Cyclic AMP-stimulated Phosphorylation Research Articles

Evaluation of Kemptide, a synthetic serinecontaining heptapeptide, as a phosphate acceptor for the estimation of cyclic AMP-dependent protein kinase activity in respiratory tissues

In the search for a phosphate acceptor to estimate cyclic AMP-dependent protein kinase (A-kinase) activity in respiratory tissues devoid of the disadvantages inherent with the use of histones, we compared and contrasted a conventional substrate, histone IIa, with that of a novel heptapeptide phosphate acceptor, Kemptide (Kemp et al., J Biol Chem 252: 4888–4894, 1977). The specific activities of soluble A-kinase isolated from guinea-pig lung parenchyma and from bovine tracheal smooth muscle were significantly (12–16-fold) greater when Kemptide was used as substrate when compared to histone IIa. Moreover, studies with a specific inhibitor of A-kinase demonstrated that whilst the cyclic AMP-stimulated phosphorylation of Kemptide was catalysed exclusively by A-kinase, only ~89% of the phosphate incorporated into histone IIa was attributed to the activity of this enzyme. The activation constant ( K act) of cyclic AMP for A-kinase was estimated to be 1.5 μM when Kemptide was used as substrate, 25-fold higher than that obtained using histone IIa (60 nM) under identical conditions. In a complementary series of experiments it was found that both the basal and isoprenaline-stimulated A-kinase activity ratios were significantly higher when histone IIa was used as substrate when compared with that obtained using Kemptide. Elevating the ionic strength of the homogenization buffer or assay cocktail with NaCl produced a reversible (following dialysis), concentration-dependent, mixed uncompetitive, inhibition of cyclic AMP-stimulated histone phosphorylation; phosphokemptide formation was unaffected. The ability of salt to inhibit A-kinase activity also affected the elution profile of A-kinase isoenzymes following their separation by DEAE-cellulose chromatography. With histone IIa as substrate the Type I: Type II isoenzyme ratio in guinea-pig lung and in bovine tracheal smooth muscle was 9:91 and 35:65, respectively. Changing the phosphate acceptor to Kemptide reduced these respective ratios to 5:95 and 29:71. Although the effect on the isoenzyme ratio was small, the specific activities of the two isoenzymes isolated from both tissues were markedly attentuated by ca. 9% (Type I) and 36% (Type II). It is concluded that Kemptide is without many of the disadvantages inherent with histone IIa and is, thus, a preferable phosphate acceptor for estimating soluble A-kinase activity and determining the isoenzyme ratio in guinea-pig lung and in bovine tracheal smooth muscle.

Cyclic AMP-dependent Phosphorylation of Glycoprotein Ib Inhibits Collagen-induced Polymerization of Actin in Platelets

Platelet function is inhibited by agents such as prostaglandin E1 (PGE1) that elevate the cytoplasmic concentration of cyclic AMP. Inhibition presumably results from the cyclic AMP-stimulated phosphorylation of intracellular proteins. Polypeptides that become phosphorylated are actin-binding protein, P51 (Mr = 51,000), P36 (Mr = 36,000), P24 (Mr = 24,000), and P22 (Mr = 22,000). Recently, we identified P24 as the beta-chain of glycoprotein (GP) Ib, a component of the plasma membrane GP Ib.IX complex. The existence of Bernard-Soulier syndrome, a hereditary disorder in which platelets selectively lack the GP Ib.IX complex, enabled us to examine whether the phosphorylation of GP Ib beta (P24) is responsible for any of the inhibitory effects of elevated cyclic AMP on platelet function. Exposure of control platelets to PGE1 increased phosphorylation of actin-binding protein, P51, P36, GP Ib beta, and P22. Prostaglandin E1 induced the same phosphorylation reactions in Bernard-Soulier platelets, except that of GP Ib beta, which is absent. In control platelets, PGE1 inhibited collagen-induced phosphorylation of myosin light chain, phosphorylation of P47 (an unidentified Mr 47,000 cytoplasmic protein that is phosphorylated by protein kinase C in stimulated platelets), aggregation, and the secretion of granule contents. Despite the absence of GP Ib beta, PGE1 also inhibited these collagen-induced responses in Bernard-Soulier platelets. However, while PGE1 inhibited collagen-induced polymerization of actin in control platelets, it did not inhibit actin polymerization in Bernard-Soulier platelets. These results suggest that cyclic AMP-induced phosphorylation of GP Ib inhibits collagen-induced actin polymerization in platelets. Because actin polymerization is required for at least some of the functional responses of platelets to an agonist, phosphorylation of Gp Ib beta may be one way in which cyclic AMP inhibits platelet function.

