Neurochern'ical Research (2) 533-548 (1977)

DISTRIBUTION OF E N D O G E N O U S L Y PHOSPHORYLATED P R O T E I N S I N S U B C E L L U L A R FRACTIONS OF RAT CEREBRAL CORTEX YIGAL H. EHRLICH, LEONARD G. DAVIS, THOMAS GILFOIL, AND ERIC G. BRUNNGRABER Biochemistry Laboratory Missouri Institute of Psychiatry School of Medicine University of Missouri, Columbia 5400 Arsenal Street St. Louis, Missouri 63139

Accepted April 27, t977

The cerebral cortex from adult rats was separated into several subcellular fractions by using established methods of differential and sucrose density gradient centrifugation. Aliquots from each fraction were incubated with y-azPATP, in the presence and absence of adenosine 3',5'-monophosphate (cyclic AMP), and its protein constituents were separated by means of SDS-slab gel electrophoresis. Fractions containing nuclei, synaptosomes, myelin, microsomes, and soluble proteins each showed a characteristic pattern of protein staining and of endogenously phosphorylated proteins detected by autoradiography of the gels. Cyclic AMP-stimulated phosphorylafion of proteins with MW 78K and 84K can serve as markers for membranes of synaptic origin, while cyclic AMP-independent phosphorylation of low-molecular-weight proteins (15K-20K) is characteristic of myelin. The finding of different phosphoproteins in various subcellular fractions may be related to the diversity of cellular functions known to be regulated by phosphorylative activity.

INTRODUCTION Considerable evidence has accumulated that indicates that the phosphorylation of proteins and its regulation by adenosine 3',5'-monophosphate (cyclic AMP) may play an important role in brain function (1-6). 533

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534

EHRL~r ET AL.

Mechanisms involving cyclic nucleotide-regulated protein phosphorylation have been implicated in the regulation of such diverse cellular processes as membrane permeability, synaptic transmission, secretion, transport, enzyme activation, transcription, cell division, and cell adhesion, as well as cell growth and differentiation (7-12). Different phosphoproteins and their kinases may be involved in the regulation of the biochemical activities that underlie the foregoing processes (13,14). Since several subcellular localizations may be involved, it is of importance to identify the specific proteins that can serve as endogenous substrates for naturally occurring protein kinases in various subcellular organelles from brain tissue. In the present study, subcellular fractions from rat cerebral cortex were incubated in vitro with y-szp-ATP and cyclic AMP under conditions that permit endogenous phosphorylation. The specific proteins that incorporate labeled phosphate under these conditions were separated by means of SDS-gel electrophoresis and identified by autoradiography of the gel slabs. The results indicate that electrophoretically separated proteins of the cytosol, myelin, synaptosomes, microsomes, and nuclei show characteristic patterns of protein staining and protein phosphorylation. The distribution of labeled phosphate among specific proteins can be used as a sensitive marker which may serve to identify membrane fragments of unknown origin. EXPERIMENTAL PROCEDURE

Preparation of Subcellular Fractions Male albino rats (200-300 g, National Labs, O'Fallon, Missouri) were decapitated and the cerebral cortices were homogenized in l0 vol (w/v) of 0.32 M sucrose containing 1 mM Tris chloride (pH 7.4) and I mM MgCI2. The homogenate was fractionated by differential centrifugation to provide the crude nuclear pellet (P1, 8000g • min), crude mitochondrial pellet (P2, 240,000g • min.), microsomal pellet [P3, (6 • 106)g • mini, and cytosol (Sz). Nuclei were prepared from the P1 fraction as previously described (15) with 0.25% Triton X-100 added to the 2.4 M sucrose solution. A preparation of soluble proteins ($4) was obtained by dialyzing the cytosol (Sz) against water (pH adjusted to 7.0) to remove sucrose. The contents of the dialysis bag were centrifuged at (6 x 106)g x min to remove a small pellet (P4). Fraction $4 was concentrated by lyophilization and dissolved in 1 mM Tris chloride (pH 7.4) containing 1 mM MgCI2 (TM buffer). Subfractionation of the crude mitocbondrial fraction, P2, to produce P2A, P2B, and P2C was performed on a discontinuous sucrose density gradient (0.32 M/0.8 M/1.2 M sucrose) according to the method of Whittaker (16). Each of the postnuclear particulate fractions was washed 3 times by suspension in TM buffer (i.e., hypoosmotic conditions) and centrifuged at the g • min used for their original preparation. The fractions were analyzed for protein concentration (17) and stored at - 7 0 ~ Preparations enriched in synaptosomes and myelin but low in mitochondria were obtained by method 2 of Brunngraber et al. (18) (fraction A + B) or by

