [y-“2PlATP (specific activity 1 mCi/~mol), a purified protein kinase (from various sources), and different amounts of tissue preparations containing the protein kinase inhibi- tor. The phosphorylating reaction was carried out at 30” for 10 min and was terminated by pipetting 30 /*l of the incubation mixture onto phosphocellulose paper. The amount of 32P incorporated into histone was measured according to Witt and Roskoski (10). When the syn- thetic Leu-Arg-Arg-Ala-Ser-Leu-Gly peptide (kemptide) was used in place of histone, the phosphocellulose paper was washed with 30% acetic acid (10). When casein or proteins present in various tissue preparations were used as the :raP acceptors, the reaction was termi- nated by pipetting 50-~1 aliquots of the incubation mixture on What- man No. 1 filter paper discs. The discs were washed as described by Wastila et al. (11). Each assay was run in duplicate using an incuba- tion mixture containing the boiled protein kinase preparation as a blank.

Preparation of Crude Protein Kinase Inhibitor -The cerebellum or other tissues of male Sprague-Dawley rats (150 to 175 g) were homog- enized in 3 volumes of ice cold 5 mM potassium phosphate buffer, pH 7.0. The homogenate was centrifuged for 20 min at 20,000 x g at 0 to 4” and the resulting supernatant was placed at 90 to 95” for 10 min. The mixture was cooled on ice and centrifuged for 20 min at 20,000 x g. This supernatant will be referred to as “crude inhibitor.”

Purification of Different Types of Protein Kinases - CAMP-depend- ent protein kinase from beef heart and cGMP-dependent protein kinase from lobster tail were purified using ammonium sulfate precipitation and ion exchange DEAE-chromatography as described by Kuo and Greengard (12).

cGMP-dependent protein kinase from rat cerebellum was purified by a similar procedure except that a linear gradient of NaCl (60 to

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400 mM) was used to elute protein kinase from a DE52 column. Each fraction from the DE52 column was assayed for protein kinase activ- ity in the presence of CAMP and cGMP and in the presence or absence of purified endogenous inhibitor of CAMP-dependent protein kinase. The inhibitor was prepared according to Donnelly et al. (6). Those fractions of the DE52 eluate which were activated by cGMP even in the presence of the CAMP-dependent protein kinase inhibi- tor, were pooled and used as a source of cGMP-dependent protein kinase.

TABLE I

Inhibition ofprotein kinases from various sources by cerebellar crude

inhibitor and its 5% trichloroacetic acid precipitate

Arginine-rich histone (333 pg/ml) was used as nsP acceptor for cGMP- and CAMP-dependent protein kinase. Activity of nuclear protein kinase was measured in the presence of cu-casein (333 pgiml). The activity of cyclic nucleotide-independent protein kinase from rat adrenals was measured using the endoeenous 32P acceotors.

Cyclic nucleotide-independent protein kinase was extracted from the 20,000 x g pellet of rat adrenals with 0.2% Triton X-100 and then purified on Sephadex G-200 (13). This protein kinase preparation included an endogenous phosphate acceptor; therefore, exogenous phosphate acceptor protein was not added in these experiments.

5% Trichlo-

Protein kinase SOurce No inhib- Crude in- Inhibi- acf,yr$$pi- Inhibi- itor hibitm” tion tate of crude tion

inhibit&

Types I and II CAMP-dependent protein kinases were partially purified from rat adrenal medulla cytosol by DE52 ion exchange chromatography using a linear concentration gradient of NaCl (0 to 0.5 M) (14). Two types (types A and B) of nuclear cyclic nucleotide- independent protein kinases’ were isolated from bovine adrenal medulla nuclei by ammonium sulfate precipitation and ion exchange DE52 chromatography using a linear concentration gradient of am- monium sulfate (0 to 0.4 M). Casein was a preferential phosphate acceptor for nuclear protein kinase type A, whereas histone was the preferential phosphate acceptor for type B enzyme.