Distribution of Calmodulin‐ and Cyclic AMP‐Stimulated Protein Kinases in Synaptosomes

The subcellular location of calmodulin- and cyclic AMP stimulated protein kinases was assessed in synaptosomes which were prepared on Percoll density gradients. The distribution of the protein kinases between the outside and the inside and between the soluble and membrane fractions was determined by incubating intact and lysed synaptosomes, as well as supernatant and pellet fractions obtained from lysed synaptosomes, in the presence of [gamma-32P]ATP. Protein kinase activity was assessed by the labelling of endogenous proteins, or exogenous peptide substrates, under conditions optimized for either calmodulin- or cyclic AMP-stimulated protein phosphorylation. When assessed by calmodulin-stimulated autophosphorylation of the alpha subunit of calmodulin kinase II, 44% of this enzyme was on the outside of synaptosomes, and 41% was in the 100,000 g supernatant. Using an exogenous peptide substrate, the distribution of total calmodulin-stimulated kinase activity was 27% on the outside and 34% in the supernatant. The high proportion of calmodulin kinase II on the outside of synaptosomes is consistent with its known localization at postsynaptic densities. The proportion of calmodulin kinase II which was soluble depended on the ionic strength conditions used to prepare the supernatant, but the results suggest that a major proportion of this enzyme which is inside synaptosomes is soluble. When assessed by cyclic AMP-stimulated phosphorylation of endogenous substrates, no cyclic AMP-stimulated kinase activity was observed on the outside of synaptosomes, whereas 21% was found with an exogenous peptide substrate. This suggests that if endogenous substrates are present on the outside of synaptosomes, then the enzyme does not have access to them. The cyclic AMP-stimulated protein kinase present inside synaptosomes was largely bound to membranes and/or the cytoskeleton, with only 10% found in the supernatant when assessed by endogenous protein phosphorylation and 25% with an exogenous substrate. The markedly different distribution of the calmodulin- and cyclic AMP-stimulated protein kinases presumably reflects differences in the functions of these enzymes at synapses.

Identification of glycoprotein Ib beta as one of the major proteins phosphorylated during exposure of intact platelets to agents that activate cyclic AMP-dependent protein kinase.

Platelet function is inhibited by prostaglandin E1, prostaglandin I2, or forskolin, agents that increase the intracellular concentration of cyclic AMP. The inhibition appears to result from cyclic AMP-stimulated phosphorylation of specific intracellular proteins. One of the major increases in phosphorylation occurs in a polypeptide of Mr = 24,000 (P24). In this study, an effort was made to identify P24. Platelets prelabeled with [32P]phosphate were incubated with prostaglandin E1, prostaglandin I2, or forskolin. Proteins that became phosphorylated were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several lines of evidence indicated that P24 was the beta-subunit of the plasma membrane glycoprotein (GP) Ib, a glycoprotein that is essential for the adhesion of platelets to damaged subendothelium, for the rapid response of platelets to thrombin, and for the attachment of the membrane skeleton to the cytoplasmic face of the plasma membrane. P24 co-migrated with GP Ib beta on reduced gels (Mr = 24,000) and also on nonreduced gels (when GP Ib beta is disulfide-linked to GP Ib alpha and migrates with Mr = 170,000). Like GP Ib beta, P24 was associated with actin filaments in Triton X-100 lysates. Like GP Ib beta, it was selectively associated with filaments of the membrane skeleton and was released from filaments when the Ca2+-dependent protease was active. Antibodies against GP Ib immunoprecipitated P24 from platelet lysates. Finally, exposure of Bernard-Soulier platelets (which lack GP Ib) to prostaglandin E1 resulted in phosphorylation of other polypeptides, but not of P24. These studies show that P24, one of the major polypeptides phosphorylated when platelets are exposed to agents that inhibit platelet function by increasing the concentration of cyclic AMP, is the beta-subunit of GP Ib.

