Depolarization-Dependent Protein Phosphorylation in Rat Cortical Synaptosomes: Characterization of Active Protein Kinases by Phosphopeptide Analysis of Substrates

Depolarization of synaptosomes is known to cause a calcium-dependent increase in the phosphorylation of a number of proteins. It was the aim of this study to determine which protein kinases are activated on depolarization by analyzing the incorporation of 32Pi into synaptosomal phosphoproteins and phosphopeptides. The following well-characterized phosphoproteins were chosen for study: phosphoprotein "87K," synapsin Ia and Ib, phosphoproteins IIIa and IIIb, the catalytic subunits of calmodulin kinase II, and the B-50 protein. Each was initially identified as a phosphoprotein in lysed synaptosomes after incubation with [gamma-32P]ATP. Mobility on two-dimensional polyacrylamide gels and phosphorylation by specific protein kinases were the primary criteria used for identification. A technique was developed that allowed simultaneous analysis of the phosphopeptides derived from all of these proteins. Phosphopeptides were characterized in lysed synaptosomes after activating cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases in the presence of [gamma-32P]ATP. Phosphoproteins labelled in intact synaptosomes after incubation with 32Pi were then compared with those seen after ATP-labelling of lysed synaptosomes. As expected from previous work, phosphoprotein "87K," and synapsin Ia and Ib were labelled, but for the first time, phosphoproteins IIIa, IIIb, and the B-50 protein were identified as being labelled in intact synaptosomes; the calmodulin kinase II subunits were hardly phosphorylated. From a comparison of the phosphopeptide profiles it was found that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases are all active in intact synaptosomes and their activity is dependent on extrasynaptosomal calcium. The activation of cyclic AMP-stimulated protein kinases in intact synaptosomes was confirmed by the addition of dibutyryl cyclic AMP and theophylline which specifically increased the labelling of phosphopeptides in synapsin Ia and Ib and in phosphoproteins IIIa and IIIb. On depolarization of intact synaptosomes, a number of phosphopeptides showed increased labelling and the pattern suggested that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases were all activated. No new peptides were phosphorylated, suggesting that depolarization simply increased the activity of already active protein kinases and that there was no depolarization-specific increase in protein phosphorylation.     

Adenosine 3',5'-monophosphate-dependent protein kinases of myocardial non-histone nuclear proteins.

Myocardial acidic non-histone nuclear proteins (NHPs) contain endogenous protein kinase activity. Phosphocellulose chromatography of purified NHPs identifies nine separate peaks of protein kinases which can phosphorylate both endogenous and exogenous substrates to a variable degree; endogenous NHPs are the best substrates. Cyclic AMP-stimulated protein kinase induced phosphorylation of endogenous and exogenous substrates; the extent of this stimulation varied according to the protein kinase fraction and substrate used. Cyclic AMP also enhanced NHP-induced stimulation of RNA polymerase activity. This enhancement was dependent on protein kinase-induced phosphorylation of NHPs since it was prevented by alkaline phosphatase pretreatment. It is concluded that nuclear protein kinases regulate myocardial RNA synthesis by enhancing phosphorylation of NHPs and that this regulation is under cyclic AMP control.     

Cyclic AMP-stimulated protein kinases at brain synaptic junctions.

We have examined endogenous cyclic AMP-stimulated phosphorylation of subcellular fractions of rat brain enriched in synaptic plasma membranes (SPM), purified synaptic junctions (SJ), and postsynaptic densities (PSD). The analyses of these fractions are essential to provide direct evidence for cyclic AMP-dependent endogenous phosphorylation at discrete synaptic junctional loci. Protein kinase activity was measured in subcellular fractions using both endogenous and exogenous (histones) proteins as substrates. The SJ fraction possessed the highest kinase activity toward endogenous protein substrates, 5-fold greater than SPM and approximately 120-fold greater than PSD fractions. Although the kinase activity as measured with histones as substrates was only slightly higher in SJ than SPM fractions, there was a marked preference of kinase activity toward endogenous compared to exogenous substrates in SJ fractions but in SPM fractions. Although overall phosphorylation in SJ fractions was increased only 36% by 5 micron cyclic AMP, there were discrete proteins of Mr = 85,000, 82,000, 78,000, and 55,000 which incorporated 2- to 3-fold more radioactive phosphate in the presence of cyclic AMP. Most, if not all, of the cyclic AMP-independent kinase activity is probably catalyzed by catalytic subunit derived from cyclic AMP-dependent kinase, since the phosphorylation of both exogenous and endogenous proteins was greatly decreased in the presence of a heat-stable inhibitor protein prepared from the soluble fraction of rat brain. The specific retention of SJ protein kinase(s) activity during purification and their resistance to detergent solubilization was achieved by chemical treatments which produce interprotein cross-linking via disulfide bridges. Two SJ polypeptides of Mr = 55,000 and 49,000 were photoaffinity-labeled with [32P]8-N3-cyclic AMP and probably represent the regulatory subunits of the type I and II cyclic AMP-dependent protein kinases. The protein of Mr = 55,000 was phosphorylated in a cyclic AMP-stimulated manner suggesting autophosphorylation as previously observed in other systems.     