pm01 ,=P incorpo rated/sample

Estimation of Molecular Weight -A Sephadex G-100 column (0.2 x

70 cm) was calibrated according to Andrews (15) using ribonuclease A, chymotrypsinogen A, and ovalbumin. The column was equili- brated with 50 mM potassium phosphate buffer, pH 7.0, containing 100 rnM NaCl and was operated at 22”.

cGMP-dependent from 25 rat cerebellum

cGMP-dependent from 31 lobster tail

CAMP-dependent from 32 beef heart

Cyclic nucleotide-inde- 15 pendent from rat ad- I-en&

Nuclear type A from 43 beef adrenal medulla

12

15

7.1

7.3

23

w pmoz ,-P Ln- 9% corporated/

SUVZpZC2 52 25 0

52 29 6.5

78 21 35

51 16 0

47 42 2.3

Sodium dodecyl sulfatepolyacrylamide gel electrophoresis, using 10, 15, and 20% gels, was carried out at 22” according to Weber and Osborn (16). One hundred milliliters of 20% gel solution contained 0.3 g ofN,N’-methylenebisacrylamide, 0.15 ml ofN,N,N’,N’-tetra- methylethylenediamine, and 0.075 g of ammonium persulfate. The electrophoresis buffer contained 3.9 g of NaH,PO,H,O, 19.3 g of Na,HPO,, ‘7H,O and 1 g of sodium dodecyl sulfate Coomassie brillant blue G was used for staining the proteins. Different concentrations of gel were obtained by dilution of the 20% gel solution with distilled water.

0 Each incubation mixture contained 10 pg of crude inhibitor. b The crude inhibitor preparation was precipitated with ‘/Y volume of 508

trichloroacetic acid. The precipitate was centrifuged off and dissolved in water. The pH of the solution was adjusted to 7.0 with 5 N NaOH. Acid precipitable proteins (10 pg) were used for assay.

Preparative Polyacrylamide Gel Electrophoresis - An apparatus manufactured by Shandon Scientific Co., Philadelphia, Pa., was used in these experiments. The column (11 cm in height) was pre- pared with 7.5% acrylamide gel polymerized in the presence of ammonium persulfate and 0.37 M Tris/HCl buffer, pH 9.2. To remove the polymerization catalyst, the column was washed for 1 h with the same Tris buffer by applying a current of 20 mA. A sample which included bromphenol blue as a marker was then loaded on the column. The electrophoresis was run for 1 h with a current of 25 mA using 80 rnM Trisiglycine buffer, pH 8.3, at 4”. The current was then increased to 60 mA and maintained for 20 h. The proteins were eluted from the elution chamber with a constant stream of 0.43 M

Tris/acetate buffer, pH 7.6; l-ml fractions were collected (17).

5% trichloroacetic acid precipitate of crude inhibitor inhibited CAMP-dependent protein kinase selectively (Table I). To ex- amine whether the crude inhibitor contained one or more molecular forms, this preparation was chromatographed on a Sephadex G-100 column. The elution pr6file of the inhibitory activity (Fig. 1A) showed two distinct peaks. The first peak included an inhibitory material specifically directed toward CAMP-dependent protein kinase. The second peak included a material with smaller molecular weight which inhibited pro- tein kinase activity regardless of whether the protein kinase was cyclic nucleotide-dependent or -independent.

Phosphoprotein Phosphatase Activity -Phosphoprotein phospha- tase (EC 3.1.3.16) activity was measured by the decay of 3aP incorpo- rated in the histones according to Maeno and Greengard (18). Pro- teins were assayed by the Lowry method (19).

In order to exclude the possibility that the two peaks of inhibitory activities as resolved by Sephadex G-100 chroma- tography, were due to homogenization or heating artifact, we killed the rats with focussed microwave irradiation which within 1 to 2 s inactivated the tissue enzymes including those that might be responsible for the postmortem synthesis or degradation of the inhibitory material (20). As shown in Fig. 1 (Panel B) the Sephadex G-100 chromatographic profile of the inhibitors present in the cerebellar homogenate of rats killed by microwave was identical to that of the inhibitors obtained from heat-inactivated cerebellar homogenates of decapitated rats. Identical results (Fig. 1, Panel C) were obtained by chromatographing the 20,000 x g supernatant of cerebellar homogenates and heating the various elution fractions before measuring their inhibitory activity on protein kinase.

