Implication of guanosine 3′,5′-cyclic monophosphate,adenosine 3′,5′-cyclic monophosphate,adenosine 5′-mono-, di- and triphosphate and fructose-2,6-bisphosphate in the regulation of the glycolytic pathway in hypoxic/anoxic mussel,Mytilus galloprovincialis

The change in the content of cyclic GMP, cyclic AMP, ATP, ADP, AMP and fructose-2,6-bisphosphate that occurred in the mantle of the mussel Mytilus galloprovincialis Lmk when specimens of this mollusk were subjected to a hypoxia/anoxia situation were assessed. After the early 24 h in anaerobiosis, a clear decrease was observed in the ATP content, which remained close to that value for the rest of the time. AMP content doubled during the early 24 h in anaerobiosis and, from that time on, it remained close to that value. Fructose-2,6-bisphoshate and cyclic GMP showed a similar behavior. The levels of these compounds rose significantly during the early hours in anaerobiosis, and then fell to values similar to those of aerobiosis, remaining constant for the rest of the time. Neither ADP nor cAMP showed significant variations.     

Adenosine 3'5'-m onophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum.

In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fol. Chem. 248,494-501). We have now studied the effects oc cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteinsmextraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis; The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormonemat least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the ase of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP wherease luteinizing hormone has no effect on ribosome phosphorylation.     

Adenosine 3′,5′-monophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum

In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fraction by cyclic AMP and luteinizing hormone was reported (Menon, K.M.J. (1973) J. Biol. Chem. 248, 494–501). We have now studied the effects of cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteins. Extraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis. The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormone. At least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the case of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP whereas luteinizing hormone has no effect on ribosome phosphorylation.     

Phosphorylation of ribosomal proteins in hamster fibroblasts infected with pseudorabies virus or herpes simplex virus.

In BHK cells infected with pseudorabies virus, there was a substantial increase in the phosphorylation of ribosomal protein S6. This increase occurred between 2 and 4 h after infection and persisted at least until 9 h. We estimated that in mock-infected cells S6 contained, on an average, one phosphate group per protein chain, whereas in infected cells this rose to between four and five phosphate groups per protein chain. A second ribosomal protein, either S16 or S18, was also phosphorylated after infection. No increase in cyclic AMP was found at the time of phosphorylation. We also found an increased phosphorylation of S6 in herpes simplex virus-infected BHK cells.     

Phosphorylation of translational initiation factor 3 (eIF-3) by cyclic AMP-regulated protein kinase.

Translational initiation factor 3 (eIF-3) is phosphorylated by the cyclic AMP-regulated protein kinases from rabbit reticulocytes. eIF-3 is a large molecular weight complex which facilitates binding of the ternary complex containing met tRNA_f, GTP and initiation factor 2 to 40S ribosomal subunits. A single polypeptide with a molecular weight of 130,000 is modified. The phosphorylation is dependent upon the presence of cyclic AMP and is inhibited by the inhibitor protein diagnostic for cyclic AMP-regulated protein kinase. Assuming a molecular weight of 700,000 for eIF-3, one mole of phosphate is incorporated per mole of eIF-3. Thus the phosphorylation of two interacting components of the protein synthesizing system, 40S ribosomal subunits and eIF-3, is controlled by cyclic AMP.     

Phosphorylation of multiple proteins of both ribosomal subunits in rat cerebral cortex in vivo. Effect of adenosine 3'':5''-cyclic monophosphate.

Investigations were carried out on the phosphorylation of ribosomal proteins in vivo in cerebral cortices of immature rats. Two-dimensional electrophoresis revealed that the cerebral 40S subunit contained at least four ribosomal proteins which were phosphorylated in animals given [32P]orthophosphate intracisternally. These proteins exhibited electrophoretic properties similar to those of the constitutive basic proteins S2, S3a, S5 and S6. The cerebral 60S subunit contained several proteins that were phosphorylated in vivo, including three basic proteins with electrophoretic mobilities similar to those of ribosomal proteins L6, L14 and L19. Four other proteins associated with the 60S subunit that were more acidic were also phosphorylated. Phosphorylated congeners of 40S and 60S ribosomal proteins could often be detected in distinct protein-stained spots on two-dimensional electrophoretograms. The cerebral S6 protein consisted of at least five distinct species in different states of phosphorylation. Administration of N6O-2' dibutyryl cyclic AMP increased the proportion of the more phosphorylated congeners of the S6 protein, but appeared to have little or no effect on phosphorylation of other cerebral ribosomal proteins. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated S6-protein phosphorylation; N2O2'-dibutyryl cyclic GMP had no effect on this process. These observations indicate that several ribosomal proteins of both subunits are normally phosphorylated in rat cerebral cortex in situ. The results also suggest that selective and specific alterations in the phosphorylation state of the S6 ribosomal protein of the cerebral 40S subunit may accompany the production of cyclic AMP during neural activation.     

