Proc. Natl. Acad. Sci. USAVol. 76, No. 5, pp. 2475-2479, May 1979Neurobiology

Ca2+ and cyclic AMP regulate phosphorylation of same twomembrane-associated proteins specific to nerve tissue

(protein kinase/protein phosphorylation/synaptic membranes/synaptosomes)

WERNER SIEGHART*, JAVIER FORNt, AND PAUL GREENGARDDepartment of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510

Contributed by Paul Greengard, March 8, 1979

ABSTRACT It was shown previously that addition of cyclicAMP (cAMP) to a synaptic membrane fraction incubated with1[y-32PJATP stimulated the phosphorylation of two proteins,designated proteins Ia and Ib, found only in nerve tissue. Ad-dition of Ca2+ plus veratridine to synaptosomes preincubatedwith 32p; stimu ated the phosphorvlation of two proteins withsimilar apparent molecular weights. Various techniques havenow been used to determine whether the two proteins phos-phorylated in synaptosomes in the presence of Ca2+ plus vera-tridine are the same as proteins Ia and lb phosphorylated insynaptic membranes in the presence of cAMP. The proteinsphosphorylated by the two procedures were extracted undersimilar conditions, had similar apparent molecular weights andcharges, and were digested by collagenase at similar rates andto the same radioactive intermediates and end products. Fur-thermore, the two sets of proteins were digested by three otherproteolytic enzymes to phosphopeptides with similar molecularweights. The results indicate that Ca2+ and cAMP are each ca-pable of regulating the phosphorylation of proteins Ia andlb.

Studies in several laboratories have provided evidence thatcyclic AMP (cAMP) may mediate or modulate the presynapticrelease or postsynaptic actions of neurotransmitters at certaintypes of synapses (1-5). Other studies indicate that most, if notall, of the effects of cAMP in eukaryotic cells are mediatedthrough regulation of the phosphorylation of specific proteins(for reviews, see refs. 6-8). A protein whose phosphorylationwas highly stimulated by cAMP was found in synaptic mem-brane fractions (9). This protein, designated protein I, is com-posed of two types of polypeptide with apparent molecularweights of 86,000 (protein Ia) and 80,000 (protein Ib) (10).Proteins Ia and lb have been purified and some of their mo-lecular properties have been characterized. These two poly-peptides are similar to one another and have a number ofunique properties (10).The important role of Ca2+ in the physiology of the nervous

system is well documented (11, 12). However, the biochemicalmechanisms underlying certain of its physiological effects, suchas stimulus-secretion coupling and synthesis of catecholamines,have not yet been elucidated. Veratridine and high K+ increaseCa2+ transport across synaptosomal membranes, through amechanism involving membrane depolarization (13). Recently,it has been found, by using synaptosomal preparations prein-cubated in vitro with 32P1, that veratridine and high K+ eachstimulated the incorporation of 32p into two specific proteinswith apparent molecular weights of 86,000 and 80,000 (14).This phosphorylation in intact synaptosomes was absolutelydependent on the presence of Ca2+ in the incubation mediumand was not mimicked by cyclic nucleotides. In view of thesimilarity in apparent molecular weight between protein I

(referred to in this study as [protein I]CAMP) and the proteinphosphorylated in intact synaptosomes under the influence ofdepolarizing agents plus Ca2+ (referred to in this study as[protein I]Ca2+), we compared some of the biochemical prop-erties of these proteins.

METHODSPhosphorylation in Crude Synaptosomal Fraction. A crude

synaptosomal (P2) fraction was prepared from the cerebralcortices of eight Sprague-Dawley rats as described (14).