Materials

Synthetic Leu-Arg-Arg-Ala-Ser-Leu-Gly peptide (kemptide) was purchased from Peninsula Laboratories, San Carlos, Calif.; argi- nine-rich histone was from Worthington, Freehold, N. J. All other histones were purchased from Sigma, St. Louis, MO., Sephadex G- 100 was obtained from Pharmacia, Upsala, and [a-:‘“PlATP from New England Nuclear, Boston, Mass. All the other reagents were of the purest grade available from various commercial sources.

RESULTS

Evidence for Presence of Two Molecular Forms of Endoge- nous Protein Kinase Inhibitors in Rat Cerebellum-Table I shows that the 20,000 x g supernatant of cerebellum homoge- nate incubated at 90 to 95” for 10 min (crude inhibitor) in- hibited the activity of protein kinases from various sources. A

’ R. A. Hollenbeck and D. M. Chuang, manuscript in preparation.

The two inhibitory peaks shown in Fig. 1 will be called type I and type II inhibitors of protein kinase. Type I is used to designate the inhibitor with the higher molecular weight.

Characterization of T,ype I Inhibitor ofProtein Kinase- The type I inhibitor specifically inhibited the activity of catalytic subunits of CAMP-dependent protein kinase and failed to af feet other types of protein kinases (Fig. 1). In addition, this inhibitor was precipitable by 5% trichloroacetic acid (Table I). We found that, in accordance with the report of Walsh et al. (31, the type I inhibitor (M, = 24,000 with sodium dodecyl

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01 lb ' 0 5 15 20 Okeirie- Oi 20 0 5 IO I5 20

FRACTION

FIG. 1. Sephadex G-100 elution profile of protein kinase inhibi- tory proteins from rat cerebellum. A, rats were killed by decapita- tion and the 20,000 x g supernatant was placed at 90 to 95” for 10 min. Proteins (200 pg) were applied to Sephadex G-100 column (0.2 x 70 cm) previously equilibrated with 50 mM potassium phosphate buffer, pH 7.0, containing 100 mM NaCl. Elution was performed with the same buffer. Fractions of 300 ~1 were collected. B, rats were killed in 1.5 s by focussed microwave radiation on the head (20). Crude inhibitor was prepared as in A and 200 ~1 of inhibitory proteins were applied to Sephadex G-100 column, Elution as de- scribed above. C, rats were killed by decapitation. Cerebella were

sulfate-gel electrophoresis) was destroyed by trypsin digestion and acted noncompetitively with respect to histones and ATP in the protein kinase-catalyzed phosphorylation of histones.

Purification and Characterization of Type II Inhibitor of Protein Kinase- The type II inhibitor was prepared from brains of 50 rats killed by microwave irradiation, homogenized in 3 volumes of 5 mM potassium phosphate buffer, pH 7.0, and centrifuged at 20,000 x a. The supernatant was heated at 90 to 95” for 15 min. After centrifugation, the supernatant was passed through an XM 50 Diaflow membrane (Table II). The high molecular weight (type I) inhibitor was retained while the small molecular weight (type II) inhibitor passed through the membrane. The filtrate was then concentrated on a Di- aflow UM 2 membrane and chromatographed with Sephadex G-100 column (1 x 80 cm) equilibrated with 50 mM potassium phosphate buffer, pH 7.0, containing 100 mM NaCl (Table II). Fractions of 5 ml each were collected and an aliquot of each fraction was assayed for inhibitory activity against CAMP- and cGMP-dependent protein kinases. Only one distinct peak of inhibitory activity corresponding to type II inhibitor was found. The fractions containing type II inhibitor were pooled, dialyzed against water overnight, then concentrated by lyoph- ilization and chromatographed on a Dowex 50-X8 column (0.7 x 10 cm) (Table II). The inhibitory activity was not retained by this column indicating the acidic nature of the inhibitory material. Further purification was achieved by preparative polyacrylamide gel electrophoresis. As shown in Fig. 2, the inhibitory activity was eluted from the column as a single peak immediately after the bromphenol blue marker. As shown in Table II, about 7% of the original inhibitory activity could be purified by about 1500-fold. Type II inhibitor was approximately equipotent in blocking the activity of CAMP- and cGMP-dependent protein kinases (Table II, Fig. 2). When the type II inhibitor was chromatographed on DE52 column or hydroxylapatite, the yield was extremely low.

