Ribosomal Protein Phosphorylation in Rat Cerebral Cortex · adenylate cyclase systems in the cerebral cortex respond maximally to catecholamines with increased production of cyclic

Ribosomal Protein Phosphorylation in Rat Cerebral Cortex in vitro

INFLUENCE OF CYCLIC ADENOSINE 3’:5’-MONOPHOSPHATE*

(Received for publication, August 10, 1977)

SIDNEY ROBERTS AND C. DENNIS ASHBY

From the Department of Biological Chemistry, School of Medicine, and the Brain Research Institute, University of California Center for the Health Sciences, Los Angeles, California 90024

Earlier investigations of ribosomal protein phosphorylation in rat cerebra1 cortex in vitro have been extended to include an analysis of the effects of adenosine 3’:5’-monophosphate (cyclic AMP) on this process. Cerebral cortical slices from ll-day-old rats were incubated for 2 h with [Y”Plorthophosphate, washed free of extracellular radioactivity, then incubated again in the presence of N”,O”-dibutyryl adenosine 3’:5’-monophosphate (dibutyryl cyclic AMP) or related substances. Addition of 1 m.n dibutyryl cyclic AMP consistently stimulated phosphorylation of ribosomal proteins in the cerebra1 40 S subunit, whereas overall phosphorylation of proteins in the 60 S subunit was not increased by the cyclic nucleotide. The minimum dose of dibutyryl cyclic AMP which was effective in stimulating phosphorylation of 40 S ribosomal proteins was about 0.1 mM. Stimulation was also produced with monobutyryl and other dibutyryl derivatives of cyclic AMP, as well as the phosphodiesterase inhibitor, I-methyl-3Gsobutylxanthine. N2,0”-Dibutyryl guanosine 3’:5’-monophosphate and sodium butyrate (1 mM) were without effect on ribosomal protein phosphorylation. Following electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate, the protein or proteins of the 40 S subunit which exhibited enhanced phosphorylation in the presence of dibutyryl cyclic AMP appeared to be located in a single radioactive band with a mobility which corresponded to a molecular weight of approximately 32,000. Two-dimensional electrophoresis of the ribosomal proteins on polyacrylamide gels containing urea revealed that the small subunit contained three ribosomal proteins which were phosphorylated in cerebra1 cortical tissue in uitro. The most highly labeled protein (S6) had a molecular weight of approximately 32,000 and consisted of at least five distinct species in different states of phosphorylation. Under basal conditions, the bulk of the S6 protein existed in the nonphosphorylated state.

* This work was suoported bv Grant NS-07869 from the National Institutes of Health &d by Grant R-259 from the United Cerebral Palsy Research and Educational Foundation, Inc. Preliminary reports of this work have appeared in abstract form (Roberts, S., and Ashby, C. D. (1976) Trans. Am. Sot. Neurochem. 7, 108; (1977) Trans. Am. Sot. Neurochem. 8, 262). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “‘advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

Incubation of cerebral cortical tissue with 1 rnM dibutyryl cyclic AMP increased the proportion of phosphorylated congeners of the S6 protein as indicated by their relative absorbances at 600 nm compared to the nonphosphorylated species. The radioactivity associated with each of these congeners was also increased. The other two phosphorylated proteins of the 40 S subunit had electrophoretic properties which were similar to those of ribosomal proteins S2 and S3. Phosphorylation of these proteins was considerably less than that of the S6 protein. The cerebra1 60 S subunit contained several proteins which were phosphorylated in vitro. Three of these were basic proteins with electropho- retie mobilities similar to those of ribosomal proteins L6, L13, and L14. At least three other proteins associated with the 60 S subunit which were less basic were also phosphorylated. The latter proteins have not been identified as corresponding electrophoretically to constitutive ribosomal proteins. These investigations indicate that although ribosomal protein phosphorylation in cellular preparations of rat cerebral cortex in. vitro involves proteins of both subunits, stimulation of this process by cyclic AMP is largely attrib- utable to a single ribosomal protein of the cerebral 40 S subunit. The possible relationship of these findings to alterations in brain protein synthesis accompanying the trans- mission of neural impulses is discussed.

Considerable evidence supports the concept that adenosine 3’:5’-monophosphate (cyclic AMP) plays an important role in neurotransmission (l-7). Several neurotransmitter amines

increase the accumulation of this cyclic nucleotide in nervous tissue (8) by activating adenylate cyclase systems (9-11). Catecholamine-sensitive adenylate cyclase activity is partly localized in synaptic membrane fractions of rat cerebrum (12) and corpus striatum (13). Moreover, topical application of

cyclic AMP or dibutyryl cyclic AMP to postsynaptic neurons normally responsive to catecholamines can mimic the inhibi- tory effects of these amines on neuronal firing (14-16).

Certain of the synaptic actions of the catecholamines may

be mediated by cyclic AMP-regulated protein kinases which catalyze the phosphorylation of synaptic membrane proteins (17-19). However, the cyclic nucleotides have additional actions in neural tissue which are related to neurotransmission.

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Phosphorylation of Cerebral Ribosomal Proteins 289

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis (20), appears to be both activated (21) and induced (22) by cyclic AMP. Activation may involve phospho-

rylation of the enzyme by a cyclic AMP-regulated protein kinase (23, 24). Although the mechanisms underlying cyclic AMP induction of the synthesis of tyrosine hydroxylase and other neural proteins (25) are not known, there is evidence that protein synthesis in the brain is unusually sensitive to

environmental influences and that this sensitivity is partly associated with alterations in ribosomal properties (26-28). In the course of investigations of the nature of these ribosomal

alterations, we have recently observed that several proteins which are present in purified ribosomes and ribosomal subunits from rat cerebral cortex are readily phosphorylated during incubation of cellular preparations in vitro (29). As part of continuing investigations of the possible relationship

of regulatory mechanisms in protein synthesis to specialized function in the central nervous system, an analysis of the effects of cyclic nucleotides on protein phosphorylations in this organ has been undertaken. The present report indicates that ribosomal protein phosphorylation in rat cerebral cortex

is markedly enhanced by cyclic AMP in vitro. This action appears to be highly selective for one ribosomal phosphoprotein.