Octopamine- and cyclic AMP-stimulated phosphorylation of a protein in Limulus ventral and lateral eyes

The biogenic amine octopamine (OCT) fulfills most of the criteria as a neurotransmitter of efferent fibers that project to lateral and ventral eyes of the horseshoe crab, Limulus polyphemus. OCT is synthesized by and released from the efferent fibers, and OCT mimics many of the effects of endogenous efferent activity. OCT stimulates an increase in intracellular adenosine 3',5'-monophosphate (cAMP) in both ventral and lateral eyes, and many of the physiological effects of OCT in these eyes appear to be mediated via cAMP-dependent mechanisms. Here we show that OCT, acting apparently through an OCT-specific receptor, stimulates the increased phosphorylation of a protein with an apparent molecular weight of 122 kDa in both ventral and lateral eyes. This protein is also phosphorylated in response to 8-bromo cAMP and forskolin, suggesting that its phosphorylation involves activation of a cAMP-dependent protein kinase. We present evidence that the 122 kDa protein may be widely distributed in the Limulus visual system but that its phosphorylation in intact tissue in response to OCT, or agents acting through cAMP, may be restricted to portions containing photoreceptor cell bodies. The 122 kDa protein is quantitatively a major cellular protein in the photoreceptor cell body enriched portions of the ventral eye, its isoelectric point is between pH 6.2 and 6.4, and it is associated with both cell membranes and the cytoplasm. The function of this protein is not yet known. It may be important in mediating one or more of the effects of octopamine on Limulus vision.

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.

Endogenous cyclic AMP-stimulated phosphorylation of a Wolfgram protein component in rabbit central-nervous-system myelin.

Cyclic AMP-stimulated phosphorylation of membrane proteins in central-nervous-system myelin was investigated, with rabbit brain myelin. Subfractionation of a myelin membrane preparation by sucrose-density-gradient centrifugation produced a rapidly sedimenting population of membrane vesicles containing 5'-nucleotidase and acetylcholinesterase, a light membrane fraction containing myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase, and an intermediate membrane fraction containing the highest specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase and a small proportion of myelin basic protein. Cyclic AMP stimulation of protein phosphorylation was confined to a protein of Mr 49 700, which co-electrophoresed with the upper component of the Wolfgram protein doublet. Cyclic AMP did not affect the phosphorylation of myelin basic protein. Cyclic AMP-stimulated phosphorylation of this protein followed 2',3'-cyclic nucleotide 3'-phosphodiesterase activity on subcellular fractionation and was correspondingly high in the intermediate or 'myelin-like' fraction on sucrose-density-gradient centrifugation.

Localization of an endogenous substrate for cyclic AMP-stimulated protein phosphorylation in retina

Incubation of mouse or rabbit whole retina homogenates in the presence of [γ 32P]-ATP and Mg 2+ leads to the phosphorylation of various proteins, as demonstrated using SDS-polyacrylamide gel electrophoresis and autoradiography. The phosphorylation of one protein (ca. ≈ 31 000 mol. wt) was increased by cyclic AMP in both species, with half-maximal stimulation at 5 × 10 −7 m. Cyclic GMP was also active, but much less potent. Protein phosphorylation patterns were compared in retina homogenates from normal mice (C57BL/6J), from adult C57BL/6J mice homozygous for the retinal degeneration gene (rd/rd) in which rod photoreceptor cells are absent, and from 21-day-old 020/Cpb mice homozygous for the retinal degeneration slow gene (rds/rds) in which only the outer segments of the rod photoreceptors are missing. The 31 K protein was only present in normal and in 21-day-old rds/rds mice. When rabbit retina was microdissected into outer segment, inner segment plus outer nuclear, and inner retina layers, cyclic AMP-stimulated phosphorylation of the 31 K protein was evident only in the inner segment plus outer nuclear layer. These data indicated the presence of a specific, endogenous substrate for a cAMP-dependent protein kinase which is found in the inner portions of rod photoreceptor cells.