Since the activity of the purified inhibitor was destroyed by low concentrations of either trypsin or chymotrypsin but not by RNase, DNase, or phospholipase, we concluded that the

homogenized in 3 volumes of ice cold 5 rnM potassium phosphate buffer, pH 7.0, containing 150 rnM NaCl, 10 mM ethylene glycol bis (P-aminoethyl ether)N,N-tetraacetic acid, 10 rnM phenylmethylsul- fonyl fluoride, and 0.2% Triton X-100. After centrifugation at 20,000 x g for 20 min, the supernatant was applied to Sephadex G-100 column. Fractions of 300 ~1 were collected and heated at 90 to 95” for 10 min. A 30-4 aliquot from each elution fraction was examined for the presence of inhibitory material against 20 units of cGMP-depend- ent protein kinase (O---O), 20 units of CAMP-dependent protein kinase (O- - -01, or 20 units of cyclic nucleotide-independent pro- tein kinase from bovine adrenal medulla nuclei (type A) (n---n,.

TABLE II

Purification of type II inhibitor from rat bran

Inhibition cGMP-dependent

steps Purification Yield protein kinase/In- hibition of CAMP- depen;lcL&~tein

-fold %

Homogenate 1 100 0.64

95” Treated supernatant’L 20 87 0.67 Membranes Diaflow XM 50 37 49 0.95”

and UM 2 Sephadex G-100 160 33 0.99

Dowex 50-X8 450 14 0.93

Polyacrylamide preparative 1500 7.4 0.94

gel electrophoresis -- a The supernatant was obtained by centrifuging the homogenate

at 20,000 X x 20 min. fi b The specific inhibitor of CAMP-dependent protein kinase was

removed by Diaflow XM 50 membrane filtration. The inhibitory activity for CAMP- and cGMP-dependent protein kinases became identical after the filtration.

inhibitor was a protein. The apparent molecular weight estab- lished by sodium dodecyl sulfate-polyacrylamide disc electro- phoresis and gel filtration using ribonuclease A, chymotryp- sinogen A, and ovalbumin as standards was about 14,500 (Fig. 3, A and B). Using different concentrations of sodium dodecyl sulfate-polyacrylamide gel (10, 15, and X0%), only a single major protein band was found. When the sodium dodecyl sulfate-polyacrylamide preparation with 10% gel concentra- tion was sliced and each slice was extracted according to Weber and Osborn (16), the peak of inhibitory activity corre- sponded to the major band of protein (Fig. 4).

Mechanism of Action of Type ZI Inhibitor- A kinetic study of the effect of a purified preparation of type II inhibitor on cGMP-catalyzed phosphorylation of arginine-rich histone indi- cated that the inhibitor was noncompetitive with respect to ATP, cGMP, and MgZ+ (Fig. 5). In contrast, the inhibitor

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apparently competitively antagonized the interaction between protein kinase and the phosphate acceptor protein; this com-

petitive antagonism was observed using a variety of protein kinases and phosphorylation substrates (Fig. 6). From the data plotted in Fig. 6, the K, values were almost identical for arginine-rich histone (using CAMP- or cGMP-dependent pro- tein kinases), for a-casein (using cyclic nucleotide-independent protein kinase A from nuclei of beef adrenal medulla), and for the synthetic Leu-Arg-Arg-Ala-Ser-Leu-Gly peptide (kemp- tide) (using CAMP-dependent protein kinase). Moreover, when

FRACTIONS FIG. 2. Elution profile of the type II inhibitor from preparative

polyacrylamide gel electrophoresis. About 2 mg of partially purified type II inhibitor (see Table II) was subjected to electrophoresis. Electrophoresis was run as described under “Methods.” Fractions of 1 ml each were collected. An aliquot of 10 ~1 of each fraction was examined for the presence of the inhibitor using 20 units of CAMP- dependent protein kinase (O- - - 0) or 20 units of cGMP-dependent protein kinase (0-O). The arrow indicates the fraction in which bromphenol blue emerged from the column.