Half of the synaptosome preparation was resuspended in 10ml of ice-cold modified Krebs-Ringer buffer (KRB) [compo-sition, in mM: NaCI, 132; KCI, 4.8; MgSO4, 2.4; ethylene glycolbis(f3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA),0.1; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 20;glucose, 10]. This KRB was adjusted to pH 7.4 with NaOH andoxygenated by bubbling with a stream of 02 before use. Thesynaptosome suspension was preincubated with 0.75 mCi (1 Ci= 3.7 X 1010 becquerels) of 32p per ml for 30 min at 37°C, and200-,1 aliquots of the synaptosome suspension were then addedto 100 ,l of KRB containing veratridine or veratridine plus Ca2+in final concentrations of 100 uM and 1 mM, respectively. In-cubation was carried out for 15 sec. In the standard procedure,the reactions were terminated by the addition of 10 vol of ice-cold 5 mM Zn acetate. [Zn2+ previously was shown to inhibitprotein phosphatases and, to a lesser extent, protein kinases (15,16). In preliminary experiments it was found that, in the ab-sence of Zn2+, [protein I]CAMP and [protein I]Ca2+ becamecompletely dephosphorylated during a single centrifugationaimed at collecting synaptosomes or membranes after labelingof the proteins with 32P and that dephosphorylation duringcentrifugation and subsequent extraction could be totallyprevented by terminating the reaction with Zn acetate.] In theexperiment of Fig. 1, some of the reactions were terminated bythe addition of 0.5 vol of a "sodium dodecyl sulfate (Na-DodSO4)-stop solution" (10% NaDodSO4/100 mM Tris-HCI,pH 7.4/5 mM 2-mercaptoethanol/0.1 g of sucrose per ml/0.02mg of bromphenol blue per ml).

Phosphorylation in Synaptosomal Membrane Fraction.The other half of the synaptosome preparation was resuspendedin 20 ml of distilled water and a fraction enriched in synapticmembranes (M1) was prepared as described (10). This fractionwas resuspended in 10 ml of 2 mM Tris-HCI, pH 7.4/2 mMEGTA. Aliquots were incubated in a reaction mixture (finalvolume, 100 Ml) containing 50 mM Tris-HCI at pH 7.4, 10 mM

Abbreviations: NaDodSO4, sodium dodecyl sulfate; cAMP, cyclic AMP;KRB, Krebs-Ringer buffer; EGTA, ethylene glycol bis(,B-aminoethylether)-N,N,N',N'-tetraacetic acid.* Present address: Psychiatrische Universitaetsklinik, Department ofBiological Psychiatry, University of Vienna, Vienna, Austria.

t Present address: Department of Pharmacology, University of Bar-celona, Faculty of Medicine, Barcelona, Spain.

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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Membranes SynaptosomesAfter Before Before After

extraction extraction extraction extraction

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FIG. 1. Autoradiogram showing cAMP-dependent phosphoryl-ation of proteins Ta and lb in synaptosomal membranes and Ca2+-dependent phosphorylation of proteins Ia and lb in intact synapto-somes. Synaptosomal membranes were incubated with 4 juM [,y-32P]ATP in the absence or presence of 10MqM cAMP for 15 sec. Intactsynaptosomes were preincubated with 32p; for 30 min and then wereincubated with 100yM veratridine in the absence or presence of 1 mMCa2+ for an additional 15 sec. The phosphorylation reactions wereterminated by the addition of either 0.5 vol of NaDodSO4-stop solu-

tion (lanes 3, 4, 5, and 6) or of 10 vol of 5 mM Zn acetate (lanes 1, 2,7, and 8). Proteins were extracted from the Zn acetate-treated sam-ples; to 100 ,ul of the pH 6 extract, 50 ,ul of NaDodSO4-stop solutionwas added. Aliquots (100 p1A) of all samples were then subjected toNaDodSO4/10% polyacrylamide gel electrophoresis and autoradi-ography as described (14).

MgCl2, 1 mM 3-isobutyl-1-methylxanthine, and 4 jiM [y-32P]ATP (1-3 X 107 cpm/nmol), in the absence or presence of10 ,qM cAMP. After preincubation for 30 sec at 300C, the re-

action was initiated by the addition of the ['y-32P]ATP and in-cubation was carried out for 15 see at 300C. The reaction wasterminated as described for the crude synaptosomal fraction.