Effect of Hypothyroidism on the Subcellular Distribution of Ca2+/Calmodulin-Stimulated Protein Kinase II in Chicken Brain During Posthatch Development

Abstract: In developing chicken brain Ca²⁺/calmodulin-stimulated protein kinase II (CaMPK-II) changes from being primarily cytosolic to being primarily particulate during the protracted maturation period. To investigate whether thyroid hormone levels may be involved in regulating this subcellular redistribution, we raised chickens from 1 day posthatching on food soaked in 0.15% (wt/vol) propylthiouracil (PTU) plus 0.05% (wt/vol) methimazole (MMI). This produced a mild hypothyroidism specifically during the maturation period and resulted in a 67% reduction in the levels of free triiodothyronine (T₃) at 42 days. The concentrations of α- and β-CaMPK-II in cytosol (S3) and crude synaptic membrane (P2M) fractions from forebrain were measured by three methods: Ca²⁺/calmodulin- or Zn²⁺-stimulated autophosphorylation or binding of biotinylated calmodulin. By all three methods hypothyroid animals showed a marked retardation of the redistribution of both subunits of CaMPK-II: an increase in the concentration of the enzyme in S3 and a corresponding decrease in P2M with no overall change in the total amount of enzyme and little apparent change in the concentration of other proteins. In both fractions, there was a parallel change in the Ca²⁺/calmodulin-stimulated phosphorylation of endogenous protein substrates but no change in the basal or cyclic AMP-stimulated protein phosphorylation. Supplementing the PTU/MMI-treated diet with thyroxine (0.5 ppm) prevented all of the observed changes.     

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain.

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.     

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg²⁺ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.     

The in vitro phosphorylation of actin from rat cerebral cortex

Actin was phosphorylated by a cyclic AMP-stimulated protein kinase in a lysed synaptosomal fraction incubated with [gamma-32P]ATP, while calcium had no effect on endogenous labeling of the protein. Incubation of an intact synaptosomal fraction with 32P-inorganic phosphate did not lead to any detectable phosphorylation of actin in the presence or absence of dibutyryl-cyclic AMP, or chemical depolarization. It is suggested that actin is not phosphorylated in the physiologically relevant intact synaptosomes but gains access to protein kinases on lysis.     

Protein kinases and their substrates in brown adipose tissue from newborn rats.

The 10000 X g supernatant fraction of brown fat from newborn rats catalyzed the cyclic AMP-dependent phosphorylation of both histone and a preparation of proteins from the same subcellular fraction (endogenous proteins). The apparent affinity for ATP was lower for the phosphorylation of the endogenous proteins than for the phosphorylation of histone. In order to discover whether the phosphorylation of histone and the endogenous proteins were catalyzed by different enzymes, the 100000 X g supernatant was fractionated by ion-exchange and adsorption chromatography. Three different cyclic AMP-dependent protein kinases and one cyclic AMP-independent protein kinase were separated and partially purified. Each of these enzymes catalyzed the phosphorylation of both substrates, and the difference in apparent Km for ATP remained. Neither affinity chromatography on histone-Sepharose, nor electrophoresis on polyacrylamide gels resulted in the separation of the phosphorylation of histone from that of the endogenous proteins of any of the partially purified kinases. Moreover, experiments in which the phosphorylated substrates were separated by differential precipitation with trichloroacetic acid showed that the endogenous proteins competitively inhibited the phosphorylation of lysine-rich histone. It is concluded that each of the partially purified kinase preparations contains protein kinase, which catalyzes the phosphorylation of both substrates. The difference in apparent Km for ATP was found to be due to the presence in the endogenous protein preparation of a low molecular weight compound which competes with ATP. This was not ATP nor the modulator protein. The ratio of the phosphorylation of endogenous proteins to that of histone was much higher for the cyclic AMP-independent kinase preparation than for the other enzymes. Electrophoresis of the endogenous substrates in the presence of sodium dodecyl sulphate showed that the enzyme phosphorylated a greater number of proteins than did the cyclic AMP-dependent kinases. The phosphorylation of endogenous proteins relative to that of histone was significantly lower for one of the cyclic AMP-dependent kinases than for the other two. This difference was not reflected in a different pattern of phosphorylation of the individual proteins of the endogenous mixture.     