A

I‘\ IO 1

0 01 0.2 0.3 0.4 05

RELATIVE MOBILITY

MW B ( 105

chymotrypsinogen

IO I 2 3 4 5

ELUTION VOLUME(d)

FIG. 3. Estimation of the molecular weight of type II inhibitor by sodium dodecyl sulfate.-polyacrylamide gel electrophoresis (A) and Sephadex G-100 gel filtration (B). Inhibitor (10 pg of 1500-fold puri- fied) (Table II) was subjected to electrophoresis or gel filtration. The closed circle with an arrow indicates the position of the inhibitor type II. In Panel A, the relative mobility is calculated with respect to bromphenol blue whose value is assumed to be 1.

-!- (mM-‘) Mg”

the type II inhibitor of protein kinases was tested with cGMP- dependent protein kinase isolated from lobster muscle (see Table III) with type I and type II CAMP-dependent protein kinases obtained from rat adrenal medulla cytosol, with cGMP-dependent protein kinase from rat cerebellum, or with cyclic nucleotide-independent protein kinases obtained from nuclei of rat or beef adrenal medulla, using different sub- strates, the inhibition of :s2P incorporation was always present. The action of the inhibitor was not affected by Ca*+ (0 to 200 PM) or by ethylene glycol bis (fi-aminoethyl ether)N,N-tetraa- cetic acid (0 to 40 PM). The nature and characteristics of the protein kinase inhibition remained constant across various buffering conditions: 50 mM Tris buffer, pH 7.5, 50 mM potas- sium phosphate buffer, pH 7.0, or 100 mM acetate buffer, pH 6.0. The inhibitor itself was not phosphorylated. The amount of substrate dephosphorylation was negligible in all conditions tested, and was unaffected by the presence of type II inhibitor.

Distribution of Type Z and Type ZZ Inhibitors of Protein Kinase in Various Tissues -The content of type I and type II inhibitors of protein kinase was determined in several tissues (see Table IV). The crude inhibiter was prepared from homog- enates of rat brain, skeletal muscle, pancreas, and lobster tail muscle, respectively. These preparations were chromam-

20

2

15

5 i IO

I 3 5

0 u 0 IO 20 30 40 50 60

I SLICE NUMBER

FIG. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophore- sis of inhibitor type 11;. Purified inhibitor type II (10 pg) was sub- jected to electrophoresis (as described under “Methods”) on 10% polyacrylamide gel (bottom). Then the gel was sliced into l-mm slices. Each slice was then extracted with 0.1% sodium dodecyl sulfate as described by Weber and Osborn (IS). The extracts (10 ~11 were examined for the presence of inhibitor type II (top) using 20 units of cGMP-dependent protein kinase (0-O) and CAMP-de- pendent protein kinase (0-O).

--T---

I”

L (QM-‘I cGMP

,

FIG. 5. Effect of the inhibitor type II purified from rat brain on the double reciprocal plots for initial velocity of phosphorylation of arginine-rich his- tone by cGMP-dependent protein ki- nase (20 units) versus ATP, Mg2+, and cGMP. O-O, no inhibitor; O-O, 0.5 pg/ml of inhibitor type II, Panels A and B and 1 wg/ml of inhibitor type II, Panel C.

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L

IotD

FIG. 6. Double reciprocal plots for initial velocity of phosphoryla- tion of various substrates by different protein kinases uers~s concen- trations of substrates at constant levels of purified type II inhibitor.

