Brain Protein Kinase Research Articles

Ethanol‐Induced Inhibition of Chick Brain Growth

Retarded fetal brain growth is associated with a high incidence of mental retardation among the offspring of chronic alcoholic mothers. Research using an embryonic chick model suggests that ethanol exposure suppresses fetal development including suppression of brain growth. Total brain cyclic AMP content and endogenous brain protein kinase specific activity are not altered by ethanol; however, ethanol exposure does significantly stimulate kinase catalytic activity measured in the presence of saturating amounts of exogenous cyclic AMP.

Protein kinase(s) in bovine brain coated vesicles

Purified bovine brain coated vesicles contain protein kinase activity which phosphorylates 165,54 and 50 kDa protein substrates. These phosphorylations do not seem to be induced by a unique protein kinase: indeed, the three substrates present different localizations in coated vesicles, the phosphorylation sites are either serine or threonine residues and vanadate and ATP[γS] have different effects on 32P incorporation in the substrates. Comparison of the coated vesicle protein and phosphorylation patterns from different tissues and animal origins shows that only the 50 kDa protein phosphorylation is always observed, compared to the great diversity in other minor phosphorylations which are observed or not in the various coated vesicles. The possible presence of a 50 kDa phosphoprotein phosphatase is also discussed. It is suggested that the 50 kDa protein with its connected specific kinase and phosphatase seems to constitute a regulatory system present in coated vesicles.

A rat brain protein kinase phosphorylating specifically neurofilaments

Protein kinase activities associated with neurofilament (cAMP, cGMP, Ca 2+-independent) were almost completely extracted by 0.8 M KCl. Two activities were separated by either sucrose gradient ultracentrifugation of phosphocellulose chromatography. One of them phosphorylates specifically neurofilament proteins and preferentially the triplet (200 kDA, 145 kDa and 68 kDa) but neither casein nor type II A histone. The second activity was identified as casein kinase I and does not catalyze the phosphorylation of neurofilament protein.

Ca2+, calmodulin-dependent phosphorylation of glycogen synthase by a brain protein kinase

A Ca2+, calmodulin-dependent protein kinase from brain with a Mr of 640 000 is capable of phosphorylating glycogen synthase from skeletal muscle. The reaction was inhibited by the addition of 1 mM EGTA and 50 μM trifluoperazine, but not by protein kinase inhibitor and heparin. The amount of phosphate incorporated into glycogen synthase was 1.4 mol/mol subunit. The phosphorylation sites of glycogen synthase were cyanogen bromide-treated peptides CB-1 and CB-2 and only the seryl residue was phosphorylated.

19] Use of immunological approaches to identify a brain protein kinase isozyme

Publisher Summary This chapter describes the use of immunological approach to identify a brain protein kinase isozyme. Type II cAMP-dependent protein kinases (PKs) from many mammalian tissues appear to be very closely related based on their physicochemical and kinetic properties, subunit compositions, and mode of activation by cAMP. For example, homogeneous preparations of the prototypic heart and skeletal muscle type II PKs are virtually identical in size, shape, ion-exchange properties, and isoelectric point. The immunological techniques of indirect immunoprecipitation and competitive binding immunoassay can be combined with functional assays to address the question of whether there are subclasses of type II PKs.. Antibodies raised against bovine cerebral cortex R II have been employed as highly specific and sensitive probes for identifying and characterizing tissue-specific subclasses of type II PKs.

Presence of cyclic nucleotide-Ca2+ independent protein kinase in bovine brain coated vesicles.

Coated vesicles, which are membrane vesicles enclosed by a polyhedral protein lattice, are involved in many cellular events, including intracellular membrane transport and protein secretion, in which they must be able to undergo repeated membrane fusion and fission. The icosahedral lattice of protein surrounding the core of coated vesicles is composed predominantly of clathrin, a 180,000 (180 K) molecular weight protein, and other 30K and 36K polypeptides. In native conditions, the basic subunit of the coat consists of a trimer of clathrin with probably three polypeptides of 30K and/or 36K (refs 9-11). Additional minor proteins of 100K and 55K have been reported in purified coated vesicles. We describe here the presence of cyclic nucleotide- and Ca2+-independent protein kinase activity in coated vesicles. This protein kinase phosphorylates specifically a unique 50K protein which can be co-purified with clathrin and seems to be an integral protein of coated vesicles.

