Differential Distribution of Protein Kinase C Isozymes in the Various ...

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 262, No. 32, Issue of November 15, pp. 15714-15720,1987 Printed in U.S.A.

Differential Distributionof Protein KinaseC Isozymes in the Various Regions of Brain* (Received for publication, May 26, 1987)

Freesia L. Huang, Yasuyoshi Yoshida, Hiroki Nakabayashi, andKuo-Ping HuangS From the Section onMetabolic Regulation, Endocrinology and Reproduction Research Branch, National Instituteof Child Health and Human Development, National Institutesof Health, Bethesda, Maryland20892

We have previously identified three types of protein stimulation; however, its role in the centralnervous system is kinase C (a Ca2+-activatedphospholipid-dependent ki- largely unknown. Several lines of evidence have implicated nase) isozymes, designated types I, 11, and 111, from rat protein kinase C incontrolling the release of neurotransmitter brain (Huang, K.-P., Nakabayashi, H., and Huang, F. and neuroplasticity (6-11). L. (1986) Proc. Natl. Acad. Sei. U.S. A. 83, 8535Recent studies have shown the presence of multiple protein 8539). These enzymes are different in their elution kinase C genes from different sources. Three distinct cDNA profile from hydroxylapatite column, sites of auto- sequences have been identified from rat, rabbit, bovine, and phosphorylation, andimmunoreactivitytowardtwo human brain cDNA libraries (12-18), and the corresponding types of monoclonal antibodies. Now we describe the human genes have been located on distinct human chromopurification of similar protein kinaseC isozymes from somes (15). The possibility of even greater genetic complexity monkey brain and their regional distribution in the brain. These primate enzymes all have the same molec- of this gene family was suggested by Northern and Southern ular weight of 82,000, and each type of isozyme cross- hybridization analyses (15). All the protein kinase C cDNA reacts with the purified monospecific antibodies examined so far display regions of similarity in the sequences against its corresponding rat brain counterpart iso- of these kinase families. Two major domains encoding for the zyme. These purified antibodies wereused to quantify kinase and phospholipid/phorbol ester binding have been the relative contents of three types of protein kinaseC predicted from the deduced amino acids sequences. Indeed isozymes in various regionsof rat andmonkey brains. both domains of the kinase have been identified and separated In rat brain,cerebellum contained a high level of the following tryptic digestion of the native protein kinase C (19type I isozyme; cerebral cortex, thalamus, and corpus 24). The resultant proteolytic fragments consist of a Ca2+/ callosum were high in the typeI1 enzyme; and olfactory phospholipid-independent protein kinase andaCa2+-indebulb was highest in the type I11 enzyme. In monkey pendent, phospholipid-dependent phorbol ester-binding probrain, the type I isozyme was found to be enriched in tein (23). cerebellum, hippocampus, and amygdala; the type I1 Even though multiple kinase C genes have been identified enzyme was at very high level in caudate, frontal and from several sources, so far the multiple protein kinase C motor cerebral cortices, substantia nigra, and thalaisozymes have only been isolated from rat brain (25). These mus; and the typeI11 enzyme was at the highest level enzymes, designated type I, 11, and I11 protein kinase C, have in olfactory bulb. These results indicate that protein been purified to near-homogeneity. All three isozymes have kinase C isozymes are differentially distributed in var- similar molecular weight of 82,000and allpossess kinase and ious regions of rat and monkey brains and suggest a phorbol ester-binding activities dependent on Ca2+and phosunique role for each isozyme in controlling the differ- pholipid. However, they have different sites of autophosphoent neuronal functions in the brain. rylation and different immunoreactivities toward monoclonal antibodies, indicating their differences in the primary structures. The role of these kinases in the regulation of cellular Protein kinase C (a Ca2+-activatedphospholipid-dependent function and their genetic origin are still unknown. In this kinase) is thought to play an important role in controlling report we provide evidence demonstrating that these three several cellular processes. This enzyme has also been identi- types of protein kinase C isozymes are also present in monkey fied as a receptor for tumor-promoting phorbol esters which brain. In addition, we have identified the predominant preselicit pleiotropic physiological responses including differentia- ence of the various isozymes in distinct regions of the rat and tion, secretion, metabolism, sensory transduction, and gene monkey brains. These findings will facilitate greatly in preexpression (1, 2). In viuo, protein kinase C is believed to be paring a specific type of protein kinase C isozyme for bioactivated by diacylglycerol, an intracellular second messenger, chemical study and identifying the physiological substrates generated from the signal-induced breakdown of phosphati- for these kinases. dylinositol 4,5-bisphosphate (3). Protein kinase C is present EXPERIMENTALPROCEDURES ubiquitously in a variety of tissues and is most concentrated in the brain (4, 5 ) . In peripheral tissues protein kinase C is Materials-The following materials were obtained from the indiknown to mediate cellular responses to cell surface receptor cated sources: histone IIIs, EGTA,’ and poly(L-lysine)-agarosefrom