Extraction of Phosphorylated Proteins. After terminationof the phosphorylation reaction by the addition of 10 vol of Znacetate, the samples were centrifuged at 3000 rpm for 10 min.The supernatant was discarded and the pellet was resuspendedin 3 ml of 0.01 M citric acid. The pH of the suspension was

adjusted to 3.0 by addition of 0.1 M citric acid, and the sus-

pension was centrifuged at 14,000 rpm for 20 min. The pelletwas discarded and the supernatant was adjusted to pH 6.0 byaddition of 1 M Tris-maleate (pH 7.4). The precipitate was

removed by a 10-min centrifugation at 6000 rpm, and EDTAwas added to the supernatant to a final concentration of 1 mMto complex any Zn2+ remaining in the extract. This extract isreferred to as the "pH 6 extract."Two-Dimensional Electrophoresis. Nonequilibrium pH

gradient electrophoresis was performed essentially as describedby O'Farrell et al. (17). The gels to be used for the first di-mension were poured to a height of 12 cm in glass tubes (130X 4 mm inside diameter) with a gel mixture composed of 9.2M urea, 2% Nonidet P-40, 4% acrylamide (from a 30% stocksolution of 28.4% acrylamide and 1.6% bisacrylamide), and 2%Ampholine (pH 3.5-10). This mixture was polymerized in thetubes by addition, immediately before use, of 14 !l of

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FIG. 2. Autoradiogram showing results of two-dimensionalelectrophoresis of proteins Ia and Ib after phosphorylation in thepresence of cAMP in synaptosomal membranes and in the presence

of Ca2+ in intact synaptosomes. Synaptosomal membranes were in-cubated with 4 ,uM [y-32P]ATP in the presence of 10MgM cAMP for15 sec. Intact synaptosomes were preincubated with 32Pi for 30 minand then incubated with 100 ,uM veratridine in the presence of 1 mMCa2+ for an additional 15 sec. The phosphorylation reactions were

terminated by the addition of Zn acetate and the proteins were ex-

tracted. Aliquots (100 Al) of the extracts were subjected to nonequi-librium pH gradient electrophoresis in the first dimension and Na-DodSO4polyacrylamide gel electrophoresis in the second dimension.(a) Extract from synaptosomal membranes phosphorylated in thepresence of cAMP. (b) Extract from intact synaptosomes phos-phorylated in the presence of veratridine plus Ca2+. (a + b) Mixtureof extracts obtained from membranes phosphorylated in the presenceof cAMP and synaptosomal fraction phosphorylated in the presenceof veratridine plus Ca2+. The spots originating from 32P-labeled[protein 'ICAMP, 32P-labeled [protein IJCa2+, or both are encircled.

N,N,N',N'-tetramethylethylenediamine and 20 jil of 10%ammonium persulfate per 10 ml. The gels were overlayed withwater and polymerized for 1-2 hr. To 100 ,l of the pH 6.0 ex-

tract, 100 mg of urea, 20 mg of sucrose, 10 til of 40% Ampho-line, 26 ,l of Nonidet P-40, and 10 IA of 2-mercaptoethanolwere added. This mixture was layered on top of the gel and thetube was filled with a mixture containing 8 M urea and 1%Ampholine. Electrophoresis was then performed at 400 V for3 hr as described by O'Farrell et al. (17). The gels were removedfrom the glass tubes and equilibrated with 20 ml of 2.3% Na-DodSO4/62.5 mM Tris-HCl, pH 6.8/0.001% bromphenolblue/5% 2-mercaptoethanol for 2 hr. They were then placedon top of an NaDodSO4/polyacrylamide gel consisting of a2-cm-long 5% stacking gel and a 13-cm-long 7% separating gel.The electrophoresis, staining of the gel, and autoradiographywere performed as described (14).

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Proc. Natl. Acad. Sci. USA 76 (1979)

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Ca2+ cAMP Ca2+ cAMP Ca2+ cAMP Ca2+ cAMPFIG. 3. Autoradiogram showing effect of collagenase on phosphorylated proteins present in extracts of membranes and intact synaptosomes.