S U B C E L L U L A R DISTRIBUTION OF CORTICAL PHOSPHOPROTEINS

535

the method of Koenig (19) (fraction LM). The total postnuclear particulate fraction from the line NBP2 of neuroblastoma cells grown in culture was provided by Dr. K. N. Prasad from the University of Colorado Medical Center (12).

Standard Assay of Endogenous Phosphorylation Aliquots from each fraction were preincubated in a shaking water bath (30 ~ for 5 rain. The reaction was initiated by addition of ATP, with or without cyclic AMP. The reaction mixture, in a final volume of 0.06 ml, contained 50 mM sodium acetate (pH 6.5), 10 mM MgC12, 3 /xM y-3zP-ATP (ICN, Irvine, California, diluted with nonradioactive ATP from Sigma Chemical Co. to give (3-9) • 107 cpm/nmol), 5 tzM (or equal volume of water) cyclic AMP, and 20 tzg protein from each fraction. After incubation for 1 min, the reaction was terminated by adding 10 /zl of "'stop solution" containing 21% sodium dodecyl sulfate (SDS), 35 mM Tris acetate buffer (pH 8.0), 7 mM EDTA, 14%/3-mercaptoethanol, 42% sucrose, and 0.07% bromophenol blue (tracking dye). Samples were heated in a boiling water bath for 2 min prior to application to the gel.

Electrophoretic Separation Linear gradients (7-16% or 7-18%) of acrylamide were polymerized in the Hoefer (California) SE-500-1.5 slab gel electrophoresis unit. Aliquots (35 /zl) of the SDSsotubilized reaction mixture were applied to each well of the gel, and electrophoresis was performed using a discontinuous buffer system (20) and the Ortec 4100 pulsed constant power supply as described (13,14). Under the conditions used 26-h electrophoresis at 160 V, 0.1 /xF, and 150 pulses/s/gel---ATP, inorganic phosphate, and lipid-bound phosphate migrated ahead of the phosphoproteins and did not interfere with their separation. DNA of nuclear preparations remained in the stacking gel. The gels were stained with Coomassiebrilliant blue-R for protein and dried in the Hoefer SE-540 gel drier. Autoradiographs were obtained by placing the gels in close contact with Kodak-X-Omat X-ray film. Molecularweight estimations were obtained by electrophoresis of marker proteins under the same conditions. Densitometric tracing of the autoradiograms were obtained by scanning at. 649 nm using a Beckman Acta CV Spectrophotometer equipped with a linear transport device. The amount of radioactive phosphate incorporated into specific bands was estimated from these scans as described previously (12-14). The amount of total 10% TCA-precipitable ~ZP-phosphate incorporated into the fractions was determined by scintillation counting. The results obtained after various extraction procedures using chloroform-methanol, hot trichloroacetic acid, hydroxylamine, and NaOH to extract the precipitates indicated that 75-85% of the radioactive phosphate applied to the gel was bound to protein in a phosphoester linkage. The results obtained from endogenous phosphorylation assays depend on the activity of protein kinases, phosphoprotein phosphatases, ATPases, and phosphodiesterases present in the preparation (9,10,13,14,23,24,26,28). The main purpose of this study was to compare the pattern of 3ZP-phosphate incorporation into specific protein substrates in different suhcellular fractions assayed under identical Conditions. Agents that are used to inhibit these enzymes (i.e., fluoride, zinc ions, theophyline, etc.) were not included in our standard reaction mixture, since each inhibitor can affect more than one enzyme and may have different effects in different subcellular fractions, thereby obscuring the comparisons we wish to make.