* The costs of publication of this article were defrayed in part by

Sigma; [ T - ~ ~ P I A T[3H]phorbol P, 12,13-dibutyrate (PDBu), and ‘“1protein A from DuPont-New England Nuclear; phosphatidylserine

the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed Bldg. 6, Rm. 126, NIH, Bethesda, MD 20892.

The abbreviations used are: EGTA, (ethylenebis(0xyethy1enenitrilo)ltetraacetic acid; PS, phosphatidylserine; PDBu, phorbol 12,13-dibutyrate; SDS, sodium dodecyl sulfate; PAGE polyacrylamide gel electrophoresis.

15714

Protein Kinase C Isozymes in Brain (PS) and1,2-dioleoylglycerolfrom Avanti Polar Lipids, Birmingham, AL; horseradish peroxidase-conjugated rabbit anti-goat IgG from Cooper Biochemicals; DEAE-cellulose (DE52) and GF/C glass fiber filters from Whatman; and hydroxylapatite, sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and protein determination reagents from Bio-Rad. Methods-Protein kinase C activitywas measured in Tris-C1 buffer (30 mM), pH 7.5, containing 6 mM magnesium acetate, 0.12 mM [ y 32P]ATP,0.4 mM CaC12,1 mg/ml histone IIIS, 40 pg/ml PS, 8 pg/ml 1,2-dioleoylglycerol,and protein kinase. The Ca2+-and phospholipidindependent activity was measured under the same condition without Ca2+ andphospholipid but containing 1 mM EGTA. Measurement of 32Pincorporation into protein was performed as described previously (26). One unit of protein kinase activity is defined as the amountof enzyme catalyzing the incorporation of 1 nmol of phosphate from ATP into histone IIIS under the standard assay condition. Lipid vesicles containing PS or PSplus 1,2-dioleoylglyceroiwere prepared by evaporation of these compounds previously dissolved in chloroform to dryness under NP and resuspended in 20 mM Tris-CI buffer, pH 7.5, with sonication and vortexing. Binding of [3H]PDBuwas measured in 0.2 ml of reaction mixture containing 30 mM Tris-C1 buffer, pH 7.5, 6 mM magnesium acetate, 0.5 mg/ml bovine serum albumin, 20 pg/ml PS, 0.25 mM EGTA, 0.5 mMCaC12, 50 nM [3H]PDBu, and protein kinase C. The reaction mixture was incubated a t room temperature for 30 min and followed by incubation a t 4 “C for 30 min after adding 0.5 ml of 30% DEAEcellulose in 20 mM Tris-C1 buffer, pH 7.5. Bound [3H]PDBu was separated from free ligand by filtering through Whatman GF/C glass fiber filter and washed five times each with 1 ml of 20 mM Tris-C1 buffer, pH 7.5. The nonspecific binding was determined under the same condition with the addition of 100 pM nonradioactive PDBu. Monkey brain (male Macaca fascicularis, 3-4 years of age) protein kinase Cwas purified by a procedure similar to that described for the rat brain enzyme (25,27). Brain was removed immediately after monkey was killed under deep pentobarbital anesthesia. One hemisphere of fresh cerebrum and whole cerebellum (40 g total wet weight) were homogenized with Polytron in 200 ml of Buffer A (20 mM TrisC1 buffer, pH 7.5, containing 1 mM dithiothreitol, 0.5 mM EDTA, 0.5 mM EGTA, 10% glycerol, 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and 2 pg/ml each of leupeptin, chymostatin, and pepstatin A). The homogenate was centrifuged a t 34,000 rpm for 45 min in a Beckman 35 rotor. The resulting pellet was extracted once with 150 ml of the same buffer. The combined high speed supernatant fluid was applied to a DEAE-cellulose column (2.5 X 20 cm) equilibrated with Buffer B (20 mM Tris-C1, pH 7.5, containing 1 mM dithiothreitol, 0.5 mM EDTA, 0.5 mM EGTA, and 10% glycerol). Protein kinase C was eluted by a 0-0.3 M KC1 gradient in Buffer B. Subsequent purification by phenyl-Sepharose (2.0 X 9 cm, using a 1.5-0 M KC1 gradient), Sephacryl S-200 (2.5 X 95 cm), polylysineagarose (1.5 X 12 cm, using a 0-0.8 M KC1 gradient), and hydroxylapatite (1.5 X 6 cm, usinga 0.02-0.3 M KPO, gradient) column chromatography was as described previously (25,27). C isoMonospecific antibodies against different protein kinase zymes were purified from the polyclonal goat anti-rat brain protein kinase C antibodies (5) according to the method of Olmsted (28). Briefly, purified rat brain isozymes were transferred to nitrocellulose membrane following SDS-PAGE. These membranes were incubated with goat anti-rat brain protein kinase C antibodies a t room temperature for 2 h. The specific antibodies againsteach isozyme were eluted from the membrane with 0.1 M acetic acid and immediately neutralized with 2 M Tris. Various tissues from rat (male Sprague-Dawley, 200-250 g) and monkey (male M. fasciclllaris) brains were dissected immediately after the animals were killed under pentobarbital anesthesia. Tissueswere quickly kept in dry ice and stored a t -70 “C until use. Homogenates were prepared by sonication of the tissue in 5 volumes (v/w) of Buffer A without Nonidet P-40 and followed by heating a t 90 “C for 5 min in SDS-PAGE sample buffer. Immunoblotting was camed out by 2 3 1gel) 2 3and 1 2 initial separation of proteins (50 pg) by SDS-PAGE1(10% followed by electrophoretic transfer to nitrocellulose membrane (29). The membrane was incubated successively with 3% gelatin in TBS (20 mM Tris-C1 buffer, pH 7.5, containing 500 mM NaCI) for 30 min, with various monospecific antibodies or unfractionated antibodies in TBS containing 1%gelatin for 90 min, with affinity-purified rabbit anti-goat IgG (2000-fold dilution) for 1h, and with “’1-protein A (0.4 pg, 4-6 pCi/2O ml) for 1 h. Following each incubation the membrane was washed extensively with TBS containing 0.05% Tween 20. The immunoreactive bands were visualized by autoradiography. The po-