EXPERIMENTAL PROCEDURES

Incubation of Cerebral Cortical Slices-Young male rats of an inbred Sprague-Dawley strain, 13 to 15 days old and weighing about 30 g, were used in these studies. At this stage of development, adenylate cyclase systems in the cerebral cortex respond maximally to catecholamines with increased production of cyclic AMP (30). The animals were decapitated, and the brains were rapidly removed and placed on filter paper moistened with cold incubation medium in a Petri dish kept on ice. Cerebral cortical slices (0.3 mm thick) were prepared with a McIlwain tissue chopper (311 and washed briefly by suspension in cold modified Krebs-Ringer bicarbonate buffer, pH 7.4, containing 10 rnM glucose. The buffer was prepared as described by Cohen (32), except that KH,PO, was replaced by KCl. Following a brief centrifugation period (15 sl, the medium was decanted and the slices (about 600 mg) were transferred to a plastic beaker containing 7 ml of the same medium. [“‘PlOrthophosphate, neutralized with NaOH, was added to the medium at concentrations ranging from 115 to 300 @J/ml and incubation was carried out for 2 h at 37” under an atmosphere of 95% 0,, 5% CO, in a Dubnoff metabolic incubator at 90 oscillations/min. The contents of each beaker were then washed with 7 ml of fresh medium at room temperature to remove [““Plorthophosphate that had not been taken up by the slices. The cerebral cortical slices were resuspended in 7 ml of modified Krebs-Ringer bicarbonate buffer in a plastic beaker in the presence of dibutyryl cyclic AMP or other related substances. Incubation was continued for 1 additional hour. Variations from these conditions of incubation are described under “Results.” After incubation, the cerebral slices were collected on Whatman No. 1 filter paper in a Buchner funnel and washed with 100 ml of cold 0.9% NaCl.

Preparation of Cerebral Ribosomes and Ribosomal Subunits - Free ribosomes and ribosomal subunits were prepared by two procedures from cerebral cortical slices following incubation. These procedures differed principally in the ionic strength of the media employed during ribosome isolation (29). The use of these two isolation methods was predicated upon earlier findings that, although media containing high concentrations of KC1 were most effective in removing initiation factors and other ribosomal contam- inants which may be phosphorylated in uitro, the use of these media also results in loss of proteins which appear to be integral constituents of the ribosomal structure (see below). Moreover, media of lower ionic strength removed nonproteinaceous :i2P-labeled material that contaminated subunits isolated from cerebral free ribosomes in the presence of high concentrations of KC1 (29).

In the “high salt” procedure for preparing ribosomal subunits from rat cerebral cortex, the postmitochondrial supernatant fraction (291 was layered over 2 M sucrose containing 12 rnM MgCl,, 500 rnM

KCl, 6 rnM /3-mercaptoethanol, 50 rnM Tris/HCl, pH 7.6 (4”), and ribonuclease inhibitor protein (2.5 units/ml) from rat liver (33). This preparation was centrifuged for 20 h at 105,000 x g and 0” in a Spinco 40 or 50 Ti rotor to pellet the free ribosomes. The pellet was then resuspended in the same medium containing 0.25 M sucrose and centrifuged at 105,000 x g and 0” ior 4 h. These purified, salt- washed ribosomes were dissociated as described by Leader and Wool (34). The ribosomal subunits were then separated by centrifugation for 17 h at 64,000 x g and 20” on a linear sucrose gradient (10 to 30%) containing 5 rnM MgCl,, 580 rnM KCl, 20 rnM P-mercaptoethanol, and 10 rnM Tris/HCl, pH 7.5 (22”). The subunits were individually isolated by collecting those fractions of the 40 S and 60 S bands which exhibited absorbances above the half-heights of the peaks on the density gradient recording. For two-dimensional gel electrophoresis, the pooled fractions of each subunit were diluted to a final concentration of 3 rnM MgCl,, and pelleted by centrifugation for 20 h at 226,400 x g and 0” in a Spinco 50 Ti rotor. Although the 40 S subunit obtained by the single dissociation and fractionation procedure described above appeared to be free of 60 S subunits, the 60 S subunits were significantly contaminated with 80 S ribosomes or 40 S subunits, as judged by the appearance of radioactive congeners of the S6 ribosomal protein of the small subunit on autoradiographs of two-dimensional electrophoretograms. Redisso- ciation and refractionation of these 60 S subunit preparations by the high salt procedure described above completely eliminated this contamination. Substitution of 2 rnM MgCl, for 5 rnM MgCl, in the gradients used to isolate cerebral ribosomal subunits produced poorer separation of the 40 S and 60 S species. In contrast, 2 rnM MgCl, was superior to 5 rnM MgCl, for separation of rabbit reticulocyte subunits (35).

In the “low salt” procedure, cerebral free ribosomes were obtained as described above, except that the 2 M sucrose medium contained 100 rnM KC1 instead of 500 rnM KCl. Without further purification, these ribosomes were dissociated by (a) suspension in medium containing 1.5% sucrose, 30 rnM EDTA, 100 rnM KCl, and 50 mM Tris/HCl, pH 7.4 (4”) or (b) incubation at 37” for 20 min in the same medium containing 0.2 rnM puromycin instead of EDTA. Ribosomal subunits prepared by either low salt procedure contained similar complements of radioactive and nonradioactive proteins (29).