Synaptosomal membranes were phosphorylated in the presence ofcAMP and intact synaptosomes were phosphorylated in the presence of ver-atridine plus Ca2+', as described in the legend to Fig. 2. The phosphorylation reactions were terminated by the addition of Zn acetate, and theproteins were extracted and incubated with collagenase. At the indicated times after the addition of collagenase, aliquots were removed, thecollagenase reaction was terminated by the addition of 0.5 vol of NaDodSO4-stop solution, and the samples were subjected to NaDodSO4/poly-acrylamide gel electrophoresis and autoradiography. Lanes labeled Ca2+ and cAMP correspond to samples prepared from intact synaptosomesand synaptosomal membranes, respectively. Arrows indicate radioactive bands corresponding to proteins Ia and lb and their digestion products:fragment A (70,000 daltons), fragment B (66,000 daltons), and fragment D (50,000 daltons).

Treatment with Collagenase. The pH 6.0 extract was ad-justed to pH 7.4 with 0.5 M Hepes at pH 8.3. To 1.2 ml of thiswas added 1800 units of collagenase (Advance BiofacturesCorp., Lynbrook, NY, collagenase form III) freshly dissolvedat a concentration of 10 units/Aul in 25 mM Tris-maleate, pH7.4/200 mM CaCl2; the mixture was incubated at 30°C. Ali-quots (160jul) were removed and added to 80 ul of an Na-DodSO4-stop solution at different times after the addition ofcollagenase. The samples were boiled for 2 min and subjectedto NaDodSO4/polyacrylamide gel electrophoresis in 10%polyacrylamide gels as described (14).

Peptide Mapping after Limited Proteolysis. This was

performed essentially as described by Cleveland et al. (18). Asample (100 ,ul) of the pH 6.0 extract of membranes or synap-tosomes was diluted with 50 jul of NaDodSO4-stop solution andboiled for 2 min. A 100-jul aliquot was then subjected to Na-DodSO4/gel electrophoresis on a 6% polyacrylamide gel. Thegels were stained for 30 min and destained for 1 hr as recom-

mended (18) and, after drying, were subjected to autoradi-ography. Proteins Ta and Tb were cut out separately and the gelslices were swollen for 1-2 hr in NaDodSO4 buffer (0.125 MTris-HCl, pH 6.8/0.1% NaDodSO4/1 mM EDTA). A secondNaDodSO4 gel was prepared consisting of a 5-cm-long 5%polyacrylamide stacking gel and a 10-cm-long 15% separatinggel. The gel slices were pushed to the bottom of the wells of thisgel with a spatula and were overlaid with 20 jul of a mixture ofNaDodSO4 buffer and glycerol, 4:1 (vol/vol). Finally, 20 ul ofa 9:1 mixture of NaDodSO4 buffer and glycerol and either 0.1jug of Staphylococcus aureus protease, 0.1 jug of papain, or 10

jAg of chymotrypsin were placed over each sample and elec-trophoresis was performed as described (14) at 60 V for 14 hr.The gels were dried without staining and subjected to autora-diography.

RESULTSWhen synaptosomal membrane fractions were incubated with[y-32P]ATP, the phosphorylation of several proteins was foundto be stimulated by the presence of cAMP (Fig. 1, lanes 3 and4). In agreement with previous results (9, 19), the phosphoryl-ation of proteins Ta and Tb ([protein IICAMP) was highly stimu-lated by cAMP. When crude synaptosomal fractions werepreincubated with 32p; for 30 min and then incubated in thepresence of veratridine, the phosphorylation of several proteinswas found to be stimulated by the presence of Ca2+ (Fig. 1,lanes 5 and 6). In agreement with previous results (14), the mostprominent effect of Ca2+ was on [protein I]Ca2+, two proteinsthat had apparent molecular weights similar to those of [proteinI]CAMP The electrophoretic mobilities of [protein I]Ca2+ and[protein I]CAMP were further compared by NaDodSO4/poly-acrylamide gel electrophoresis in a gel containing 7% acryl-amide and in a gradient gel ranging from 5 to 15% acrylamide.In these electrophoretic systems, in which a better resolutionof proteins Ta and Tb is obtained, the [protein I]CAMP bandscomigrated with the [protein I]Ca2+ bands (data not shown), inagreement with the results obtained with the 10% acrylamidegel.