15715

sitions of M,marker proteins were localized by brief staining with 0.1% Amido black in 25% isopropyl alcohol containing 10% acetic acid. Three types of protein kinase Cin thevarious tissue homogenates were quantified by the binding of‘?-protein A to the antigenantibody complex following immunoblotting (30) using monospecific antibodies. The standardcurves were constructed by using 20-200 ng of purified type I, 11, and 111 rat brain protein kinase C. The amount of IZSI-proteinA bound,as determined by densitometric tracing of the autoradiograms, was proportional to the quantityof protein kinase C applied to the gel. Routinely, a t least three amounts of the purified kinase were included as standardto quantify the level of each isozyme in the tissue homogenates. RESULTS

Characterization of Momspecific Antibodies-The antibodies purified by immunoabsorption (see “Methods”) were specific to each type of isozyme used as immunogen (Fig. l), whereas the unfractionated polyclonal antibodies recognized all three forms of protein kinase C isozymes, though with different potency. The immunoreactive protein band detected by immunoblot analysis was the 82-kDa protein of the native kinase. However, the purified monospecific antibodies also recognize the proteolyzed fragments of 45-48-kDa catalytic unit and 33-38-kDa phorbol ester-binding unit of each isozyme. The immunoreactive signal as quantified by 1261-protein A binding with each monospecific antibody was proportional to theamount of each corresponding isozyme (from 20 to 200 ng) applied even in the presence of other isozyme (data not shown). Addition of each individual isozyme to the purified monospecific antibodies competed away the corresponding immunoreactive signal detectable on the nitrocellulose. Therefore, these monospecific antibodies were suitable for quantifying the relative content of each protein kinase C isozyme in various tissues. Differential Distribution of Protein Kinase C Isozymes in Various Regions of Rat Brain-Rat brain was dissected into various regions, and theywere analyzed for the threetypes of protein kinase C by immunoblotting with monospecific antibodies. Type I protein kinase C (Fig. 2A) was found to be enriched in cerebellum ( l a n e 2) and paraflocculus ( l a n e 8); the type I1 enzyme(Fig. 2B) in cerebral cortex ( l a n e I ) , thalamus ( l a n e 5),and corpus callosum ( l a n e 7);and thetype