Electrophoresis of Ribosomal Proteins on Polyacrylamide Gels Containing Sodium Dodecyl Sulfate -Ribosomal proteins were prepared for electrophoresis on sodium dodecyl sulfate gels as described earlier (29). Samples of the resulting preparations were taken for determination of protein (36) and total radioactivity. The remainder of the ribosomal protein preparation (100 to 200 pg of protein) was then subjected to electrophoretic analysis (37, 38). Following electrophoresis, the gels were stained with Coomassie brilliant blue (R- 2501, destained, and scanned at 600 nm in a Gilford spectrophotome- ter equipped with a linear transport attachment or frozen on dry ice and cut into l-mm sections. The slices were prepared for scintillation counting as described earlier (29). Molecular weights of the separated ribosomal proteins were estimated by concurrent electropho- retie analysis of a standard protein mixture (29).

Two-dimensional Electrophoresis of Ribosomal Proteins -Two-dimensional electrophoresis of ribosomal proteins on polyacrylamide gels containing urea was carried out by a modification of the procedure of Howard and Traut (39). The proteins were isolated from cerebral ribosomal subunits essentially as described by Barri- tault et al. (40). The ribosomal pellet was homogenized in medium composed of 10 mM triethanolamine/HCl (pH 7.2, 4”), 10 mM MgCl,, and 6 rnM ,&mercaptoethanol. To this suspension at 0” was added 1 M MgCl, (to a final concentration of 0.1 M) and 2 volumes of glacial acetic acid with constant stirring. After 45 min, the mixture was centrifuged at 5,000 x g and 0” for 10 min. The ribosomal pellet was extracted again in a similar fashion and the combined supernatants were stirred at 0” while 7 volumes of cold acetone were added dropwise to precipitate the ribosomal proteins. This suspension was then allowed to remain at -20” overnight prior to centrifugation at 15,000 x g and -15” for 20 min to collect the precipitate. This precipitate of ribosomal proteins was washed with acetone and dried in uacuo. For application to polyacrylamide gels containing urea, ribosomal proteins were allowed to dissolve for at least 6 h at room temperature with frequent stirring in a small volume (about 100 to 150 ~1) of medium composed of 8 M urea and 10 rnM fi- mercaptoethanol. Samples were taken for measurements of total protein and radioactivity. The remainder of the ribosomal protein preparation (200 to 600 pg) was applied to a cylindrical polyacrylamide gel measuring 105 x 5 mm. This first dimensional gel


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290 Phosphorylation of Cerebral Ribosomal Proteins

contained 8% acrylamide and 6 M urea and was buffered at pH 8.6 (39). The proteins were stacked for 1 h with a current of 3 mA/gel in a Bio-Rad model 151 tube-gel electrophoresis apparatus maintained at 15” with circulating coolant. Electrophoresis of ribosomal proteins placed at the anode was allowed to proceed toward the cathode at 6 mA/gel for 1’7 h; electrophoresis from cathode to anode was conducted at 1.7 mA/gel for 17 h. Upon completion of the first dimensional run, the gels were removed from the tubes and cut in half length- wise. The two halves of the gel were dialyzed against 8 M urea, 40 rnM acetic acid, and 10 rnM KOH, pH 5.2 (22”) for 1 h with two to three changes of buffer and then incorporated into polyacrylamide slab gels (1.5 mm thick) containing 18% acrylamide and 6 M urea and buffered at pH 4.5. After the ribosomal proteins were stacked at 40 V for 1 h, electrophoresis in the second dimension was allowed to proceed toward the cathode at 400 V for 6 h in a Bio-Rad model 220 slab-gel electrophoresis apparatus cooled by circulating refrig- erant at O-4”. The slabs were stained with Coomassie brilliant blue and then destained. In some instances, the gel slabs were then dried and subjected to autoradiography against Kodak No-Screen medical x-ray film for 3 to 10 days. In other cases, the region of the gel slabs corresponding to the major phosphorylated protein of the 40 S subunit was excised, scanned at 600 nm, and then cut into l- mm sections for scintillation counting. In yet another procedure, individual radioactive spots on the gels of 40 S and 60 S ribosomal proteins were cut out and macerated in a small tube with 1 ml of a solution containing 1% sodium dodecyl sulfate, 8 M urea, and 10 mM /3-mercaptoethanol. The samples were left overnight at room temperature, then prepared for electrophoresis on sodium dodecyl sulfate gels as described above. This third dimensional gel was sliced for scintillation counting to provide a measure of the molecular weights of the radioactive proteins.

Materials -Sucrose (ribonuclease-free), urea, and Tris (all ultra- pure) were obtained from Schwarz/Mann. Urea solutions were further purified by passage through a column of Bio-Rad AG 501. X8(D) Dowex resin prior to use. Ribonuclease (Hirs component A, A grade homogeneous) and puromycin dihydrochloride were purchased from Calbiochem. [“2P10rthophosphate (carrier-free) in 0.02 N HCl, 285 Ci/mg) was obtained from ICN. Reagents for preparation of acrylamide gels were supplied by Bio-Rad, except that sodium dodecyl sulfate was from Sigma. Sigma was also the source of bovine serum albumin (Cohn’s Fraction V), the standard proteins except trypsin, and the sodium salts of N”,02’-dibutyryl adenosine 3’:5’-monophosphate, NG-monobutyryladenosine 3’:5’-monophosphate, and N2,02’-dibutyryl guanosine 3’:5’-monophosphate. Tryp- sin (twice crystallized) was obtained from Nutritional Biochemicals. Sodium butyrate was from Matheson, Coleman and Bell; l-methyl- 3-isobutylxanthine was from Aldrich Chemical Co. Inorganic reagents were Mallinckrodt analytical reagent grade or Baker “Ana- lyzed.” Scintillation counting solutions were supplied by Mallinck- rodt (Handifluor) and Research Products Incorporated (3a70B). N”- Monobutyryl-8-methylthioadenosine 3’:5’-monophosphate at N6, O*‘-dibutyryl-8-hydroxyadenosine 3’:5’-monophosphate were gener- ously supplied by Dr. Eric Nelson of Nelson Research and Develop- ment Company, Irvine, Calif.