The role of protein phosphorylation in the regulation of cyclic nucleotide phosphodiesterases

The cyclic nucleotide phosphodiesterases constitute a complex superfamily of enzymes responsible for catalyzing the hydrolysis of cyclic nucleotides. Regulation of cyclic nucleotide phosphodiesterases is one of the two major mechanisms by which intracellular cyclic nucleotide levels are controlled. In many cases the fluctuations in cyclic nucleotide cAMP-specific, calmodulin-stimulated and cGMP-binding phosphodiesterases have been demonstrated to be substrates for protein kinases. Here we review the evidence that hormonally responsive phosphorylation acts to regulate cyclic nucleotide phosphodiesterases. In particular, the cGMP-inhibited phosphodiesterases, which can be phosphorylated by at least two different protein kinases, are activated as a result of phosphorylation. In contrast, phosphorylation of the calmodulin-stimulated phosphodiesterases, which coincides with, a decreased sensitivity to activation by calmodulin, results in decreased phosphodiesterase activity.     

Topography of protein kinases and phosphoproteins in the plasma membrane of 3T3 cells

The plasma membrane of 3T3 cells contains at least two different endogenous cyclic AMP-dependent protein kinase systems. One catalyzes the phosphorylation of endogenous protein substrates, i.e., PP24 and PP14, whereas the other catalyzes the phosphorylation of exogenous substrates. In this paper the topography of these cyclic AMP-dependent phosphorylation systems is described. The results show that the kinases which phosphorylate only exogenous substrates are primarily localized to the outer plasma membrane surface whereas the endogenous cyclic AMP-dependent protein kinase and its two endogenous substrates are localized to the cytoplasmic plasma membrane surface. The data also establish that neither the cytoplasmically orientated kinase nor its substrates has a transmembrane orientation even though factors acting on the outer plasma membrane can affect these proteins. This suggests that functional modulation of the cytoplasmically localized cyclic AMP-dependent phosphorylation system can be mediated by a transmembrane regulatory mechanism. The importance of determining the topography of such plasma membrane phosphorylation systems is emphasized by recent studies which show that neoplastic transformation can be mediated at least in part by protein kinases and/or phosphoproteins which are localized on the cytoplasmic surface of the plasma membrane.     

Phosphorylation of membranes from the rat gastric mucosa

Gastric mucosal membranes derived primarily from parietal cells were found to contain endogenous protein kinase systems as well as several phosphate-accepting substrates. One specific membrane protein with a molecular weight of 88 000 was phosphorylated only in the presence of calcium, while the degree of phosphorylation of three other membrane proteins was similarly increased. The activity of the calcium-dependent protein kinase was found to be totally inhibited in the presence of trifluoperazine, a phenothiazine known to specifically inactivate calmodulin. These results suggest that a calmodulin- and calcium-dependent phosphorylation system may be a component of the parietal cell membrane. Phosphorylation of the membrane proteins was not affected by either cyclic AMP or cyclic GMP. The heat-stable inhibitor protein of cyclic AMP-dependent protein kinase did not inhibit the endogenous protein kinase activity suggesting that the membrane enzyme is not similar to the cytosolic protein kinase. However, the catalytic subunit of the soluble enzyme was capable of phosphorylating a number of membrane proteins indicating that after maximal autophosphorylation of the gastric membranes, phosphate-acceptor sites are still available to the cytosolic cyclic AMP-dependent protein kinase.     

Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Partition of substrates and protein kinase activities with triton X-100.

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase activity catalysing the phosphorylation of endogenous substrates. Extraction of membrane fragments with Triton X-100 solubilized less than 20% of the kinase activity and left the major part of the endogenous substrates in the insoluble fraction.     