B

A

C

D

-45K -31K “dye front 31 2 3

FIG.1. Characterizationof monoepecific antibodiesagainst type I. and I11 rat brain protein kinase C. Approximately 0.1 pg each of the purified type I ( l a n e I ) , I1 ( l u n e 2), and 111 ( l a n e 3) protein kinase Cwas electrophoretically transferred to nitrocellulose membrane after SDS-PAGE. Eachpiece of membrane was incubated with monospecific type I (pane A ) , type I1 (panel B ) , type 111 (panel C), or unfractionated ( p a n e l D ) antibodies. The immunoreactive bands were detected by 4-chloro-1-naphthol after incubation with horseradish peroxidase-conjugated rabbit anti-goat IgG.

II.

Protein Kinase C Isozymes in Brain

15716

A

B

C 12345678

D 94K 82K

-

- _" 45K 68K

-0 Fraction Number

FIG. 3. Separation of three monkey brain proteinkinase C isozymes by hydroxylapatite column chromatography.Protein kinase C purified from polylysine-agarose column chromatography 31K was concentrated in Amicon ultrafiltration cells and dialyzed against 0.02 M KPO, buffer, pH 7.5, containing 0.5 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, and 10% glycerol before applying to a hydroxylapatite column (1.5 X 6 cm). The enzymes were eluted with dye front -. a KPO, gradient (0.02-0.3 M) delivered by a LKB Ultrograd gradient FIG.2. Identification ofdifferent protein kinase C isozymes maker a t a flow rate of 10 ml/h for 16 h. Fractions of 3 ml were in the various regions of rat brain. Samples (50 pg of protein) collected and 3 pl of the fraction was used for the measurements of from frontal cerebral cortex (lane I ) , cerebellum (lane 2), brain stem protein kinase activity withCa2+,PS, and 1,2-dioleoylglycerol (0)or (lane 3), olfactory bulb (lane 4 ) , thalamus (lane 5), hypothalamus with EGTA (0)for 5 min. [3H]PDBu bindingwas measured with 20 (lane 6).corpus callosum (lane 7), and paraflocculus (lane 8) were pl of fraction with Ca2+and PS (A)or with PS and EGTA (A). The specific activity of [3H]PDBu was 9000 cpm/pmol and counting subjected to SDS-PAGE (10% gel) and electrophoretic transfer to nitrocellulose membrane. Immunoblot analysis was carried out by efficiency was 40%. The conductivity (- - -) of the effluent solution using monospecific antibodies against type I @anelA ) , type I1 (panel was determined by an on-line conductivity meter. The threeactivity B ) , and type I11 ( p a n e l C ) protein kinase C or unfractionated anti- peaks were designated type I, 11, and I11 protein kinase C. bodies (panelD).The immunoreactive bands were detected by lZ5Iprotein A binding. kinase C isozymes is shown in Table I. Three types of the