536

EHRLICH ET AL.

RESULTS SDS-polyacrylamide gel electrophoretic analysis of as little as 10/~g protein obtained from the total cerebral homogenate (TH), purified nuclei (Nuc), and the crude primary fractions P2, P3, $3, and $4 provided protein profiles that were characteristic for each fraction (Figure 1). As judged by their electrophoretic mobility, the molecular weights of the proteins ranged from about 10,000 to values greater than 200,000. The staining patterns were reproducible, and the use of gel slabs permitted a direct comparison between fractions. Proteins present in the homogenate were unequally distributed among the subcellular fractions derived from it. Only the patterns of the cytosol (S~) and soluble ($4) fractions were similar. $3 contains small postmicrosomal particulate matter that had been removed by high-speed centrifugation after dialysis to remove the sucrose. The amount of protein in this particulate fraction (P4) was small relative to the total soluble protein, accounting for the similarity of the electrophoretic patterns of $3 and $4. Aliquots of all these fractions were tested for endogenous protein phosphorylation activity. Incubation of aliquots from these primary fractions with labeled ATP prior to electrophoretic analysis did not produce any detectable alterations in the patterns of protein staining obtained. Aliquots of each fraction were incubated with 3~p-ATP, in the presence and absence of added cyclic AMP, prior to solubilization in SDS and electrophoretic analysis. After incubation of the homogenate, 16 phosphorylated bands were detected by autoradiography (Figure 2); addition of cyclic AMP had little or no effect on the phosphorylation of these bands. Six prominent bands were seen. Four migrated to a position corresponding to molecular weights ranging from 40,000 to 80,000, and two were of low molecular weight (15,000-20,000). The distribution of 3~p-phosphoproteins was also characteristic of each fraction. The nuclei showed phosphorylation of 26 protein bands (Figure 2, Nuc). Many of the phosphorylated bands identified in nuclei were not detected in the autoradiograms of the total homogenate. It should be noted that the proteins in the nuclear preparation constituted less than 3% of the total protein of the homogenate. The major phosphorylated bands in nuclei had mobilities corresponding to molecular weights greater than 80,000. Thus, these would not appear to be histones, since all of the bands obtained by electrophoresis of a commercially available preparation of calf thymus histories (Sigma, Type II) migrated to positions corresponding to molecular weights below 50,000. The addition of cyclic AMP (10-9-10 -5 M) to the reaction mixture had no significant

SUBCELLULAR DISTRIBUTION OF CORTICAL PHOSPHOPROTEINS

Protein

537

Staining

i.W, Scale

TH

200

K-

100

K-

50

25

Nuc

P2

P3

S3

S4

K

K .

10 K ~

~i)~!~!~i~i~i~i!~!i~I!~i!!~!~

Fig. 1. Electrophoretic separation of proteins in the total homogenate (TH), nuclear (Nuc), mitochondrial (P2), microsomal (P~), cytoplasmic ($3), and soluble ($4) fractions of the rat cerebral cortex. The fractions were prepared and solubilized in SDS as described in the "Methods" Section. Aliquots containing 10 ~g protein were electrophoresed in 7-16% polyacrylamide gel gradients and stained with Coomassie brilliant blue. Incubation with AT3~P prior to SDS-electrophoresis did not produce changes that could be detected in the staining pattern. effects on phosphate incorporation into any of the phosphoproteins of t h e p u r i f i e d n u c l e i . T h e s e r e s u l t s a r e in a c c o r d a n c e w i t h s e v e r a l s t u d i e s ( r e v i e w e d in R e f e r e n c e 10) w h i c h d e m o n s t r a t e d t h a t t h e p r o t e i n k i n a s e s in n u c l e i a r e p r e d o m i n a t e l y c y c l i c A M P - i n d e p e n d e n t a n d a p p e a r to prefer nuclear nonhistone proteins as the phosphate acceptors.