[Protein I]cAMP can be extracted from brain membranes byadjustment of the pH of the membrane suspension to 3.0 (10).

Neurobiology: Sieghart et al.

SIMS

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2478 Neurobiology: Sieghart et al.

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FIG. 4. Effect of collagenase on proteins Ia and lb after phos-phorylation in the presence of cAMP in synaptosomal membranes(Upper) and in the presence of veratridine plus Ca2+ in intact sy-naptosomes (Lower). Synaptosomal membranes and intact synap-tosomes were phosphorylated, extracted, incubated with collagenasefor various periods, and subjected to NaDodSO4/polyacrylamide gelelectrophoresis and autoradiography, as described in the legend toFig. 3. The autoradiograms were scanned with a Canalco G-II mi-crodensitometer and the amount of 32p incorporated into proteinsIa (0) and lb (@) and into the three digestion products, fragmentsA (A), B (o), and D (-), was estimated by measuring the relative peakheights of the densitometric tracings.

Upon readjustment of the pH of the extract to 6.0, [proteinI CAMP remains in the supernatant, although many of the otherproteins extracted at pH 3 are precipitated by the neutralizationstep. As illustrated in Fig. 1, [protein IICa2+ is extracted by thesame pH 3-pH 6 steps used to extract [protein IIcAMP (comparelanes 1, 2, 7, and 8).The isoelectric points of pure proteins Ta and Tb have been

shown to be 10.3 and 10.2, respectively (10). Recently, a high-resolution two-dimensional electrophoresis technique usefulfor the resolution of highly basic proteins has been described(17). This technique uses nonequilibrium pH gradient elec-trophoresis in the first dimension and NaDodSO4 gel electro-phoresis on a 7% polyacrylamide gel in the second dimensionto separate proteins. [Protein &ICAMP (Fig. 2a) and [protein I]Ca2+(Fig. 2b) present in the pH 6.0 extracts were found to run in thesame position in this two-dimensional system. Moreover, co-electrophoresis of a mixture of [protein I]CAMP and [proteinIICa2+ demonstrated identical electrophoretic mobilities of theseproteins in this high-resolution electrophoretic system (Fig.2a+b).

Previous studies with highly purified proteins Ta and lb in-

Protein lb"'IO' IIV,"p

,

10 10 Mg

cAMP Ca2+ cAMP Ca2+FIG. 5. Autoradiogram showing patterns of radioactive peptides

derived from proteins Ia and lb by digestion with S. aureus protease.Synaptosomal membranes were phosphorylated in the presence ofcAMP and intact synaptosomes were phosphorylated in the presenceof veratridine plus Ca2+. The phosphorylated proteins were extractedand subjected to NaDodSO4/polyacrylamide gel electrophoresis andautoradiography. Proteins Ia and lb were cut out of the gels separatelyusing the autoradiogram as a guide. The proteins in the gel pieces werethen subjected to a second NaDodSO4/polyacrylamide gel electro-phoresis, this time in the presence of 0.1 jig of S. aureus protease. Thegels were dried without staining and then subjected to autoradi-ography. Lanes labeled cAMP and Ca2+ correspond to samples pre-pared from synaptosomal membranes and intact synaptosomes, re-spectively.