-

-

-

I11 enzyme (Fig. 2C) in olfactory bulb (lane 4 ) . Among the various brain regions tested, the brain stem (lane 3 ) and hypothalamus (lane 6 ) contained relatively low level of each type of protein kinase C isozyme. Immunoblot analysis with unfractionated antibodies (Fig. 2 0 ) indicated that cerebral cortex, cerebellum, and corpus callosum contained higher level of protein kinase C than those in brain stem andhypothalamus. Although the antibodies could detect the proteolytic fragments of 45-48-kDa catalytic unit and33-38-kDa phorbol ester-binding unit of protein kinase C, the major immunoreactive band detected in all these tissue extracts was the 82kDa protein, which corresponded to thenative enzyme. These results indicated that the preparation of the various tissue extracts did not cause a gross degradation of the kinase. Purification of Protein Kinase C Isozymes from Monkey Bruin-To determine that protein kinase C also appears in isozymic forms in the monkey(M. fmcicularis) brain, we purified the kinases following the method described for rat brain enzymes (25). Three protein kinase C activity peaks were identified by hydroxylapatite column chromatography (Fig. 3). All these kinase activity peaks also correspond with the [3H]phorbol 12,13-dibutyrate binding. Both the kinase and phorbol ester-binding activities were dependent on Ca2+ and phospholipid. The slight Ca2+/PS-independent kinase and Ca2+-independentphorbol ester-binding activities in the type I enzyme (first activity peak) is most likely due to the presence of protease-degradedenzyme in these fractions. The purified monkey brain protein kinase C isozymes all had a molecular weightof 82,000 as determined by SDS-PAGE (Fig. 4). The major Coomassie Blue-stained protein bands corresponded well with the kinase and phorbol ester-binding activities, indicating the near-homogeneity of the purified enzymes. A summary of the purification of monkey brain protein

kinase were purified to 300-600-fold with an overall yield of near 20%. The specific activities were ranging from 3000 to 6000 units/mg, with type I1 and I11 enzymes roughly twiceas active as thetype I enzyme. The three types of monkey brain protein kinase C were cross-reactive with the polyclonal antibodies raised against a mixture of rat brain isozymesas evidenced by immunoblotting (Fig. 5A). Small quantity of a 45-kDa protein, presumably the proteolytic fragment of the kinase, was also detected in fractions containing the type Ienzyme. The three types of monkey brain protein kinase C could also be detected by purified monospecificantibodies against each type of rat brain isozyme (Fig. 5, B-D). Even the 45-kDa proteolyzed fragment (fractions 22, 24) was also detected by type I antibody (data not shown). These results demonstrate that the immunoreactive determinants of the corresponding protein kinase C isozymes in rat andmonkey brains aresimilar. Furthermore, incubation of the monkey brain extract with the unfractionated polyclonal antibodies resulted in theimmunoprecipitation of greater than 90% of the total protein kinase C activity, indicating that themajority of monkey brain enzymes can be recognized by the goat anti-rat brainprotein kinase C antibodies. Distinct Distribution of Protein Kinase C Isozymes in Various Regionsof Monkey Brain-Immunoblot analysis for the relative levels of the protein kinase C isozymes in thedifferent regions of monkey brain is shownin Fig. 6. The type I enzyme is more enriched in cerebellum (lane 2), hippocampus (lane 7), and amygdala (lane10)and the type I1 enzyme in thalamus (lane 5), substantia nigra (lane 91, caudate (lane 111, and frontal andmotor cortices of cerebrum (lanes 12 and 13).The type I11 enzyme was present in comparable quantity in most of the regions tested, with olfactory bulb (lane 1 ) having the highest level and pons (lane 3 ) and medulla oblongata (lane 4 ) the lowest. The total levels of protein kinase C in pons,

Kinase Protein

C Isozymes Brain in

15717

94K82K68K-

45K -

31K-

Dye Front-

2224262830323436341 45505558606264666871 Fraction Number FIG. 4. SDS-PAGE of the effluentfractions from hydroxylapatite column. Fractions (40 pl each) were analyzed by SDS-PAGE (10%gel), and thegel was stained with Coomassie Blue. The fraction number corresponded to thatof Fig. 3.