In Vitro Protein Phosphorylation in Head Preparations from Normal and Mutant Drosophila melanogaster

We have characterized protein phosphorylation in vitro in subcellular fractions from Drosophila melanogaster heads. Optimal conditions for the incorporation of 32P into proteins, and its dependence on ATP, divalent cations, and cyclic nucleotides have been determined, as well as the effect of inhibitors of ATPase, protein phosphatase, and protein kinase on protein phosphorylation. Among these inhibitors, Zn2+ was found to affect the incorporation of 32P into specific bands and p-hydroxymercuribenzoate was found to be most suited for freezing the activity of both kinases and phosphatases. Cyclic AMP-dependent protein kinase (cAMP-dPK) activity was present in both supernatant (S2) and particulate (P2) fractions, with the majority (60-85%, depending on the homogenization medium) being associated with S2, as determined by phosphorylation of exogenous synapsin I. cAMP-dPK catalyzed the phosphorylation of at least 18 endogenous polypeptides in S2 and at least 10 endogenous polypeptides in P2. These proteins could be classified on the basis of the extent of stimulation of phosphorylation by cyclic nucleotides, dependence on cyclic nucleotide concentration, and rate of phosphorylation. A phosphoprotein of 51 kilodaltons (pp51) was a major component of the S2 and P2 fractions and displayed properties expected from the regulatory subunit of the cAMP-dPK, R-II. A phosphoprotein doublet of approximately 37 kilodaltons (pp37) was stimulated to the largest extent by cAMP in the P2 and S2 fractions. The phosphorylation of several proteins in both fractions was significantly lowered by the mammalian Walsh inhibitor of cAMP-dPK, whereas in some cases the stimulation of phosphorylation of the same proteins by exogeneous cAMP was relatively small. Phosphoproteins from two learning mutants known to be deficient in cAMP metabolism, dnc and rut, were analyzed for their extent of phosphorylation in the presence of a stable cAMP analogue; no significant differences from normal were detected, suggesting that the genetic defect in cAMP metabolism is not accompanied by constituent abnormalities in phosphorylated substrates in the adult fly, and that the physiological defects in these mutants result from aberrations in the interaction of the cAMP cascade with normal substrates. The majority of Ca2+/calmodulin kinase activity (80-90%, depending on the homogenization procedure) was associated with S2, as revealed by phosphorylation of exogenous synapsin I. Two endogenous substrates for this kinase in P2 had molecular masses of approximately 45 and 87 kilodaltons. At least 11 substrates for the Ca2+/calmodulin-dependent kinase were detected in S2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+ and Calmodulin-Dependent Phosphorylation of Endogenous Synaptic Vesicle Tubulin by a Vesicle-Bound Calmodulin Kinase System

Endogenous synaptic vesicle alpha- and beta-tubulin were shown to be the major substrates for a Ca2+-calmodulin-regulated protein kinase system in enriched synaptic vesicle preparations from rat cortex as determined by two-dimensional gel electrophoresis and peptide mapping. The activation of this endogenous tubulin kinase system was dependent on Ca2+ and the Ca2+ binding protein, calmodulin. Under maximally stimulated conditions, approximately 40% of the tubulin present in enriched synaptic vesicles was phosphorylated within less than 50 s by the vesicle Ca2+-calmodulin kinase. Evidence is presented indicating that the Ca2+-calmodulin tubulin kinase is an enzyme system distinct from previously described cyclic AMP protein kinases. alpha-Tubulin and beta-tubulin were identified as major components of previously designated vesicle phosphorylation bands DPH-L and DPH-M. The Ca2+-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. Ca2+-activated synaptic vesicle tubulin phosphorylation correlated with vesicle neurotransmitter release. Depolarization-dependent Ca2+ uptake in intact synaptosomes simultaneously stimulated the release of neurotransmitters and the phosphorylation of synaptic vesicle alpha- and beta-tubulin. The results indicate that regulation of the synaptic vesicle tubulin kinase by Ca2+ and calmodulin may play a role in the functional utilization of synaptic vesicle tubulin and may mediate some of the effects of Ca2+ on vesicle function and neurosecretion.     

Inhibition by adriamycin of calmodulin-sensitive and phospholipid-sensitive calcium-dependent phosphorylation of endogenous proteins from heart.