538

EHRLICH ET AL.

Autoradiogram MW TH

D

Nuc

P2

P3

$3

S4

(xl0

~83

-

E--

~55

F--

--

47

G--

--

34

- - 17

H --

cAMP

--3)

--~"

--+

--+

--+

--+

--+

FIG. 2. Endogenously phosphorylated proteins of the major subcellular fractions from rat cerebral cortex. Aliquots from each fraction were incubated with y-z~P-ATP(3 • 107cpm/ nmol) in the presence (+) or absence (-) of 5 /~M added cyclic AMP under the standard assay conditions used in this study (see "Methods" section). The reaction was terminated by solubilization of the proteins in SDS (13,14), and aliquots were separated in polyacrylamide gels as described in the legend to Figure 1. The bands containing labeled phosphate were detected by autoradiography of stained and dried gels. TH denotes total homogenate; Nuc = nuclei purified from a P, fraction; P~ = crude mitochondrial fraction; P3 = microsomal fraction; $3 = cytosol; and $4 = soluble proteins. Autoradiography for these fractions was carried out for 3 days. The mitochondrial fraction Pz (which contained myelin and synaptosomes in addition to mitochondria) was assayed after osmotic shock, and the 3ZP-phosphoprotein distribution (Figure 2, P2) provided an autoradiographic pattern similar to that previously reported (14). Modifications of the assay conditions used (increase in specific activity of 3zp_ A T P and the electrophoretic separation of aliquots containing only 10/zg protein or less) improved the resolution of the previously reported major bands D, E, and H. This improved resolution is observed in the D, E, and H regions of the electropherogram of P2 (Figure 2), and the resolved bands are clearly shown and noted in Figure 3 for P2B and P2A. D and E were each resolved into two bands (D-l, D-2, E - I , and E-2) and band H

SUBCELLULAR DISTRIBUTION OF CORTICAL PHOSPHOPROTEINS

539

was resolved into 3 bands (H-l, H-2, and H-3). Most of the label incorporated into P~ proteins appeared in the H group, but the greatest effects of exogenous cyclic AMP were noted in D-l, D-2, E-I, and E-2. Band F appeared as a single band as before (13, 14). The microsomes (P3) revealed prominent phosphorylated bands corresponding in mobility to phosphoproteins D and F noted in P2; bands corresponding to E and H were low in activity (Figure 2, P3). The major band in the cytosol ($3), which comigrated with band E, was but a minor band in P3. Major bands in P3 (for example, a band comigrating with F) were low in $3. 3~P-phosphate incorporation into specific bands of the microsomal and cytosol fractions was high even prior to the addition of cyclic AMP. Exogenous cyclic AMP had little or no stimulatory effects on phosphoprotein phosphorylation in P3 and $3 (Figure 2). The pattern of protein phosphorylation in the postmicrosomal particulate matter (P4) resembled that of P3, and phosphorylation was not appreciably affected by the addition of cyclic AMP. However, the incorporation of 3~P-phosphate, per mg protein, in P4 was 2 times greater than that observed in Ps. The soluble fraction ($4) obtained after removal of P4 by high-speed centrifugation was lyophilized; the preparation retained its enzymatic activity (Figure 2, $4). Phosphorylation of proteins with apparent molecular weights of 17,000, 20,000, 34,000, 56,000, 69,000, and 84,000 daltons, as well as a predominent band near the gel origin with a molecular weight greater than 200,000, was readily detected. In addition to these bands, the phosphorylation of which was greatly stimulated by cyclic AMP, fraction $4 also contained bands whose phosphorylation was cyclic AMP independent. The differences in patterns between S a and $4 may be caused by the removal of inhibitors and/or activators during the dialysis, as well as by the removal of the particulate fraction P4. P4 had high activity in the absence of exogenous cyclic AMP. P2 was fractionated by sucrose density gradient centrifugation to yield a myelin-enriched fraction (P2A), a synaptosome-enriched fraction (P2B), and the mitochondria (P2C). The protein patterns obtained by SDSelectrophoresis of the three fractions were clearly different (Figure 3). P~A showed five prominent protein bands that are characteristic of myelin preparations (21). They corresponded in mobility to the basic myelin proteins (MW = 15,000 and 17,000), protein DM20 (20,000), lipoprotein (24,000), and Wolfgram protein (55,000). Lysed particles of this fraction were assayed for incorporation of aZP-phosphate from 3zp_ ATP into endogenous protein substrates. The four bands that accounted for most of the incorporated radioactive phosphate were among the prominent bands in the protein pattern. Three of these (H-1, H-2, and H-