TABLEI Summary of the purification of monkey brain protein kinase C

DISCUSSION

The results presented in this report demonstrate, for the first time, the presence of three protein kinaseC isozymes in Relative Fraction primate brain and regional the enrichment of different protein protein activity purification kinase C isozymes in both rat and monkey brains. The protein mg unitslmg % -foM kinase C isozymes from the monkey brain were similar to Crude extract 1560 11.2 100 1 their rat brain counterparts in the chromatographicbehavior DEAE-cellulose 31.6 420 2.8 76 during purification, the molecular weight (82,000), the dePhenyl-Sepharose 59 173 58 15 pendence of Ca2'/PS for the kinase and phorbol ester-binding Sephacryl S-200 27 317 28 49 Polylysine 4490 1.6 41 401 activities, the regional enrichment of different isozymes, and Hydroxylapatite their immunological properties. These findings suggest that Type I 0.12 3250 290 2.2 each type of the monkey brain protein kinase C isozyme is Type I1 0.27 6110 9.4 546 By using immunoblot homologous to its rat brain counterpart. T w e I11 0.25 6760 9.6 604 analysis with monospecific antibodies, we have also detected these isozymes in fish (rainbow trout) and chicken brains. It medulla oblongata, and hypothalamus as detected with unappearsthattheseproteinkinase C isozymes are widely of vertebrates. Recently, Worley et al. fractionated antibodywere also lower than thosefound in the distributed in the brain (42) mapped the phorbol ester-binding sites and also sugother regions. The relative content of three types of protein kinase C gested the wide distribution of protein kinase C in rat brain. isozymes in different regions of monkey and rat brains was Although phorbol ester binding could not differentiate these determined by '2sI-protein A binding following immunoblot- isozymes, brain regions found high in binding (42), such as cerebral cortex, cerebellum, amygdala, hippocampus,caudate, ting using purifiedisozymes as standards (Table 11). The and substantia nigra, were also found to be high in either or pattern of distribution of these isozymes in different brain all types of protein kinase C isozyme in the presentstudy. regions is quite similar between these two animals. In cereAmong the various rat peripheral tissuesanalyzed, we have bellum and olfactory bulb the typeI and 111 enzymes, respec- identified type I1 protein kinase C as the major enzyme in tively, accounted for greater than 50% of all three protein thymus and spleen. Generally, type I1 enzyme has the most kinase C isozymes togetherinthese tissues. The cerebral widespread presence among various tissues, while the type I cortex contained relativelyhigh level of all three types of enzyme has been identified only in brain.2 The presence of isozymes with the type I1 enzyme being the most abundant. different protein kinaseC isozymes in the various tissues and regions of brain is likely a result of differential expression of In monkey brain, hippocampus, substantia nigra, amygdala, and caudate are also high in most types of isozymes; these * Y. Yoshida, F. L. Huang, H. Nakabayashi, and K.-P. Huang, regions were not analyzed for rat brain. manuscript in preparation. i$ozVms ~~. Total Specific ~

d

~~

15718


A 94K82K-

68K45KFIG. 5. Immunoblot analysis of the effluent fractions from hydroxylapatite column by polyclonal and isozyme-specific antibodies. Fractions (10 pl each) were separated by SDS-PAGE (10% gel) and electrophoretically transferred to nitrocellulose membrane. The immunoreactive proteins were revealed by using goat antirat brain polyclonal ( p a n e l A ) , type Ispecific ( p a n e l B ) , type 11-specific(panel C ) , or type 111-specific (panel D ) antibodies. The immunoreactive bands were detected by using '251-proteinA.

31K-

22242628303234363841455055586062~666871 Fraction Number

B 82K-

C 82K+

D 82Kmultiple protein kinase C genes. Recent studies from many laboratories have identified the presence of at least three cDNA codingfor protein kinase C (12-18). Brandt et al. (43) have demonstrated that these various protein kinase C gene transcripts areheterogeneously distributed in various regions of rat brain. In rat cerebellum, these authors identified the PKC-I transcripts in Purkinje cells andthe alternatively spliced PKC-I1 and -111 transcripts in granular cells (43). Preliminarily, by immunocytochemistry, we detected type I isozyme in Purkinje cells and type I1isozyme in granular c e k 3 Furthermore, we.detected a high level of the type I1 isozyme inspleen which had been shownto express the highly homologous PKC-I1 and PKC-I11 mRNAs (16, 43). It seems likely that the type I1 protein kinase C, which is the main isozyme in cerebellar granular cells and spleen, is encodedby the PKC-I1 or PKC-I11 genes of Knopf et al. (16) and the type I protein kinase C by the PKC-I gene. Distinct enrichment of protein kinase C isozymes in different brain regions may reflect important differences in the functions of individual isozyme. These enzymes may respond to different stimulators or inhibitors in modulating cellular F. L. Huang, Y. Yoshida, H. Nakabayashi, W. S. Young, and K.P. Huang, unpublished data.