dicated that both polypeptides are composed of a collage-nase-resistant fragment and a collagenase-sensitive fragment(10). When 32P-labeled purified proteins Ta and Tb were incu-bated with highly purified bacterial collagenase, these proteinswere degraded through a series of radioactive intermediatepeptides, designated fragments A, B, and C, to what appearedto be a single stable radioactive peptide (fragment D) (10). Inthe present investigation, the sensitivity of proteins Ta and Tbto collagenase was used to compare [protein I]Ca2+ with [proteinI]cAMP and purified protein I. When an extract containingphosphorylated [protein ICAMP or phosphorylated [proteinI]Ca2+was incubated with highly purified bacterial collagenase,these proteins were degraded through radioactive intermediatepeptides of 70,000 daltons (fragment A) and 66,000 daltons(fragment B) to a 50,000-dalton peptide (fragment D) (Fig. 3).[The molecular weights of these digestion products were slightlydifferent from those reported earlier (10), owing to the differentgel electrophoresis system used. ] Moreover, the time course ofdisappearance of precursors and formation of products for[protein I]Ca2+ was similar to that for [protein I]cAMP (Fig. 4) andfor purified protein I (data not shown). Because of backgroundradioactivity, fragment C, which was found only in smallamounts in collagenase digests of pure protein I (10), was notdetected in the pH 6 extracts containing [protein I]CAMP or[protein I]Ca2+. The pattern of the other radioactive bands seenin the autoradiograms of the pH 6 extracts of [protein I]cAMPand [protein I]Ca2+, as well as the protein staining pattern forother proteins in the gels, was not affected by this collagenasetreatment, indicating that the collagenase preparation used wasnot significantly contaminated by other proteolytic enzymesand that the effect of collagenase was specific for protein I. A

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slow general loss Of 32p was observed during incubation withcollagenase; this loss seemed to be due to traces of phosphataseactivity.To compare further the structures of [protein '[cAMP and

Iprotein I[Ca2+, we used a technique that involves partial en-zymatic proteolysis of proteins in the presence of NaDodSO4and analysis of the cleavage products by polyacrylamide gelelectrophoresis (18). The presence of NaDodSO4 during thedigestion prevents a complete degradation of the proteins andthus allows intermediate phosphopeptides to be detected. It hasbeen shown that the pattern of peptide fragments producedunder a given set of conditions is characteristic of the proteinsubstrate and the proteolytic enzyme and is highly reproducible(18). The pH 6.0 extracts containing [protein 1[cAMP or [proteinI ]Ca2+ were subjected to NaDodSO4/polyacrylamide gel elec-trophoresis on a 6% polyacrylamide gel to ensure good sepa-ration of proteins Ia and lb. The gels were briefly stained anddestained to avoid unnecessary degradation of proteins by theacetic acid present in the staining solution and, after drying,were subjected to autoradiography. Gel slices containing pro-teins Ta and Tb were cut out separately by using the autoradio-gram as a guide, and, after swelling, were placed in the wellsof a second NaDodSO4/polyacrylamide gel and overlayed witheither S. aureus protease, chymotrypsin, or papain. The proteinswere digested by the enzymes during the electrophoresis. Eachenzyme yielded a characteristic pattern of radioactive peptides.In agreement with the finding of Cleveland et al. (18), thepattern of radioactive peptides obtained by using the threeenzymes was highly reproducible and rather insensitive to smallvariations in protease or substrate concentrations. The peptidepattern obtained by digestion of the [protein I]CAMP polypep-tides was similar to that obtained by digestion of the [proteinI](:a2+ polypeptides, when 0.1 gug of S. aureus protease (Fig. 5),chymotrypsin, or papain (results not shown) was used for thedigestion. Furthermore, the peptide pattern generated fromprotein Ta was similar to that generated from protein Tb, forIprotein I[CAMP, [protein I](Ca2+, and purified protein I, with allthree proteolytic enzymes (Fig. 5 and unpublished data).

DISCUSSIONIn synaptic membrane fractions, cAMP stimulates the phos-phorylation of two proteins with apparent molecular weightsof 86,000 and 80,000 ([protein 1[cAMP). In intact synaptosomes,Ca2+ influx stimulates the phosphorylation of two proteins([protein I]Ca2+) with similar molecular weights. [Protein I[CAMPand [protein IICa2+ have the same apparent molecular weightsin several NaDodSO4/polyacrylamide gel electrophoresissystems and are extracted by the same pH 3.0-pH 6.0 proce-dure. These proteins also appear to have the same isoelectricpoints because they show identical migration in a high-reso-lution two-dimensional gel system involving nonequilibriumpH gradient electrophoresis and NaDodSO4/polyacrylamidegel electrophoresis. [Protein 1[CAMP and [protein I]Ca2+ are se-lectively degraded by collagenase at similar rates and to thesame intermediate and final peptides. The results obtained withcollagenase provide evidence for considerable sequencehomology of these proteins. This conclusion is strongly sup-ported by the similarity of the peptide pattern obtained when[Protein '[CAMP and [Protein I]Ca2+ were subjected to limitedproteolysis with any of three different proteases. This method

has previously been shown to provide different peptide patternseven in the case of proteins with similar amino acid compositionand related NH2-terminal sequence, such as a- and f-tubulin(18).