A, CAMP-dependent protein kinase uusus arginine-rich histone; B, CAMP-dependent protein kinase uersus Leu-Arg-Arg-Ala-Ser-Leu- Gly (kemptide); C, cGMP-dependent protein kinase uersus arginine- rich histone; D, cyclic nucleotide-independent protein kinase type A versus cu-casein.

TABLE III

Substrate specificity for type II inhibitor using cGMP-dependent protein kinase

Purified rat brain type II inhibitor (0.05 pg) was used for each determination. Assay was run with 20 pg of each substrate/sample with exception of kemptide (10 pg). Other assay conditions as under “Methods.”

-cGMP +cGMP

Substrate -Inhibitor +Inhibitor -Inhibitor +Inbibitor II II II II

pmoi-“P incorporated/sample

ff-Casein 0.7 -0.3 1.2 -0.3 Protamine chloride 10 4.7 19 7.3 Lysine-rich histone F, 2.4 1.4 35 13

(type VI Slightly lysine-rich his- 2.3 1.0 14 5.5

tone F,, Slightly lysine-rich his- 13 3.9 41 18

tone F,,, Histone from calf thymus 1.2 -0.3 17 8.6

type II Histone mixture 2.8 0.9 29 13

Arginine-rich histone 1.0 0.4 i8 13

Leu-Arg-Arg-Ala-Ser- 2.4 0.7 25 12

Leu-Glyckemptide)

graphed on Sephadex G-100 columns and the relative potency of type I and type II protein kinase inhibitor estimated by taking advantage of the different mechanisms of action of the two inhibitors. Since type I inhibitor acts noncompetitively with respect to the phosphate acceptor, its potency was deter- mined using a system in which the limiting factor was the

TABLE IV

Tissue distribution of t,ype I and type II inhibitors

Crude inhibitor (250 pg) from each tissue was passed through Sephadex G-100 column (0.2 x 70 cm) as described in Fig. lA; 30-~1 aliquots of each fraction were assayed for type I and II inhibitory activitv.

Tissues Type II inhibi- Tpe I inhibi- Relative po-

to?’ tor” tency ratio’ type IIitYPe I

unitslmg of inhddory proteins

Rat skeletal muscle 540 740 1

Rat pancreas 26 21 1.8

Rat cerebellum 1100 620 2.6

Rat corpus striatum 1000 910 1.6 Lobster tail muscle 750 180 6.0

’ Type II inhibitor was determined in the presence of 20 units of cGMP-dependent protein kinase from lobster tail and 20 pg of argi- nine-rich histone/sample. Other assay conditions are described un- der “Methods.” One unit of inhibition was defined as the amount of type II inhibitor that produced 20% inhibition of phosphorylation of arginine-rich histone.

” Type I inhibitor was determined in the presence of 20 units of CAMP-dependent protein kinase from beef heart and 80 +g of argi- nine-rich histone as substrate. Other assay conditions are described under “Methods.” Under these conditions 1 unit of inhibitor was defined as a amount of type I inhibitor that produced 20% inhibition of phosphorylation.

’ The ratio of type II/type I inhibitory activity for rat skeletal muscle was assumed to be 1.

number of free catalytic subunits of protein kinase. Since type II inhibitor competes with protein substrates, its potency was determined with concentrations of the phosphate acceptor in the range of the K,,, value. As shown in Table IV, homogenates of rat cerebellum and corpus striatum contained high activity of both inhibitors. Rat skeletal muscles contained less type II inhibitor than did brain, while the amount of type I activity in skeletal muscles was comparable to that of brain. Rat pan- creas possessed very low activity of both inhibitors. The lob- ster tail muscle contained more type II than type I inhibitory activity. As shown in Table IV, if the type II/type I activity ratio of rat skeletal muscles is made equivalent to 1, the ratio in the lobster tail muscle was equal to 6.0, and that of rat cerebellum was 2.6.