Protein phosphorylation in isolated synaptic junctional structures: changes during development

Endogenous phosphorylation in subcellular fractions enriched in synaptic plasma membranes (SPM) and purified synaptic junction (SJ) has been examined in vitro at various stages of postnatal development. Protein kinase activity was measured using both endogenous and exogenous (histones) proteins as substrates. Protein phosphorylation that is regulated by cyclic AMP also was investigated. SPM and SJ fractions displayed large increments in kinase activities and cyclic AMP-stimulated protein phosphorylation between postnatal days 5 and 15. SJ fractions exhibited a dramatic age-dependent change in the cyclic AMP-stimulated phosphorylation of endogenous proteins of molecular weights 73,000 (1a), 68,000 (1b), 65,000 (1c) and 55,000. Experiments in which isolated SJs were phosphorylated with soluble cyclic AMP-dependent kinase showed that the phosphorylation of proteins 1a, 1b and 1c resulted from their de novo appearance in newborn SJ fractions and not from a maturation-dependent coupling of kinases and substrates that were already present in newborn SJ fractions. The levels of regulatory (R) subunits of cyclic AMP-dependent kinases in synaptic fractions were measured by [ 3H]cyclic AMP binding and photoaffinity labeling with [ 32P]8-N 3-cyclic AMP and were observed to change little during postnatal development. These findings show that there is a strong correlation between the in situ appearance of synapses and the enzymatic maturation of endogenous, cyclic AMP-stimulated protein phosphorylation in SJ fractions isolated at different stages of development.

Characteristics of magnesium-dependent, Ca 2+-stimulated endogenous protein kinase-catalyzed phosphorylation of basic proteins in myelin isolated from rat brain stem white matter

Purified myelin fraction isolated from rat brain white matter contained Mg 2+-dependent protein kinase capable of phosphorylation of myelin basic proteins. The Mg 2+-supported kinase was markedly stimulated (two- to fivefold) by micromolar concentrations of free Ca 2+ with and without Triton X-100 in the assay, the degree of stimulation being greater with the detergent present. Cyclic AMP, on the other hand, failed to show any effect on phosphorylation of myelin in the absence of Triton X-100 and in the presence of Triton caused only 25–30% stimulation. The phosphorylation reaction was temperature dependent and exhibited a pH optimum at pH 6.5. Apparent affinity toward MgATP 2− was found to be about 70 μ m and Ca 2+ had no effect on this parameter. Dependence on MgCl 2 of myelin phosphorylation indicated the presence of high- and low-affinity sites toward Mg 2+; Ca 2+ appeared to influence the low-affinity site. Maximal level of phosphorylation was attained by 10–15 min at 30 °C and it declined at longer incubation times due to phosphatase activity present in the preparation. Stimulatory effect of Ca 2+ on phosphorylation was not due to inhibition of phosphatase activity. Dephosphorylation experiments showed that neither cyclic AMP nor Ca 2+ influenced the myelin phosphatase activity. Autoradiographic analysis revealed that phosphorylation of myelin basic proteins accounted for nearly 90% of total myelin phosphorylation. This was supported by the observation that the HCl extract of myelin contained 85% of total activity and comigrated with purified myelin basic proteins. Basal and Ca 2+-stimulated phosphorylation of basic proteins were due to phosphorylation of serines mainly, although threonine was phosphorylated to a minor extent. Within myelin, Ca 2+ and cyclic AMP kinases are differentially bound. It appears that the myelin kinase (studied in vitro) is primarily influenced by Ca 2+ rather than cyclic AMP. Inhibitors (Type I and Type II) of cyclic nucleotide-stimulated protein kinases had no effect on the Ca 2+-stimulated phosphorylation although basal and cyclic AMP-stimulated phosphorylation was inhibited, indicating that the Ca 2+ kinase is a separate and distinct enzyme from the cyclic AMP-stimulated and basal kinase(s). Also, leupeptin, a protease inhibitor, did not influence basal, cyclic AMP-stimulated, or Ca 2+-stimulated myelin phosphorylation, indicating that under the conditions used protease(s) did not alter the myelin kinase activity. The potential significance of phosphorylation of myelin basic proteins and the stimulatory action of Ca 2+ on this reaction are discussed.