Thus, various pieces of evidence indicate that the two sets ofproteins are similar if not identical. This conclusion is furthersupported by the recent finding (unpublished data) that both[protein I[CAMP and [protein I[lC a2+ are precipitated by a highlyspecific antibody prepared against pure protein I, using animmunological double precipitation technique. In an extensionof the present study, it has recently been found (unpublisheddata) that cAMP and Ca2+ stimulate the phosphorylation ofdifferent amino acid residues on protein I.

Biochemical (9, 10) and cytochemical (unpublished) studieshave demonstrated that protein I is located only in nerve tissue.Within the nervous system, protein I has been demonstratedto occur predominantly in neurons (19) and to be highly en-riched in synaptic vesicles and postsynaptic densities (unpub-lished experiments carried out in collaboration with the labo-ratories of P. Siekevitz and F. E. Bloom). These and other (16,20) results support the view that protein I has an important rolein the functioning of synapses. The regulation of the phos-phorylation of protein I by two substances, cAMP and Ca2 ,known to function as second messengers in the nervous system,is of great interest.

This work was supported by U.S. Public Health Service GrantsDA-01627, MH-17387, and NS-08440 and a grant from the McKnightFoundation. W.S. was the recipient of a Max Kade Foundation Fel-lowship.

1. Bloom, F. E. (1975) Rev. Physiol. Biochem. Pharmacol. 74, 1-103.

2. Daly, J. W. (1975) in Handbook of Psychopharmacology, eds.Iversen, L. L., Iversen, S. D. & Snyder, S. H. (Plenum, New York),Vol. 5, pp. 47-130.

3. Nathanson, J. A. (1977) Physiol. Rev. 57, 157-256.4. Kandel, E. R. (1978) A Cell-Biological Approach to Learning

(Society for Neuroscience, Rockville, MD).5. Greengard, P. (1978) Cyclic Nucleotides, Phosphorylated Pro-

teins and Neuronal Function (Raven, New York).6. Krebs, E. G. (1972) Curr. Top. Cell. Regul. 5,99-133.7. Rubin, C. S. & Rosen, 0. M. (1975) Annu. Rev. Biochem. 44,

831-885.8. Greengard, P. (1978) Science 199, 146-157.9. Ueda, T., Maeno, H. & Greengard, P. (1973) J. Biol. Chem. 248,

8295-8305.10. Ueda, T. & Greengard, P. (1977) J. Biol. Chem. 252, 5155-

5163.11. Baker, P. F. (1972) Prog. Biophys. Mol. Biol. 24, 177-223.12. Rubin, R. P. (1972) Pharmacol. Rev. 22, 389-428.13. Blaustein, M. P. (1975) J. Physiol. 247, 617-655.14. Krueger, B. K., Forn, J. & Greengard, P. (1977) J. Biol. Chem.

252, 2764-2773.15. DeLorenzo, R. J. & Greengard, P. (1973) Proc. Natl. Acad. Sci.

USA 70, 1831-1835.16. Forn, J. & Greengard, P. (1978) Proc. Natl. Acad. Sci. USA 75,

5195-5199.17. O'Farrell, P. Z., Goodman, H. W. & O'Farrell, P. H. (1977) Cell

12, 1133-1142.18. Cleveland, D. W., Fischer, S. G., Kirschner, M. W. & Laemmli,

U. K. (1977) J. Biol. Chem. 252, 1102-1106.19. Sieghart, W., Forn, J., Schwarcz, R., Coyle, J. T. & Greengard,

P. (1978) Brain Res. 156,345-350.20. Lohmann, S. M., Ueda, T. & Greengard, P. (1978) Proc. Natl.

Acad. Sci. USA 75, 4037-4041.

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