" 0

-

"

functions. Detailed characterization of these enzymes is essential to understanding their regulatory functions. The purification method described here may apply to similar enzymes from other sources. However, separation of these closely resembledenzymes is very time-consuming. By selecting a starting material highly enriched in a particular type of enzyme should greatly facilitate the purification and characterization of individual isozyme and the identification of their physiological substrates. Based on the immunoblot analysis, we predict that cerebellum would be a good source for the type I enzyme, cerebral cortex for the type 11, and olfactory bulb for the type I11 enzyme. In the peripheral tissues, both thymus and spleen are good sources for the type I1 enzyme. Recent studies have provided ample evidence to link the modulation of neuronal activity to the activation of protein kinase C by either triggering the inositol phospholipids signaling pathway or addition of phorbol esters (6-11). In particular, this kinase is thought to play a role in controlling the release of neurotransmitter and registration of long term memory. The enrichment of different protein kinase C isozymes in different regions of brains does not seem to coincide with any one type of neurotransmitter system (31). However, it is likely that each protein kinase C isozyme mayparticipate


15719

TABLE I1 Contents ofprotein kinuse C isozymes in different regwns of monkey and rat brains Quantitation of protein kinase C isozymes was carried out by densitometric tracing and integrating of the autoradiograms similar to those shown in Figs. 2 and 6. Routinely, three increasing amounts of purified rat brain isozymes I, 11, or 111, determined by the dye-binding method of Bradford (44)with bovine plasma albumin as standard, were also included for quantitation of each isozyme. The amount of each isozyme in different monkey brain regions was obtained by two duplicate determinations using samples from the left hemisphere of two monkeys. Protein kinase C content in ratbrain regions was estimated by three determinations using samples each Dooled from two rats. brain Monkey Rat brain Region I

I1

111

I

I1

ng/mg protein Olfactory bulb 218 f 56 364 f 118 690 f 138 380 Cerebellum 838 f 180 408 f 84 146 f 1176 72 a 138 f 28 64 f 38 Pons -a 48 f 8 52 f 34 Medulla oblongata Thalamus 302 f 58 631 f 184 288 f 140 204 Hypothalamus 226 f 136 44 f 260 24 f 84 86 Hippocampus 1178 f283 76 f 40 384 f 50 Corpus callosum 116 f 46 433 f 76 236 f 110 Substantia nigra 222 f 66 839 f 120 412 f 202 Amygdala 902 f 260 568 k 406 122 f 178 Caudate 530 f 980 104 f 422 100 f 178 Frontal cortex 316 f 78 622 f 38 346 f 196 316 Motor cortex 270 f 112 592 f 110 354 f 184 a The kinase level was too low to be accurately determined.

-

A

B

c Bib”

m

1 2 3 4 5 6 7 8 910111213

D

82K + 68K 94K

45K

-

31K

-

dye front

-

C isozymes in the various regions ofmonkey brain. Samples (50 pg of protein) from olfactory bulb (lane I ) , cerebellum (lane 2 ) , pons (lane 3 ) , medulla oblongata ( l a n e 4 ) , thalamus (lane 5), hypothalamus (lane 6),hippocampus ( l a n e 7),corpus callosum (lane 8),substantia nigra (lane 9),amygdala (lane IO), caudate (lane II), frontal cortex (lane I2), and motor cortex ( l a n e 13) were analyzed by immunoblotting using monospecific antibodies against type I(panelA ) , type I1 ( p a n e l B ) , and type I11 (panel C ) proteinkinase C, proteinkinase or unfractionated antibodies(panel D).The immunoreactive bands were detected by 1251-proteinA. FIG. 6. Analysis of different protein kinase

111

ng/mg protein f 16 f 124 344

-a f 572 16 k153 16

380 f 56 672

f 28 897

188 f 48 897 f 30 324

f 126 f 76

69 k 120 15 f 112 321 f 245 44

f 40 f 126 f 62

f 123 360

f 80

f 90

488 f 98

in specialized regulatory functions by virtue of its predominance in a specific brain region. For instance, the presence of highlevel of thetype I protein kinase C in cerebellum, hippocampus, and amygdala may be indicative of its importance in learning and memory. So far, this isozyme has been identified only in brain and not in any peripheral tissues.’ Further study to correlate the expression of neuronal functions and mode of activation of protein kinase C isozymes by translocation (32-36), protease-mediated activation (37-41), and autophosphorylation (27) are essential to define the role of these isozymes in the central nervous system. Acknowledgments-We would like to thank Drs. Leslie G. Ungerleider and David P. Friedman for their help in dissecting the monkey brain. REFERENCES

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