540

EHRLICH ET AL. Autoradiogram

Prot.

Staining MW

p

A 2

D E

P

B 2

PC 2

P2 A

P B 2

P

C 2

I2-

_ -

84 78

t-

-

59 56 47

|.

-

20

23-

-

17

-

15

2.-

F

H

(xlO

cAMP

--+

--+

--+

--+

--+

--3

)

--+

FIG. 3. Distribution of proteins and endogenously phosphorylated proteins in the crude mitochondrial (P~) fraction. The P2 preparation from rat cerebral cortex was further fractionated according to the method of Whittaker (16). The resultant P ~ (myelinenriched), P2B (synaptosome-enriched), and P2C (mitochondria) fractions were washed under hypotonic conditions and then incubated with 7-azP-ATP and cyclic AMP and separated electrophoreticaUy as described in the legends to Figures l and 2. Autoradiography time for these fractions was 18 h. A longer autoradiographic time obscured the resolution of D1 and Dz in P2B, but revealed the presence of bands comigrating with bands D1, D2, E, and F in P~C. 3) showed exceedingly high levels of a~P-phosphate e v e n in the a b s e n c e of added cyclic AMP. T h e fourth phosphoprotein, designated E-2 (Figure 3), had low levels of basal p h o s p h o r y l a t i o n and its phosphorylation was greatly stimulated b y the addition of cyclic AMP. The s y n a p t o s o m e - e n r i c h e d fraction P2B showed four p r o m i n e n t bands which differed f r o m those o b s e r v e d in P2A. The phosphorylation o f bands D-1 and D-2 ( M W = 84,000 and 78,000) was stimulated by cyclic AMP. E-1 and F s h o w e d only a small r e s p o n s e to added cyclic A M P (Figure 3) u n d e r the standard conditions used in this study.

S U B C E L L U L A R DISTRIBUTION OF CORTICAL PHOSPHOPROTEINS

541

Incorporation of 3Zp-phosphate into P2C was low and may have been caused by the contamination of this fraction by particles derived from the synaptosomes. Evidence for this view is presented in densitometric tracings of the autoradiograms obtained for P2A, P~B, and P2C (Figure 4), which shows that the phosphoprotein bands observed in P2C are

II

o2

!tol

F

~

i'

P2A

, H1

I

I

I

I

I

I

I

I

I

I

10

9

8

7

6

5

4

3

2

I

D i s t a n c e of m i g r a t i o n

0

( cm 1

FIG. 4. Densitometric tracing of the autoradiograms obtained for the myelin-enriched (P2A), synaptosome-enriched (P~B), and mitochondrial (P~C) fractions. The autoradiograms presented for these fractions in Figure 3 were scanned as described in the " M e t h o d s " section. The effects of cyclic AMP on the phosphorylation of specific bands can be evaluated best by the use of these scans (12-14,28) as previously described. The scans detected bands in fraction P2C that were difficult to see on visual observation of the gel, and which were poorly resolved by counting l-ram gel slices. Scanning had a marked practical advantage, since the six scans depicted replaced the counting of 600 vials, each containing a l-ram gel slice. The quantitative results of the scanning method, however, depend not only on the specific activity of the T-32P-ATP used, but also on autoradiography time. Therefore, samples that are to be compared should be assayed in parallel and separated on the same gel. - - , 5 p~M cyclic AMP added; . . . . , equal volume of water added instead.