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 Mg 2+ 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.

Protein phosphorylation in rat caudate homogenate stimulatory effects of dopamine and enhancement of dopamine response following 6-hydroxydopamine treatment

Dopamine (3-hydroxytyramine) stimulates the incorporation of 32P into proteins endogenous to a homogenate of rat caudate nucleus when 10 μM [γ- 32P]-ATP is used as a substrate following preincubation with 400 μM ATP. The increase in 32P incorporation has pharmacological characteristics similar to those observed for the dopamines-sensitive adenylate cyclase system present in caudate tissue. Chronic depletion of striatal dopamine in vivo by stereotaxic injection of 6-hydroxydopamine in the nigrostriatal pathway results in a significant enhancement of the dopamine stimulation of 32P incorporation in vitro. Cyclic AMP-stimulated phosphorylation of caudate proteins remains unchanged following 6-hydroxydopamine treatment.

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.

Distribution of endogenously phosphorylated proteins in subcellular fractions of rat cerebral cortex

The cerebral cortex from adult rats was separated into several subcellular fractions by using established methods of differential and sucrose density gradient centrifugation. Aliquots from each fraction were incubated with γ-(32)P-ATP, in the presence and absence of adenosine 3',5'-monophosphate (cyclic AMP), and its protein constituents were separated by means of SDS-slab gel electrophoresis. Fractions containing nuclei, synaptosomes, myelin, microsomes, and soluble proteins each showed a characteristic pattern of protein staining and of endogenously phosphorylated proteins detected by autoradiography of the gels. Cyclic AMP-stimulated phosphorylation of proteins with MW 78K and 84K can serve as markers for membranes of synaptic origin, while cyclic AMP-independent phosphorylation of low-molecular-weight proteins (15K-20K) is characteristic of myelin. The finding of different phosphoproteins in various subcellular fractions may be related to the diversity of cellular functions known to be regulated by phosphorylative activity.

Cyclic amp-stimulated phosphorylation of a high molecular weight endogenous protein substrate in sub-cellular fractions of molluscan nervous system

Cyclic AMP stimulated the incorporation of radioactive phosphate from ATP into endogenous phosphoproteins in a 20,000 × g supernatant, as well as in a 150,000 × g supernatant, prepared from Helix nervous system homogenates. No effect of cyclic AMP was observed in the respective pellet fractions. A part (but not all) of the cyclic AMP-stimulated activity in both supernatants was due to phosphorylation of an endogenous protein substrate, of apparent molecular weight 120,000 daltons as determined from its mobility in polyacrylamide gels. Little phosphorylation of 120,000 dalton material was observed in the particulate fractions, both in the presence and absence of cyclic AMP. When the 20,000 × g pellet was incubated with radioactive ATP and an exogenous calf ovary protein kinase, cyclic AMP stimulated the phosphorylation of the 120,000 dalton material. The results suggest that both membranes and cytosol from Helix nervous system contain 120,000 dalton substrates for cyclic AMP-dependent protein kinase. However, no kinase catalyzing phosphorylation of these substrates is recovered in membrane fractions following homogenization of the tissue.