ACTIVITY OF CYCLIC AMP-DEPENDENT MICROSOMAL PROTEIN KINASE AND PHOSPHORYLATION OF RIBOSOMAL PROTEIN IN RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— Microsomes from rat brain exhibited protein kinase activity which was stimulated by cyclic AMP when assayed in the presence of exogenous protein substrate, such as thymus histone. In the absence of exogenous substrate some phosphorylation of microsomal protein occurred, but no stimulation by cyclic AMP could be discerned, probably because of limitations of substrate. The maximal activity of microsomal protein kinase observed in the presence of saturating concentrations of histone and the optimal concentration (5 μ m ) of cyclic AMP remained essentially unchanged from birth to early adulthood, but the magnitude of the stimulation by cyclic AMP was significantly higher at birth than at 30 days of age. Brain ribosomal proteins could be phosphorylated by the cyclic AMP-dependent brain protein kinase. Their total capacity for acceptance of phosphate by means of this phosphorylation reaction remained unchanged throughout the postnatal development of the brain. Our results are consistent with the possibility that phosphorylation of ribosomal protein mediated by cyclic AMP-dependent protein kinase may play a a role in the postnatal regulation of cerebral protein synthesis, as a result of the changes in the levels of cyclic AMP known to occur in brain during postnatal maturation.     

Protein-bound histidine, as well as protein-bound serine, residues are sites of phosphorylation in the synaptic plasma membrane.

When synaptic plasma membrane fragments are incubated with ATP in the presence of Mg2+, phosphate is transferred, not only to protein-bound serine, but also to protein-bound histidine. The phosphorylation of protein-bound serine is stimulated by cyclic AMP and has a Km for ATP of about 0.12 mM, both in the presence and absence of cyclic AMP. By contrast, the phosphorylation of protein-bound histidine is unaffected by cyclic AMP and does not follow Michaelis-Menton kinetics since a non-linear double reciprocal plot is given when activity is measured at various ATP concentrations.     

Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex

Suspensions of renal cortical tubules were incubated with ³³P_i and exposed to parathyroid hormone (40 μg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [γ-³²P]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 1500 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.     

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.     

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.     

Differences in the cyclic AMP-dependent phosphorylation of plasma membrane proteins of differentiated and undifferentiated L6 myogenic cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L6 myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L6 myoblasts was stimulated more than three fold by 5 x 10(-5) M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L6 cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L6 cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f2b. Therefore, the data show that differentiation in L6 cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts.

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743-745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L6 myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system. We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f2b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10(-5) M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase. Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems. The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane phosphorylation as a regulator of membrane functions including those that may control cellular differentiation.     

The effect of dibutyryl cyclic AMP and butyrate on F9 teratocarcinoma cellular retinoic acid-binding protein activity

F9 teratocarcinoma cells contain a cellular retinoic acid-binding protein (CRABP) that may mediate the retinoic acid-induced differentiation of this cell line. Specific [3H]retinoic acid binding to CRABP in F9 stem cell cytosol is protein-dependent, reaches equilibrium within 4 h at 4 degrees C, and yields 643 +/- 105 fmol of [3H]retinoic acid per mg of protein with an apparent dissociation constant of 9.2 +/- 1.1 nM. When F9 stem cells are grown in the presence of either dibutyryl cyclic AMP or sodium butyrate, CRABP activity is stimulated 2-4-fold. The effect of these drugs on CRABP activity is both time and concentration-dependent, resulting in an increase in the number of binding sites for [3H]retinoic acid with no change in their affinity. The new [3H]retinoic acid-binding sites have a sedimentation coefficient of 2 S and are not displaced by excess retinol. When F9 stem cells are grown in the presence of cyclic 8-bromo-AMP or cholera toxin, no increase in CRABP activity is observed. We conclude that the stimulation of CRABP activity by dibutyryl cyclic AMP may result from the action of butyrate. In addition, the stimulation of retinoic acid-induced F9 cell differentiation by cyclic AMP analogs (Strickland, S., Smith, K.K., and Marotti, K.R. (1980) Cell 21, 347-355) and the inhibition of this differentiation by butyrate (Levine R. A., Campisi, J., Wang, S.-Y., and Gudas, L. J. (1984) Dev. Biol. 105, 443-450) are not correlated with increases or decreases, respectively, in the level of CRABP activity.     

Cyclic AMP-Dependent Protein Phosphorylation in Chemosensory Neurons: Identification of Cyclic Nucleotide-Regulated Phosphoproteins in Olfactory Cilia

Chemosensory dendritic membranes (olfactory cilia) contain protein kinase activity that is stimulated by cyclic AMP and more efficiently by the nonhydrolyzable GTP analog guanosine-5'-O-(3-thio)triphosphate (GTP gamma S). In control nonsensory (respiratory) cilia, the cyclic AMP-dependent protein kinase is practically GTP gamma S-insensitive. GTP gamma S activation of the olfactory enzyme appears to be mediated by a stimulatory GTP-binding protein (G-protein) and adenylate cyclase previously shown to be enriched in the sensory membranes. Protein kinase C activity cannot be detected in the chemosensory cilia preparation under the conditions tested. Incubation of olfactory cilia with [gamma-32P]ATP leads to the incorporation of [32P]phosphate into many polypeptides, four of which undergo covalent modification in a cyclic nucleotide-dependent manner. The phosphorylation of one polypeptide, pp24, is strongly and specifically enhanced by cyclic AMP at concentrations lower than 1 microM. This phosphoprotein is not present in respiratory cilia, but is seen also in membranes prepared from olfactory neuroepithelium after cilia removal. Cyclic AMP-dependent protein kinase and phosphoprotein pp24 may be candidate components of the molecular machinery that transduces odor signals.     

Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex.

Suspensions of renal cortical tubules were incubated with 33Pi and exposed to parathyroid hormone (40 mlg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [gamma-32P]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 150 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.     

Inhibition of cerebral protein kinase activity and cyclic AMP-dependent ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia

Studies were carried out to elucidate the mechanisms underlying the diminished phosphorylation of cerebral ribosomal protein in experimental hyperphenylalaninaemia [Roberts & Morelos (1980) Biochem. J.190, 405-419]. Administration of N(6),O(2)'-dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine, which increased phosphorylation of the S6 protein of cerebral 40S ribosomal subunits in control infant rats, did not counteract the decreased phosphorylation of this ribosomal protein resulting from intraperitoneal administration of a loading dose of l-phenylalanine. N(2),O(2)'-Dibutyryl cyclic GMP had no effect on cerebral ribosomal-protein phosphorylation in either control or hyperphenylalaninaemic animals. The phenylalanine-induced decrease in ribosomal-protein phosphorylation was associated with decreased protein kinase activity in cerebral cytosolic and microsomal preparations. However, the maximal protein kinase response to cyclic AMP added in vitro was unaltered by prior administration of phenylalanine in vivo. The heat-stable protein inhibitor of cyclic AMP-dependent protein kinases decreased the activity of these enzymes by about 90% and eliminated the phenylalanine-induced difference in protein kinase activity in the absence of added cyclic AMP. Intracisternal administration of doses of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine which increased the cyclic AMP-dependent protein kinase activity ratio in control infant rats was without effect on this index in phenylalanine-treated animals. Dibutyryl cyclic GMP had no effect on the protein kinase activity ratio in either group of animals. These results suggest that inhibition of cerebral cyclic AMP-dependent protein kinases by abnormally high concentrations of phenylalanine may contribute to the decrease in cerebral ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia.     

Influence of extracellular phosphate concentrations on the regulation of hepatic glucose output

Experiments were carried out to investigate the role of extracellular phosphate in the hormonal regulation of glycogenolysis in perfused fed-rat liver. Omission of phosphate from the perfusate did not affect the ATP, ADP and AMP contents of the tissue and the basal glucose output from the perfused liver. However, it inhibited significantly the glycogenolysis induced by glucagon, cyclic AMP, phenylephrine and vasopressin but not that induced by 2,4-dinitrophenol. In the absence of perfusate phosphate, the increase in phosphorylase a activity caused by the addition of glucagon, phenylephrine and vasopressin was significantly less than that observed in the presence of perfusate phosphate. Insulin inhibition of the glucagon- or cyclic AMP-induced glycogenolysis was abolished when the perfusion was carried out with the phosphate-free buffer. However, the inhibitory effect of insulin on phenylephrine-induced glycogenolysis was clearly demonstrated even when the perfusate contained no phosphate. These data indicate that in the phosphate-depleted liver, the hormonal control of phosphorylation and dephosphorylation of phosphorylase is impaired. The difference in the phosphate dependency of insulin action on glucagon-and alpha-adrenergic agonist-induced glycogenolysis suggests that the mechanism or site of insulin action on glucagon and phenylephrine is different.     

Protein phosphorylation and dephosphorylation in liver plasma membrane. Effect of inorganic phosphate

When a plasma membrane preparation isolated from rat liver was incubated with [gamma-32P]ATP and Mg2+, protein-bound 32P increased rapidly, followed by a gradual decrease. The time course suggested the existence of membrane-bound kinase(s) and phosphatase(s) phosphorylating and dephosphorylating endogenous proteins. The extent of phosphorylation was not affected by inclusion of cyclic AMP in the reaction mixture. The extent of the maximum phosphorylation was dependent on membrane concentration, owing to rapid hydrolysis of ATP by the membrane-bound ATPase activity. Thus, phosphorylation proceeded further on repeated addition of ATP. Both phosphorylation and dephosphorylation were stimulated by Mg2+, an effective rate of phosphorylation being obtained at 15 mM. Pi up to 20 mM stimulated phosphorylation with little effect on the rate of dephosphorylation. At higher phosphate concentrations, the maximum 32P-incorporation decreased again, and at 100 mM, dephosphorylation was prevented significantly. Autoradiography after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea revealed six main phosphorylated bands, two of which (Band 3 and 5) were partly extractable with 1 M NaCl. In the presence of 100 mM Pi, very strong phosphorylation of Band 5 (about 23,000 daltons) was noted, and a new strongly labeled band (Band P, about 20,000 daltons) was observed. It was concluded that the phosphoproteins in the membrane may be turned over at different rates and high concentrations of Pi may affect the turnover rate of some phosphoproteins, probably through interference with the phosphatase.     

Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.