542

EHRL~CH ET AL.

identical in mobility to the major peaks seen in P2B. These tracings also serve to show the effects of added cyclic AMP. The addition of 5 t~M cyclic AMP stimulated phosphate incorporation into E-2 of P2A approximately threefold. In P2B, bands D-1 and D-2 showed an interesting relationship. In the absence of cyclic AMP, a2P-phosphate incorporation into D-I was greater than that into D-2; in the presence of added cyclic AMP, phosphorylation of D-2 was greater than D-1. In agreement with our previous report (13), cyclic AMP had small effects on 32P-phosphate incorporation into bands E and F of the synaptosomal fraction and into bands of group H in the myelin fraction at the standard reaction time used here (1 min). Phosphorylation of specific protein bands may serve as markers to identify subcellular membranes. Three subcellular preparations were studied to illustrate this point. "Light mitochondrial" fractions were prepared by using two different procedures: that of Brunngraber et al. (18) and that of Koenig (19). These preparations are enriched in acetylcholinesterase and sialidase activities but relatively poor in mitochondrial enzymes. The protein staining and phosphorylation patterns of the two preparations were similar; the pattern is shown in Figure 5 (under "RAT"). These preparations demonstrated the presence of two bands the phosphorylation of which was stimulated by cyclic AMP and which comigrated with D-1 and D-2 of P2B. This suggests that such preparations contain membranes of synaptic origin. The high levels of radioactivity incorporated into the H-bands when cyclic AMP was not added suggested that the preparations contained myelin fragments. The presence of E-1 (P2B) and E-2 (P2A) also indicated that these preparations contained both myelin and synaptosomes. A third subcellular fraction studied was the postnuclear particulate fraction obtained from differentiating neuroblastoma cells. Neuroblastoma cells, grow n in culture, do not contain myelin. When these cells are treated with inhibitors of phosphodiesterase, the cells are induced to differentiate (22). Although long neurites develop, synaptic junctions are not formed. As expected from this information, no evidence for the presence of bands D-l, D-2 (characteristic of P~B), H-2, or H-3 (characteristic of P2A) was found (Figure 5, NB). On the other hand, neuroblastoma membranes exhibited phosphorylation of bands that were low or absent in " R A T " (Figure 5). Two of the bands in neuroblastoma (MW = 100,000-110,000) showed high levels of ~ZP-phosphate incorporation in the absence of added cyclic AMP, and seven others (MW = 20,000-80,000) were stimulated by added cyclic AMP (Figure 5). The latter included a band that comigrated with band E-2 of the rat samples and a cyclic AMPstimulated band that comigrated with the cyclic AMP-independent H-I

SUBCELLULAR DISTRIBUTION OF CORTICAL PHOSPHOPROTEINS

Autoradiogram NB

Prot.