Complexity of cyclic AMP‐dependent phosphoproteins in membranes from brain tissue containing synapses

FEBS LettersVolume 75, Issue 1-2 p. 61-64 Full-length articleFree Access Complexity of cyclic AMP-dependent phosphoproteins in membranes from brain tissue containing synapses M. Reddington, Corresponding Author M. Reddington Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGDr M. Reddington, Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGSearch for more papers by this authorE. Mehl, E. Mehl Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGSearch for more papers by this author M. Reddington, Corresponding Author M. Reddington Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGDr M. Reddington, Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGSearch for more papers by this authorE. Mehl, E. Mehl Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGSearch for more papers by this author First published: March 15, 1977 https://doi.org/10.1016/0014-5793(77)80053-7Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 F.E. Bloom, Rev. Physiol. Biochem. Pharmacol., 74, (1975), 1– 103. 2 M. Reddington, R. Rodnight, M. Williams, Biochem. J., 132, (1973), 475– 482. 3 M. Williams, R. Rodnight, Biochem. J., 154, (1976), 163– 170. 4 M. Williams, Brain Res., 109, (1976), 190– 195. 5 I. Levitan, C. Madsen, S. Barondes, J. Neurobiol., 5, (1974), 511– 525. 6 M. Weller, R. Rodnight, Biochem. J., 124, (1971), 406– 511. 7 M. Weller, R. Rodnight, Biochem. J., 132, (1973), 483– 492. 8 T. Ueda, H. Maeno, P. Greengard, J. Biol. Chem., 248, (1973), 8295– 8305. 9 A. Routtenberg, Y.H. Ehrlich, Brain Res., 92, (1975), 415– 430. 10 M. Weller, I. Morgan, Biochim. Biophys. Acta, 436, (1976), 675– 685. 11 P. Dunkley, H. Holmes, R. Rodnight, Biochem. J., 157, (1976), 661– 666. 12 U.K. Laemmli, Nature, 227, (1970), 680– 685. 13 E.M. Reimann, D.A. Walsh, E.G. Krebs, J. Biol. Chem., 246, (1971), 1986– 1995. 14 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 275. 15 E. Miyamoto, J. Neurochem., 26, (1976), 573– 577. 16 C.S. Rubin, J. Biol. Chem., 250, (1975), 9044– 9052. 17 O.M. Pinkett, R.L. Pertman, Biochim. Biophys. Acta, 372, (1974), 379– 387. 18 J. Schultz, B. Hamprecht, J.W. Daly, Proc. Natl. Acad. Sci. USA, 436, (1972), 1266– 1270. 19 Blas A. De, H.R. Mahler, Biochem. Biophys. Res. Commun., 72, (1976), 24– 32. 20 M. Weller, I. Morgan, Biochim. Biophys. Acta, 391, (1976), 162– 169. Citing Literature Volume75, Issue1-2March 15, 1977Pages 61-64 ReferencesRelatedInformation

Cyclic AMP-stimulated phosphorylation of bovine tracheal smooth muscle contractile and non-contractile proteins

1. 1.|Various proteins isolated from bovine tracheal smooth muscle were examined as phosphate acceptor substrates for a cyclic AMP-dependent protein kinase isolated from the same tissue. A fraction prepared in a manner similar to that of skeletal muscle troponin was the best substrate of the presumptive contractile proteins isolate. Actomyosin and tropomyosin were relatively poor substrates. 2. 2.|An assay was developed for the rapid detection in a large number of samples of the muscle specific substrate for the protein kinase on which we reported previously. 3. 3.|Using this assay, the muscle specific substrate found in bovine tracheal smooth muscle was partially purified resulting in a preparation which when resolved by polyacrylamide gel electrophoresis showed a single peak of 32P incorporated, and which could be further characterized. 4. 4.|Our findings suggest that the substrate contains a protein subunit of molecular weight 19 000, which can be phosphorylated at serine and threonine residues, in the presence of cyclic AMP and protein kinase. The phosphate is in a covalent ester linkage with these residues. 5. 5.|A phosphoprotein phosphatase was isolated from the bovine tracheal smooth muscle. 6. 6.|Bovine tracheal smooth muscle contains cyclic AMP dependent protein kinase and phosphoprotein phospahatase activity as well as the muscle specific substrate, suggesting that these elements may be part of a mechanism which regulates smooth muscle tone.