RESULTS

Influence of Dibutyryl Cyclic AMP on Ribosomal Protein Phosphorylation in Rat Cerebral Cortex in Vitro -Cerebral cortical slices from immature rats have been shown to in- corporate radioactivity from [32P]orthophosphate into proteins

of both ribosomal subunits (29). In the present experiments, addition of dibutyryl cyclic AMP to cerebral cortical slices which had previously been incubated for 2 h with [3”P]orthophosphate consistently resulted in stimulation of protein phosphorylation in the 40 S subunit (Table I). In 10 experiments, this stimulation averaged 69% 1 h after the

addition of 1 mM dibutyryl cyclic AMP. In contrast, 32P labeling of proteins present in the cerebral large subunit was not increased under the same conditions. Specific radioactivity

of the large subunit proteins was uniformly greater than that of the small subunit proteins when the ribosomes and subunits were prepared by the high salt procedure from cerebral cortical slices incubated in the absence of dibutyryl cyclic

TABLE I

Influence of dibutyryl cyclic AMP on phosphorylation of proteins in ribosomal subunits of rat cerebral cortex in vitro

Cerebral cortical slices were incubated for 2 h in medium containing 300 &i of [“*Plorthophosphate/ml, washed to remove [3”Plorthophosphate that had not been taken up by the tissue, and then incubated 1 additional hour in fresh medium in the presence or absence of 1 rnM dibutyryl cyclic AMP. Free ribosomes were isolated and dissociated in the presence of high concentrations of KCl. The 60 S subunits were redissociated under the same conditions. Purified ribosomal proteins were subjected to one-dimensional electrophoresis on polyacrylamide gels containing 0.1% sodium dodecyl sulfate.

Specific activity

40 s 60 s

cpmlClg protein

Experiment 1” Control 42.1 57.3 Dibutyryl cyclic AMP 73.6 59.1

Experiment 2” Control 71.8 95.1 Dibutyryl cyclic AMP 123.7 87.3

” Total radioactivity in gel determined on l-mm slices following solubilization in 30% hydrogen peroxide.

b Radioactivity determined directly on ribosomal protein preparations.

AMP. In contrast, when the ribosomes and subunits were prepared by the low salt procedure, proteins associated with the small subunit incorporated considerably more radioactiv-

ity than proteins of the large subunit during incubation periods which varied from 1 to 3 h (29). Since exposure of ribosomes and ribosomal subunits to high concentrations of

KC1 tends to remove initiation factors and proteins adventi- tiously associated with the ribosomes (41, 421, the present results indicate that this treatment was more effective in removing proteins associated with the cerebral 40 S subunit than those attached to the 60 S subunit.

Increased phosphorylation of protein of the cerebral 40 S subunit in the presence of dibutyryi cyclic AMP was associated with a single protein band separated by electrophoresis on

polyacrylamide gels containing sodium dodecyl sulfate (Fig. 1). This protein band had the electrophoretic rnbbility of the S,,, protein species with a molecular weight of about 32,000 which has been described earlier (29). The selectivity of dibutyryl cyclic AMP for the S,,, protein species was observed

with 40 S ribosomal subunits isolated in media of low ionic strength (Fig. la), as well as with comparable preparations obtained after exposure to high concentrations of KC1 (Fig. lb). A lag period of about 15 min was noted after addition of 1 mM dibutyryl cyclic AMP. Thereafter, phosphorylation of the

S,,, ribosomal protein species was markedly enhanced for at least 3 h.

In addition to the S,,, ribosomal protein species, two other

radioactive bands consistently appeared on sodium dodecyl sulfate gels of ribosomal proteins from 40 S subunits prepared

in high salt media (Fig. lb). These bands contained materials with molecular weights of approximately 10,000 and 62,000. The component with the higher molecular weight (S,,) is

protein in nature (29) and was also observed when the ribosomal subunits were isolated in low salt media (Fig. la). The band with a molecular weight of about 10,000 is nonproteinaceous (29) and was removed by the low salt procedure. Phos- phorylation of proteins in the S,, band was not significantly


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FIG. 1. Polyacrylamide gel electrophoresis of radioactive proteins present in the 40 S ribosomal subunit prepared from rat cerebral cortical slices following incubation for 1 h in the presence (A-A) or in the absence (0-O) of 1 rn~ dibutyryl cyclic AMP, after a preliminary incubation of 2 h with 170 &i of [32Plorthophosphate/, ml of medium. Ribosomes were isolated and dissociated in the presence of (a) media of low ionic strength or (b) media containing high concentrations of KCl. Ribosomal proteins were subjected to electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. Radioactivity was determined in each l-mm slice of the gel and divided by the total protein applied to the gel to obtain the relative specific activity.

enhanced by dibutyryl cyclic AMP. Specificity and Sensitivity of Stimulatory Action of Dibu-

tyryl Cyclic AMP on Ribosomal Protein Phosphorylation in

Rat Cerebral Slices -The minimum effective concentration of dibutyryl cyclic AMP in stimulating phosphorylation of the

S,,, protein band of the 40 S ribosomal subunit of rat cerebral cortex was about 0.1 mM (Table II). This stimulatory action could be attributed to cyclic AMP per se. Thus, the monobutyryl-8-methylthio derivative of cyclic AMP, as well as l- methyl-3-isobutylxanthine, a potent phosphodiesterase inhibitor which favors the endogenous accumulation of cyclic AMP (431, also exhibited considerable stimulatory activity (Table II). Monobutyryl derivatives of cyclic AMP were less effective than dibutyryl cyclic AMP in stimulating phosphorylation of the S,,, protein species. The 8-hydroxy derivative of dibutyryl cyclic AMP was even more effective than dibutyryl cyclic AMP in this regard (not shown). Sodium butyrate, formed from butyryl derivatives of cyclic AMP in brain tissue (44), and dibutyryl cyclic GMP, each in 1 mM concentration, were devoid of activity (Table II).