DISCUSSION

A thermostable, small molecular weight protein which in- hibits nucleotide-dependent and -independent protein kinases has been differentiated from the heat-stable (3-5) inhibitor of CAMP-dependent protein kinase. When the 20,000 x g super- natant from different tissues was incubated at 95” and then chromatographed on Sephadex G-100, the inhibitory activity against protein kinase could be resolved into two distinct molecular forms. These two inhibitors have been termed types I and II and have been separated and characterized. The type I has a molecular weight of 24,000, is heat-stable, inhibits spe- cifically CAMP-dependent protein kinase, is precipitated by 5%

trichloroacetic acid, and noncompetitively antagonizes the phosphate acceptors. Therefore, the type I inhibitor is similar to the protein kinase inhibitor described by Walsh et al. (3-5)

and to the protein kinase modulator described by Donnelly et al. (6, 7).

The type II inhibitor of protein kinase is heat-stable and has a molecular weight of 15,000. It is not precipitated by 5% trichloroacetic acid, inhibits the activity of CAMP- or cGMP-

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dependent protein kinases, and also inhibits the activity of cyclic nucleotide-independent protein kinases. Type II inhibi- tor competitively antagonizes the phosphorylation of different phosphate acceptor proteins. Although this type of inhibitor has a molecular weight very similar to that of the thermosta- ble activator of phosphodiesterase (21, 22), type II inhibitor is not Ca’+-dependent and fails to activate cyclic nucleotide phos- phodiesterase (data not shown).

activity of type II inhibitor, but not that of type I inhibitor, can be decreased or increased by drugs (harmaline and diazepam) which increase (harmaline) or decrease (diazepam) the activ- ity of Purkinje cells and the cGMP content (26). These data support the working hypothesis that type I and type II inhibi- tors are present and functional in living cells, where they may serve as regulatory factors for phosphorylation of endogenous protein substrates.

Recently, Kemp et al. (23, 24) have shown that arginine residues determine the specificity of the phosphate acceptor function in the protein kinase reaction. In a hexapeptide (Arg- Gly-Tyr-Ser-Leu-Gly) used as a model phosphate acceptor, the replacement of arginine with other amino acids resulted in a dramatic reduction in V,,;,, of the CAMP-dependent protein kinase. Moreover, the replacement of serine with alanine transformed this hexapeptide into a competitive inhibitor for the phosphorylation of the original synthetic peptide sub- strate. Our type II inhibitor also interacts competitively with histone, ol-casein, and the synthetic Leu-Arg-Arg-Ala-Ser- Leu-Gly peptide. This similarity between the action of the endogenous type II inhibitor and that of the synthetic inhibitor described by Kemp et al. (23) raises the possibility that there are local primary structures in natural proteins which func- tion as competitive inhibitor of protein kinase. Such structures may be of great importance in inhibiting specific protein phos- phorylation by cyclic nucleotide-dependent and -independent protein kinases.

A pertinent question on the significance of these low molec- ular weight, heat-stable type I and II inhibitors is whether they are really present in tissues or are generated as a post- mortem reaction (enzymatic or otherwise) to the handling of the tissue. We have presented evidence in favor of a natural occurrence of type I and type II inhibitors in tissues. The possibility of postmortem enzymatic formation of these inhibi- tors was ruled out by data obtained from cerebella of rats killed in 1 to 2 s with a focussed microwave irradiation (20); these tissues contained an amount of type I and II inhibitors equal to that of cerebella from rats killed by decapitation (Fig. 2B). Moreoever, when 20,000 x g supernatant of cerebella were chromatographed on Sephadex G-100 before heating, the type I and II inhibitors were eluted from the column with a pattern similar to that of cerebellar homogenates which were chromatographed after heating. Furthermore, not only are type I and II inhibitors present in different amounts depending on the tissue source, but the relative amounts of the two inhibitors changes significantly from tissue to tissue. It has been reported that the inhibitor of CAMP-dependent protein kinase (type I inhibitor according to the present nomencla- ture) can change in certain physiological or pharmacological conditions (25). We have also observed that in cerebellum, the

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3. Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E. H., and Krebs, E. G. (1971)J. Biol. Chem. 246,1977-1985

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A Szmigielski, A Guidotti and E Costadistribution in different tissues.

Endogenous protein kinase inhibitors. Purification, characterization, and

1977, 252:3848-3853.J. Biol. Chem. 

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