Adriamycin, a lipid-interacting anti-cancer agent, was found to inhibit phospholipid-sensitive Ca2+-dependent phosphorylation of endogenous proteins from the cytosol of the guinea-pig heart. The drug, unexpectedly, also inhibited phosphorylation of separate endogenous proteins in the cardiac cytosol and membranes catalysed by the calmodulin-sensitive species of Ca2+-dependent protein kinase. In both phosphorylation systems, the inhibition by adriamycin was reversed by either phospholipid (phosphatidylserine or cardiolipin) or calmodulin respectively. Adriamycin also inhibited phosphorylation of histone (exogenous protein) catalysed by purified cardiac phospholipid-sensitive Ca2+-dependent protein kinase, but not that by cyclic AMP-dependent and cyclic GMP-dependent protein kinases. It appears that Ca2+-dependent protein phosphorylation systems, regulated either by phospholipid or calmodulin, may represent hitherto unrecognized sites of action of adriamycin. It remains to be seen whether inhibition by adriamycin of these systems is related to the severe cardiotoxicity, the major adverse effect of the drug that limits its clinical usefulness.     

Protein kinase activity associated with pancreatic zymogen granules.

Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.     

Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rt islets of langerhans

Activation of Ca2+ -calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic beta-cell. To study the properties of suc kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [gamma-32P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and detected by autoradiography. The phosphorylation of certain proteins could be enhanced by Ca2+ plus calmodulin or by cyclic AMP. The major effect of Ca2+ and calmodulin was to stimulate the phosphorylation of a protein (P53) of molecular weight 53,100 +/- 500 (n = 15). Maximum phosphorylation of protein P53 occurred within 2 min with 2 micrometers free Ca2+ and 0.7 micrometers calmodulin. Incorporation of label into protein P53 was inhibited by trifluoperazine or W7 but not by cyclic AMP-dependent protein kinase inhibitor. Phosphorylation of a proteins of similar molecular weight could be enhanced to a lesser extent in the absence of Ca2+ but in the presence of cyclic AMP and 3-isobutylmethylxanthine: this phosphorylation was blocked by cyclic AMP-dependent protein kinase inhibitor. Cyclic AMP also stimulated incorporation of label into polypeptides of molecular weights 55,000 and 70-80,000. The results are consistent with the hypothesis that protein phosphorylation mechanisms may play a role in the regulation of insulin secretion.     

In Vitro Phosphorylation of Bovine Adrenal Chromaffin Cell Tyrosine Hydroxylase by Endogenous Protein Kinases

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.     

Effects of Cyclic Nucleotides and Calcium/Calmodulin on Protein Phosphorylation in the CNS of Hirudo medicinalis

Protein phosphorylation plays an important role in the regulation of neural functions. We have studied the phosphorylation of proteins in homogenates of segmental ganglia of the leech Hirudo medicinalis. We describe a number of proteins whose phosphorylation is dependent on calcium/calmodulin or cyclic nucleotides. Most of the proteins whose phosphorylation is increased in the presence of calcium seem to be substrates for cyclic nucleotide-dependent protein kinases. Only two of the phosphoproteins described appear to be specific substrates for calcium/calmodulin protein kinase(s), and at least six phosphoproteins appear to be specific substrates for cyclic nucleotide-dependent kinase(s). The leech nervous system, with large and identifiable neurons, provides a good tool for studies of neural functions, such as learning. The results are discussed in the context of the role of protein phosphorylation on learning processes.     

Photoaffinity labelling of central-nervous-system myelin. Evidence for an endogenous type I cyclic AMP-dependent kinase phosphorylating the larger subunit of 2'',3''-cyclic nucleotide 3''-phosphodiesterase.

Endogenous cyclic AMP-stimulated phosphorylation of a 49700-Mr Wolfgram protein component in rabbit central nervous system was investigated by using photoaffinity labelling and 2',3'-cyclic nucleotide 3'-phosphodiesterase activity staining after electroblotting on to nitrocellulose paper. Photoaffinity labelling with 8'-azidoadenosine 3',5'-cyclic monophosphate showed a cyclic AMP-binding protein that appeared to be intrinsic to the myelin membrane and appeared to represent the R-subunit of a type I cyclic AMP-dependent protein kinase. This photoaffinity-labelled protein was of larger apparent Mr than the protein showing cyclic AMP-stimulated phosphorylation. Blotting of one-dimensional sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms followed by staining for 2',3'-cyclic nucleotide 3'-phosphodiesterase activity showed two activity bands corresponding to the two components of the Wolfgram protein doublet. Cyclic AMP-stimulated protein phosphorylation corresponded to the upper component of this doublet. Electroblotting of two-dimensional non-equilibrium pH-gradient electrophoretograms also showed co-migration of cyclic AMP-stimulated protein phosphorylation with enzyme activity. It is proposed that central-nervous-system myelin contains an endogenous type I cyclic-AMP dependent protein kinase that phosphorylates the larger subunit of 2',3'-cyclic nucleotide 3'-phosphodiesterase.