Identity of Phosphorylated Ribosomal Protein of Cerebral

40 S Subunit Responsive to Dibutyryl Cyclic AMP-Two- dimensional electrophoresis of the ribosomal proteins from the cerebral small subunit revealed the presence of at least

TABLE II

InfZuence of cyclic AMP derivatives on phosphor-y&ion of S,,, ribosomal protein in rat cerebral cortex in vitro

The conditions of incubation and gel electrophoresis were similar to those described in the legend to Fig. la. Percentage changes from the control values due to addition of the substances listed are given in parentheses.

Relative specific activity

cpml&Lg total protein 011 gel

Experiment 1 Control 26.2 Dibutyryl cyclic AMP, 0.1 rnM 29.8 (+140/o) Dibutyryl cyclic AMP, 1.0 mM 62.3 (+138%)

Experiment 2 Control 15.0 NR-Monobutyryl-8-methylthio cyclic 22.5 (+50%)

AMP, 0.5 rnM Dibutyryl cyclic AMP, 0.5 rnM 25.6 (+71%)

Experiment 3 Control 26.8 1-Methyl-3-isobutylxanthine, 1 rnM 35.3 (+32%)

Experiment 4 Control 23.9 N”-Monobutyryl cyclic AMP, 0.5 rnM 27.0 (+13%) Dibutyryl cyclic AMP, 0.5 rnM 42.4 (+77%)

Experiment 5 Control 13.5 Dibutyryl cyclic GMP, 1 rnM 12.0 (-11%) Sodium butyrate, 1 rnM 12.8 (-5%)

four distinct “2P-labeled species associated with an elongated protein-stained spot (Fig. 2). This major phosphorylated protein of the 40 S subunit from rat cerebral cortex appeared to be analogous to the S6 protein of rat liver ribosomes (45). Superposition of the autoradiographs over the corresponding electrophoretograms revealed that the radioactive spots associated with the cerebral S6 protein were located closer to the anode than the most intensely stained portion of this protein. This phenomenon was more clearly demonstrated when the region corresponding to the elongated S6 protein on the two- dimensional gel was excised, scanned for absorbance at 600 nm, then cut into l-mm wide sections for determination of radioactivity. The radioactivity in the S6 region was associated with five protein species which stained relatively lightly on the gels (Fig. 3). The major stained portion of the cerebral S6 ribosomal protein was not in itself radioactive. Incubation of cerebral cortical slices in the presence of 1 mM dibutyryl cyclic AMP for 3 h not only enhanced the radioactivity incorporated into the phosphorylated congeners of the S6 protein, but also increased the relative absorbances of these species compared to that of the unlabeled, nonphosphorylated species. When protein from different sections of the excised S6 region on two-dimensional gels was eluted and applied to sodium dodecyl sulfate gels, the radioactivity present following electrophoresis was localized exclusively in the S,,, region with a molecular weight of approximately 32,000 (see Fig. 1). Since 32P labeling of cerebral ribosomal proteins in vitro

involves almost exclusively esterification of serine and threonine hydroxyl groups (29), and the congeners of the cerebral S6 protein with slower electrophoretic mobilities in the first dimension exhibited progressively higher specific activities (Fig. 31, it may be concluded that the more acidic species of this protein ctintained an increasing number of phosphoserine or phosphothreonine residues, or both. This conclusion has also been drawn from similar studies of the S6 protein of rat


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Conlrol Dlbutyryl cyclic AMP

FIG. 2. Two-dimensional electrophoresis of radioactive proteins present in the 40 S ribosomal subunit prepared from rat cerebral cortical slices following incubation with [32Plorthophosphate in the presence or in the absence of 1 mM dibutyryl cyclic AMP. Conditions of incubation and preparation of ribosomal subunits were as described in the legend to Table I. Proteins extracted from the small subunit were subjected to two-dimensional electrophoresis on polyacrylamide gels containing urea. The amounts of protein applied to the first dimensional gels were: control, 196 Fg; dibutyryl cyclic AMP, 208 pg. a and b, electrophoretograms stained with Coomassie brilliant blue; the arrows point to the cerebral S6 ribosomal protein. c and d, autoradiographs against Kodak No-Screen medical x-ray film. Magnifications of the electrophoretograms and the autoradiographs are the same.

liver ribosomes (45, 46) and the analogous ribosomal protein (S13) of rabbit reticulocytes (35).

Two additional radioactive phosphoproteins appeared on two-dimensional electrophoretograms of ribosomal proteins isolated from the cerebral small subunit in media of high ionic strength (Fig. 2, c and d). The more prominently labeled of these ribosomal phosphoproteins traveled slightly slower in both dimensions than the slowest moving radioactive congener of the S6 protein and corresponded closely in position with the S2 protein of Sherton and Wool (47, 48) from rat liver and muscle ribosomes. The location of this protein is shown in the schematic of the cerebral 40 S ribosomal proteins in Fig. 4u. The more weakly labeled of these two additional phosphoproteins of the small subunit appeared to correspond with the upper component of the ribosomal protein species labeled S3. When the S2 and S3 protein spots were eluted from the two-dimensional gel slabs and subjected to third dimensional electrophoresis on sodium dodecyl sulfate gels, all of the detectable radioactivity was concentrated in regions of these gels which corresponded to molecular weights of approximately 35,000 and 33,000, respectively. These molecular weights are similar to those obtained for the S2 and S3 ribosomal proteins from other eukaryotic tissues under comparable conditions of analysis (see Ref. 50). Because of the relatively low level of radioactivity in the S2 and S3 proteins, we have not yet been able to determine whether dibutyryl cyclic AMP is capable of stimulating the phosphorylation of these proteins in vitro.