RAT

NB

543

Staining RAT

D E F

H

cAMP

+--

+_

+--

+_

FIG. 5. Comparison of endogenously phosphorylated proteins in membranes from rat neostriatum and neuroblastoma cells grown in culture. The P2 fraction from the caudateputamen complex (neostriatum) of rats sacrified by decapitation was separated by differential centrifugation to yield a mitochondrial fraction (8000g) (not shown in figure) and a membrane fraction (15,000g) denoted as RAT in the figure. The pattern of protein staining and the autoradiogram of this "light mitochondrial" fraction was essentially the same regardless of its method of preparation (18,19). The postnuclear particulate fraction (9 • 106g • min pellet obtained after removal of the nuclei at 8000g • min) of neuroblastoma cells (denoted NB in the figure) was provided by Dr. K. N. Prasad. The preparations used in this experiment were from cells that were induced to differentiate by treatment with a phosphodiesterase inhibitor (12). Note that bands comigrating with D and F cannot be detected in the autoradiograms of NB membranes. The samples of both preparations were assayed with the same ~ZP-ATP batch (3.6 • 107 cprrdnmole) and separated on the same gel (7-18% acrylamide gradient). o f m y e l i n . C o m p a r a b l e d a t a , n o t s h o w n h e r e , w e r e o b t a i n e d in s t u d i e s with o s m o t i c a l l y s h o c k e d P2 f r o m 1-day old rat c e r e b r a l c o r t e x . This p r e p a r a t i o n p r o v i d e d n o e v i d e n c e for the p h o s p h o r y l a t i o n o f b a n d D-2, while the p h o s p h o r y l a t i o n o f a b a n d t h a t c o m i g r a t e d with H-1 was s t i m u l a t e d t w o f o l d b y the a d d i t i o n o f 5 / x M cyclic A M P . T h u s , the gel

544

EHRLICH ET AL.

region of the H bands may contain phosphoproteins whose phosphorylation is stimulated by cyclic AMP, but which remain undetected in preparations that contain myelin.

DISCUSSION The present study has shown that specific proteins that can serve as substrates for phosphorylative activity in the central nervous system are unequally distributed among various subcellular fractions. Since phosphoproteins were determined by an in vitro phosphorylation of endogenous proteins in each fraction, 3ZP-ATP acting as the phosphate donor, it is obvious that each fraction must contain the protein kinases responsible for their phosphorylation. Several protein kinases have been reported to exist in brain, and there are strong indications that the kinases localized in different subcellular compartments differ in physical and chemical properties (23-25). Therefore, the different patterns of phosphorylated proteins in each fraction may reflect not only differences in acceptor protein species but also unique properties of the endogenous protein kinases. It is a common practice to assay for protein kinases by using exogenous substrates such as phosvitin or histones. Histones, proteins of nuclear origin, appear to be poor substrates for endogenous phosphorylative activity exhibited by purified nuclei, as studied here. Furthermore, histones and phosvitin have also been shown to be poor substrates for the membrane-bound, cyclic AMP-dependent protein kinases found in brain (24). Therefore, the identification of the endogenous substrates is essential to understand the role of protein kinases in regulating various cellular processes. Although it is possible that one protein kinase may be responsible for the phosphorylation of more than one of the phosphoproteins found in a subcellular fraction, it should be noted that Ueda et al. (26) have reported that a phosphoprotein substrate was purified as a complex consisting of the phosphoprotein along with the protein kinases and phosphoprotein phosphatase responsible for its phosphorylation and dephosphorylation. Thus, each of the membrane-bound phosphoproteins of rat brain may be tightly associated with its own kinase and phosphatase, and this association may be critical to its function. Our results indicate that such may be the case also for soluble systems. In the soluble fraction ($4), the phosphorylation of several bands was cyclic AMP independent, while others were not phosphorylated until cyclic AMP was added to the reaction mixture. The soluble cyclic AMPindependent protein kinase of brain t h u s cannot phosphorylate certain