Calcium accumulation and cyclic AMP-stimulated phosphorylation in plasma membrane-enriched preparations of myocardium

Plasma membranes were prepared from guinea pig ventricle by a procedure which involved differential centrifugation at low gravitational forces, extraction with KCl, and centrifugation in a discontinuous sucrose gradient. Adenylate cyclase was purified 10–15-fold over the starting homogenate with a yield of 75%. The membranes contained an active Ca 2+ binding and uptake system as well as Ca 2+-activated adenosine triphosphatase; protein kinase and phosphoprotein phosphatase activities were also present. The membranes could be phosphorylated by either intrinsic or exogenous protein kinase, and phosphorylation was stimulated by cyclic AMP and was reversible. Phosphorylated membranes accumulated twice as much Ca 2+ as control preparations.

Localization in the synaptic junction of the cyclic amp stimulated intrinsic protein kinase activity of synaptosomal plasma membranes

Synaptosomal plasma membrane fragments contain a tightly bound protein kinase which can catalyse the phosphorylation of endogenous protein the reaction bein stimulated by cyclic AMP. A fraction enriched in synaptic junctions, which can be isolated from Triton X-100-treated synaptosomal plasma membranes, is also enriched in the cyclic AMP stimulated intrinsic protein kinase. The location of the enzyme in the synaptic junction suggest that cyclic AMP-stimulated phosphorylation may have some role in synaptic transmission.

Cyclic Adenosine Monophosphate-dependent Phosphorylation of Specific Fat Cell Membrane Proteins by an Endogenous Membrane-bound Protein Kinase: POSSIBLE INVOLVEMENT IN THE REGULATION OF INSULIN-STIMULATED GLUCOSE TRANSPORT

Abstract The phosphorylation of specific membrane proteins by an endogenous protein kinase has been studied in purified membrane fractions from rat adipocytes using trichloroacetic acid precipitation and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. The endogenous phosphorylation of two specific membrane proteins is completely dependent on the presence of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and magnesium ions. This phosphorylation occurs very rapidly at 24°, reaching maximal levels at 1 min. The number of sites specifically phosphorylated in the presence of cyclic AMP probably does not exceed 50,000 per fat cell, requiring the use of very high specific activity (10 to 50 Ci per mmole) [γ-32P]ATP for these studies. The minimal molecular weights of the two specifically phosphorylated proteins are about 22,000 and 16,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The same two proteins are phosphorylated when intact fat cells are exposed briefly to low concentrations of exogenous ATP, a process which results in the suppression of the insulin-stimulated rates of d-glucose transport. At least 95 % of the cyclic AMP-dependent 32P incorporated into trichloroacetic acid-precipitable protein is in the form of protein-bound phosphoserine. The concentration of cyclic AMP required for maximal stimulation of phosphorylation is about 1.5 µm. Cyclic guanosine 3':5'-monophosphate has no significant effect unless its concentration is increased to 10-4 m. Phosphorylation is inhibited by calcium ions. The cyclic AMP-stimulated phosphorylation is inhibited by phloretin and by 5'-adenylyl-β, γ-methylene triphosphonate. The specific cyclic AMP-dependent membrane phosphorylation is also found in membranes from cells of obese rats, an insulin-resistant animal. This phosphorylation system, however, cannot be detected in membranes from guinea pig fat cells. The lack of phosphorylation of specific membrane proteins in guinea pig fat cell membranes is correlated with the insulin insensitivity of this tissue to glucose transport (but not lipolysis) and with the inability of ATP to inhibit the slight stimulation which insulin exerts on glucose transport. The possibility is considered that this specific cyclic AMP-dependent phosphorylating system is involved in the hormonal regulation of glucose transport and the modulation of insulin sensitivity.