When the 40 S ribosomal proteins were subjected to reverse electrophoresis in the first direction from cathode to anode, followed by the usual second dimensional procedure, no addi-

I I I I

Contro I

DISTANCE FROM ANODE (cm)

FIG. 3. Influence of dibutyryl cyclic AMP on phosphorylation of cerebral S6 ribosomal protein in vitro. Preparation of ribosomal subunits and two-dimensional electrophoresis were carried out as described in the legends to Table I and Fig. 2. Incubation was conducted for 3 h without interruption in medium containing 300 $i of [3zPlorthophosphate/m1 in the presence or in the absence of 1 mM dibutyryl cyclic AMP. The amounts of protein applied to the first dimensional gels were: control, 555 pg; dibutyryl cyclic AMP, 470 pg. After the slab gels were stained with Coomassie brilliant blue, a horizontal rectangular section corresponding to the S6 protein and its more acidic derivatives was cut out and scanned for absorbance at 600 nm (---). This section of the gel was then divided into l-mm slices for determination of radioactivity (O- - -0).

tional phosphorylated proteins were detected. This finding substantiated the conclusion that isolation of cerebral 40 S subunits in high salt media effectively removed phosphorylated proteins which were not integral constituents of the ribosome. However, the results of several studies have suggested that proteins which are part of the ribosomal structure may also be removed by exposure of ribosomes to media of high ionic strength (49, 51, 52). This possibility appeared to be supported by the present studies. Thus, protein Sl which was clearly visible on two-dimensional electrophoretograms of cerebral monomeric ribosomes and 40 S subunits isolated without repeated exposure to high concentrations of KCl, was not detected when the cerebral 40 S subunits were prepared by the high salt procedure described above, unless the gels were overloaded with ribosomal protein.’ Although a protein with the electrophoretic properties of Sl was not detected in 40 S subunits prepared from rabbit reticulocyte ribosomes in the presence of 500 mM KC1 (531, the analogous protein of rat liver or muscle 40 S subunits seemed to be less readily removed by exposure of the ribosomes to high concentrations of KC1 (48).

1 S. Roberts and B. S. Morelos, unpublished observations.


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b

FIG. 4. Schematics of the two-dimensional electrophoretograms of the proteins of cerebral ribosomal subunits. a, proteins from the 40 S subunit; b, proteins from the 60 S subunit. Certain proteins were absent or difficult to see on the stained electrophoretograms of the separated subunits unless the gels were overloaded with protein, but were clearly visible on electrophoretograms of cerebral 80 S ribosomes prepared in media of high ionic strength. These proteins are indicated by cross-hatched spots. Ribosomal proteins are labeled according to the nomenclature of Sherton and Wool (47,481. Unnum- bered spots could not be related to ribosomal proteins identified by numbers in this classification. Separation of the protein complexes designated S3 and L14, each into two or more protein-stained spots, has also been observed by Traut et al. (49).

RELATIVE MIGRATION III/l / I

90 70 50 40 30 20 IO MOLECULAR WEIGHT x lO-3

FIG. 5. Polyacrylamide gel electrophoresis of radioactive proteins present in the 60 S ribosomal subunit prepared from rat cerebral cortical slices following incubation for 1 h in the presence (A-A) or in the absence (O-O) of 1 rnM dibutyryl cyclic AMP, after a preliminary incubation of 2 h with 170 &i of P2Plorthophosphate/ ml of medium. The 60 S subunit was subjected to redissociation and refractionation in media containing high concentrations of KCl. Ribosomal proteins were analyzed by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. See legend to Table I for further explanations.

Phosphorylated Ribosomal Proteins of Cerebral 60 S Sub- unit -Earlier investigations from this laboratory had revealed the presence of several radioactive proteins in 60 S subunits obtained from cerebral cortical slices following incubation with [32Plorthophosphate (29). In the present experiments, redissociation and refractionation of the cerebral 60 S subunits in the presence of high concentrations of KC1 removed certain of these proteins, leaving two major radioactive bands which could be detected on sodium dodecyl sulfate gels (Fig. 5). These radioactive bands corresponded to the L,,, and L. ribosomal protein species previously described, with molecular weights which approximated 37,000 and 15,000, respectively (29). Addition of 1 mM dibutyryl cyclic AMP to the incubation medium did not increase phosphorylation of either of these bands.

When ribosomal proteins of the 60 S subunit from cerebral cortical slices were subjected to electrophoresis in the first direction toward the cathode at pH 8.6 on polyacrylamide gels containing urea, most of the radioactivity remained at the anode. Autoradiographs of conventional two-dimensional electrophoretograms revealed that a considerable fraction of this

First dlmenslon

b

FIG. 6. Two-dimensional electrophoresis of radioactive proteins present in the 60 S ribosomal subunit prepared from rat cerebral cortical slices following incubation with P*Plorthophosphate. Elec- trophoresis in the first dimension was from anode to cathode in a and c and from cathode to anode in b and d. a and b electrophoretograms stained with Coomassie brilliant blue. c and d, autoradiographs. See legend to Fig. 2 for additional explanations.