SUBCELLULAR DISTRIBUTION OF CORTICAL PHOSPHOPROTEINS

545

specific phosphate acceptors that are also in solution, and therefore available for phosphorylation. The specificity of such interactions may be determined, at least in part, by the nature of the substrate. Characterization of the endogenous phosphorylation and dephosphorylation of specific soluble proteins from rat cerebral cortex (manuscript in preparation) provided experimental support for this contention. The presence of phosphorylated bands with the same mobility in more than one subcellular fraction suggests, but does not prove, that the bands may be identical. In some cases, a specific band (e.g., H-1 and H2 in fractions $4 and P2) may be dependent on cyclic AMP in one fraction but not in another. While this may indicate that the two proteins are different molecules, it is equally likely that the difference in response to cyclic AMP is caused by other rate-limiting factors. Thus, various subcellular fractions may differ in the levels of several parameters that affect phosphorylation. These parameters may include the ratio of protein kinase to protein phosphatase activity, activity of ATPases which affect the concentration of ~2P-ATP, the concentration of available protein substrate, the activity of phosphodiesterases, the levels of endogenous cyclic AMP, and probable differences in membrane conformations and spatial relationships between enzymes and substrates. Subcellular fractions of rat cerebral cortex can be characterized by differences in the distribution of their constituent proteins after slab gel SDS-electrophoresis (see Figure 1). The use of this method as a criterion of purity was recently reported (27). Identification of endogenously phosphorylated phosphoproteins as described here provides an additional marker for the identification of membrane fragments. In addition to its specificity, the method provides great sensitivity. As little as 0.2 /xg protein of the P2 fraction, incubated under the standard assay conditions, was separated on our gel, and no bands could be detected by staining with Coomassie. However, all the specific bands characteristic to this fraction were detected after 9 days of autoradiography, and the results could be analyzed by densitometry (Y. H. Ehrlich, unpublished observations). The method is therefore useful for detecting specific phosphoproteins in samples from very small, but well defined, brain areas. Although the amount of 3~P-phosphate incorporated into the phosphoproteins of separate preparations of the same particulate fraction is known to vary and to depend on the specific activity of the 3~P-ATP used (14), the relative distribution of 3~P-proteins among the major phosphorylated bands of each fraction was reproducible. Furthermore, although the magnitude of the responses of the bands of differing preparations to cyclic AMP showed variations, the dependency or lack

546

EHRLICH ET AL.

of dependency of optimal phosphorylation on the presence of cyclic AMP demonstrated by a phosphoprotein band within a given subcellular fraction was reproducible and characteristic of the subcellular fraction studied. A few of the specific phosphoproteins identified here may be identical to phosphoproteins studied previously, whose role in brain function has been postulated. The band designated D in our previous studies (13,14) has properties that are identical to protein I of Ueda et al. (28). The enrichment of this band in PzB (Figure 3) and its absence in preparations of neuroblastoma and 1-day old rat support the concept of a synaptic localization for this phosphoprotein. The present investigation has shown that band D is composed of two phosphoproteins, D-1 and D-2, each of which shows a different response to added cyclic AMP (Figure 4). The role of these phosphoproteins in synaptic function remains to be elucidated. The band that we designated E was identified (14) as protein II of Ueda et al. (28). One of the E bands separated here may be the phosphorylated regulatory subunit of brain cyclic AMP-dependent protein kinase (29). The phosphorylated subunit of tubulin has a molecular weight (55,000) that would also place it among our E bands. The phosphorylation of bands F and H in lysed P2 preparations was shown to increase after a training experience (30) and to decrease after chronic morphine'treatment (31), indicating that these phosphoproteins may play a mediatory role in the responses of the central nervous system to external stimulation. Future studies should serve to identify the functional role of the various phosphoproteins identified here in relation to the activity of the numerous enzymes known to be regulated by phosphorylative processes (i0).

ACKNOWLEDGMENTS This study was supported by intramural funds from the Missouri Institute of Psychiatry and, in part, by a gra~3t from the Epilepsy Foundation of America (to Y.H.E.). The expert technical assistance of Mrs. C. L. DeClue is gratefully acknowledged.

REFERENCES I. HEALD, P. J. 1957. The incorporation of phosphate into cerebral phosphoproteins promoted by electrical impulses. Biochem. J. 66:659-663. 2. TREVOR, A. J., and RODNIGHT, R. 1965. The subcellular localization of cerebral phosphoproteins sensitive to electrical stimulation. Biochem. J. 95:889-896.

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