anodal radioactivity was actually localized in several proteins which were immobile in the first dimension, but migrated toward the cathode at pH 5.2 in the second dimension (Fig. 6, a and c). These findings suggested that these phosphorylated proteins were relatively less basic than the bulk of 60 S ribosomal proteins. This conclusion was supported by subject- ing the 60 S ribosomal proteins to one-dimensional electrophoresis in the opposite direction at pH 8.6, followed by the usual second dimensional procedure. Under these circumstances, three lightly stained protein spots were observed which moved toward the anode (Fig. 6b, arrows) and were radioactive (Fig. 6d). They appeared to correspond to phosphorylated proteins of the 60 S subunit which remained at the anodic origin at pH 8.6 (Fig. 6c). The most highly labeled protein spot, which traveled most rapidly in the second dimension, may have been composed of two or more proteins, possibly phosphorylated forms of the same protein (Fig. 6d). Electrophoresis of the eluted, radioactive material on sodium dodecyl sulfate gels revealed that its mobility corresponded to that of the cerebral L, protein species with a molecular weight of approximately 15,000. Molecular weights of the other two radioactive proteins observed on the reversed polarity two-dimensional gels ranged from 36,000 to 38,000. Thus, the proteins were components of the radioactive L,,, peak observed on sodium dodecyl sulfate gels of cerebral 60 S proteins (see Fig. 5).

The present studies also revealed that at least three basic proteins of the 60 S ribosomal subunit were phosphorylated after incubation of cerebral cortical slices with [32P10rthophosphate (Fig. 6c). These three radioactive proteins appeared to correspond closely in electrophoretic mobility to ribosomal proteins L6, L13, and L14 on stained two-dimensional gels of the cerebral 60 S subunit (Fig. 4b). If the gels were heavily loaded with protein, an additional phosphorylated basic protein was seen, corresponding in position to protein L29 (not shown). The cerebral 60 S subunit was completely free of contamination with the 40 S subunit as judged by the absence of radioactive congeners of the S6 protein on the autoradiographs (Fig. 6c). When the radioactive


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material which was associated with the L14 spot on two-

dimensional slabs was eluted and subjected to third dimensional electrophoresis on sodium dodecyl sulfate gels, this material was found to possess a molecular weight of about

26,000. Because of the low level of radioactivity in the basic 60 S ribosomal proteins which were phosphorylated in cerebral cortical tissue in uitro, they were not readily detected in slices prepared from sodium dodecyl sulfate gels (Fig. 5).

Moreover, it was not possible to determine whether their phosphorylation was individually enhanced by exposure of

the tissue to dibutyryl cyclic AMP. However, as noted earlier, overall phosphorylation of cerebral 60 S subunit proteins was not increased under these conditions (Table I).

DISCUSSION

Several reports have appeared which deal with the effects of cyclic AMP on ribosomal protein phosphorylation in intact eukaryotic cells. Roos (54) noted that addition of cyclic AMP (or adrenocorticotropic hormone) to functional tumor cell cultures of mouse adrenal origin enhanced ribosomal protein

phosphorylation. Barden and Labrie (55) demonstrated that addition of dibutyryl cyclic AMP to slices of bovine anterior pituitary gland slices stimulated phosphorylation of one major ribosomal phosphoprotein component. Subunit localization or molecular weights of the phosphorylated components were

not determined in either study. Cawthorn et al. (56) found that cyclic AMP and dibutyryl cyclic AMP increased ribosomal protein phosphorylation in intact rabbit reticulocytes. The increased phosphorylation was associated with a protein com-

ponent of the 40 S subunit with a molecular weight of approximately 27,500. This protein was probably identical to the S13 ribosomal protein of rabbit reticulocyte 40 S subunits described by Traugh and Porter (35), which undergoes en-

hanced phosphorylation in the presence of cyclic AMP-regulated protein kinases. Gressner and Wool (46) showed that phosphorylation of a single protein derived from the small subunit of rat liver ribosomes was stimulated by administra- tion in viva of cyclic AMP, dibutyryl cyclic AMP, or glucagon.

Subsequent isolation of this ribosomal protein (designated S6) revealed that it possessed a molecular weight of about 31,000 (see Ref. 50). Recently, Schubart et al. (57) demonstrated that

phosphorylation of a ribosomal protein of the 40 S subunit from hamster islet tumor cells was stimulated in vitro by the addition of glucagon, 8-bromoadenosine 3’:5’-monophosphate or 1-methyl-3-isobutylxanthine. This protein had a molecular weight of approximately 28,000.

In the present investigations with cerebral cortical tissue from immature rats, addition of derivatives of cyclic AMP or the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine, markedly enhanced phosphorylation of a single ribosomal protein of the small subunit, but did not increase phosphoryl-

ation of proteins of the large subunit. This phosphorylated protein of the cerebral 40 S subunit appeared to be analogous to the S6 protein of rat liver ribosomes (45, 46, 50) and the S13 protein of rabbit reticulocyte ribosomes (35, 53). In com- mon with the major phosphorylated protein of 40 S ribosomal

subunits from other eukaryotic cells, the cerebral S6 ribosomal protein had a molecular weight of about 32,000 and contained several serine or threonine residues, or both, which were

capable of enhanced phosphorylation in the presence of cyclic AMP. It may be noted that the more acidic (more highly phosphorylated) derivatives of the cerebral S6 protein, which are normally present only in trace amounts on stained electrophoretograms, were actively labeled even in the absence of

added cyclic AMP. This observation may reflect the fact that decapitation causes marked accumulation of cyclic AMP in

the brain (58). The cerebral S6 protein was one of the four radioactive

phosphoprotein components of the small subunit detected on sodium dodecyl sulfate gels when free ribosomes from cerebral slices, incubated in the presence of [“2P10rthophosphate, were isolated and dissociated into subunits in media of low ionic

strength (29). Exposure of the ribosomes and subunits to media containing high concentrations of KC1 did not remove the cerebral S6 protein or eliminate the response to cyclic AMP, but did result in total disappearance of all but one of

the other phosphorylated protein components present on sodium dodecyl sulfate gels following electrophoresis of the 40 S ribosomal proteins. This additional component had a molecu-

lar weight of approximately 62,000, was markedly reduced in concentration when the subunits were isolated in high salt

media, and was not affected by dibutyryl cyclic AMP. In view of the relatively high molecular weight of this component compared to the size of ribosomal proteins from the small subunit of other eukaryotic cells (50, 53, 591, and the fact that

it was largely removed by exposure to high concentrations of KCl, its partial persistence under the latter conditions may reflect residual contamination of the small cerebral subunit with nonribosomal proteins.

In addition to the S6 protein, two other phosphorylated components appeared on conventional two-dimensional gels of cerebral 40 S proteins. These components corresponded electrophoretically to the S2 and S3 proteins of rat liver ribosomes with molecular weights in the 30,000 to 35,000

range (50). 32P labeling of S2 and S3 ribosomal proteins has not been observed in rat liver in vivo (45, 46, 60, 61) even

after exposure to cyclic AMP. However, phosphorylation of a ribosomal protein with the electrophoretic properties of S2 has been noted in Krebs II ascites cells in vitro (62) and in HeLa cells infected with vaccinia virus (63). In addition, two phosphorylated proteins which trail the S3 protein complex in the first dimension have been noted in two-dimensional auto-

radiographs of 40 S ribosomal proteins from rabbit reticulocytes (35). An additional phosphorylated protein of the small ribosomal subunit, with a molecular weight of about 25,000,

has been described in cultured insulinoma cells of the hamster (57). This ribosomal protein did not exhibit alterations in phosphorylation in the presence of cyclic AMP.

The status of phosphorylated proteins in eukaryotic 60 S ribosomal subunits is less certain than that of proteins of the

40 S subunit. Several investigators have found that overall phosphorylation of proteins associated with the large subunit in uiuo or in cellular preparations in vitro proceeded at a rate

that varied from approximately 65 to 120% of the rate for 40 S ribosomal proteins (29, 35, 46, 63). In the present investigations with cerebral cortical slices incubated in the absence of dibutyryl cyclic AMP or other additives, overall phosphorylation of ribosomal proteins isolated from redissociated and

refractionated 60 S subunits was 20 to 40% greater than that of the 40 S proteins. It has been suggested that phosphorylated proteins associated with the 60 S ribosomal subunit in cellular preparations from eukaryotic cells represent mainly contamination with nonribosomal proteins (35, 45). In the present

studies, three or more phosphorylated proteins were isolated from 60 S ribosomal subunits of incubated cerebral cortical slices which exhibited relatively acidic properties on two-

dimensional polyacrylamide gels. These cerebral proteins, with molecular weights of approximately 15,000 and 37,000,


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may be analogous to phosphorylated proteins associated with 60 S ribosomes in cellular preparations of rabbit reticulocytes (35, 56) and Krebs II ascites cells (62), which have not been identified as constitutive ribosomal proteins. However, several proteins of the 60 S subunit which were more basic were also phosphorylated in cerebral cortical tissue in uitro. Three of these proteins migrated from anode to cathode at pH 8.6 with mobilities that were similar to the mobilities of the ribosomal proteins L6, L13, and L14 of Sherton and Wool (47). Phosphorylation of these proteins has not been described for cellular preparations of other eukaryotic tissues in uivo or in vitro (35, 57, 61, 62, 64).

8. 9.

10.

11.

12.

13.

Kakiuchi, S., and Rall, T. W. (1968) Mol. Pharmacol. 4, 367-378 Klainer, L. M., Chi, Y.-M., Freidberg, S. L., Rall, T. W., and

Sutherland, E. W. (1962) J. Biol. Chem. 237, 1239-1243

The present investigations support earlier findings from this laboratory which indicated that several proteins of both ribosomal subunits are capable of being phosphorylated by endogenous protein kinases in cellular preparations of rat cerebral cortex in vitro (29). However, which of these ribosomal proteins undergo phosphorylation in viuo remains to be determined. Although current investigations suggest that phosphorylation of initiation factors alters their capacity in protein synthesis (65-681, the physiological functions which may be served by phosphorylation of structural proteins of the ribosome are essentiallv unknown. It is not unlikelv that the latter phenomenon is involved in several diverse functions of the ribosome, including processes which are not directly related to protein synthesis (69). Several investigators have suggested that ribosomal protein phosphorylation may be involved in attachment of the ribosome to messenger RNA, initiation factors or other components of the protein-synthesiz- ing system, and that these processes are affected when phosphorylation is enhanced by intracellular accumulation of cyclic AMP resulting from altered cellular activity (29, 35, 46, 55, 70-72). The present results are consistent with the possibility that the accumulation of cyclic AMP in neural tissue that accompanies the release of certain neurotransmitter amines (5) may modify translational processes in protein synthesis by increasing the phosphorylation of a specific protein of the small ribosomal subunit. Moreover, in view of the strong likelihood that each 40 S cerebral ribosome contains only one congener of this protein (see Ref. 46), it is possible that neurotransmitter-induced production of cyclic AMP is capable of increasing the heterogeneity of the cerebral ribosomal pool and, thereby, perhaps selectively altering the synthesis of specific proteins which may function in neurotransmission.

Acknowledgments -We wish to thank Rita Kern and Bea- trice S. Morelos for valuable technical assistance.

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S Roberts and D Ashbycyclic adenosine 3':5'-monophosphate.

Ribosomal protein phosphorylation in rat cerebral cortex in vitro. Influence of

1978, 253:288-296.J. Biol. Chem.

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