Interaction of calmodulin with myosin light chain kinase and cAMP-dependent protein kinase in bovine brain.

Myosin light chain kinase and a fraction of type II cAMP-dependent protein kinase have been partially purified from bovine brain by affinity chromatography on calmodulin-Sepharose. The myosin kinase was purified approximately 3700-fold and has an estimated molecular weight of 130,000 +/- 10,000 by sodium dodecyl sulfate gel electrophoresis. A fraction of soluble cAMP-dependent protein kinase also bound to calmodulin-Sepharose and was purified 2300-fold. A fraction of this cAMP-dependent protein kinase after purification by glycerol gradient centrifugation was shown to contain the two subunits of calcineurin, a major calmodulin-binding protein in brain, and the two subunits of type II cAMP-dependent protein kinase in a ratio of 1:1:2:2. Its sedimentation coefficient was 8.1 S and 9.0 S when centrifuged in the absence or presence of calmodulin, suggesting the formation of a complex between calmodulin and protein kinase. Our results suggest the possibility that calcineurin may be involved in the interaction between the protein kinase and calmodulin. Furthermore, our studies imply that the regulatory subunit of the cAMP-dependent protein kinase, but not the catalytic subunit, is the site of interaction with calmodulin since the catalytic subunit of protein kinase was partially resolved from the complex by cAMP.

Immunohistochemical localization of cyclic GMP-dependent protein kinase in mammalian brain.

The distribution of cyclic GMP-dependent protein kinase in rat brain has been studied by an immunological approach involving radioimmunoassay and fluorescence immunohistochemistry. Data obtained by radioimmunoassay indicate that cyclic GMP-dependent protein kinase is 20- to 40-fold more concentrated in cerebellum than in other brain regions. Immunohistochemical experiments demonstrate that the high concentration of immunoreactivity of the protein kinase in cerebellum is attributable to Purkinje cells. Immunoreactivity in these cells is homogeneously distributed throughout the cell (perikarya, dendrites, and axons) with the exception of the nucleus. No other neurons either in the cerebellum or in other brain regions were stained by antiserum to the protein kinase. Immunoreactivity, however, was found throughout the brain on smooth muscle cells of blood vessels.

Tyrosine hydroxylase activation and inactivation by protein phosphorylation conditions.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to DOPA, Cyclic AMP-dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. In agreement with other laboratories, we find that short-term pre-incubation (3 min) of extracts under phosphorylating conditions (Mg . ATP, cAMP) increases enzyme activity two- to tenfold over control as measured during a subsequent 15-min assay. We now report that preincubation under phosphorylating conditions for longer periods (30 min) results in a loss of activity to levels equal to or below that of the control enzyme. Addition of purified bovine brain protein kinase catalytic subunit and Mg . ATP enhances activation and increases the rate of inactivation. To demonstrate that inactivation is not associated with proteolytic degradation or irreversible denaturation, the inactivated form of the enzyme can be reactivated. The protein kinase inhibitor protein decreases the activation process and prevents inactivation of the enzyme to below control values. The sedimentation coefficient is not changed by phosphorylation conditions (S = 8.8 +/- 0.1). Although the apparent Km of the enzyme for the 6-methyltetrahydropterine (6-MPH4) cofactor is reduced (0.86 mM, control; 0.32 mM, activated), it is also reduced in the inactivated form (0.38 mM). The Ki for dopamine is increased from 4.5 microM for the control to 28 microM for the activated enzyme, whereas the inactivated form of the enzyme exhibits a Ki of 10 microM. Removal of catecholamines by gel filtration fails to alter activity and the apparent cofactor Km. Moreover, both the activated and the inactivated states persist following gel filtration. It therefore appears that the activation-inactivation process is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km for 6-MPH4. We also describe a coupled decarboxylase assay in which labeled dopamine is resolved from the precursors tyrosine and DOPA by low-voltage paper electrophoresis.

Identification of endogenous substrate proteins for cAMP-dependent protein kinase in bovine brain.

Identification of endogenous substrate proteins for cAMP-dependent protein kinase in bovine brain.

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: active site directed inhibition by Cibacron Blue F3GA.

Cibacron Blue F3GA, the polycyclic blue chromophore of Blue Dextran 2000, inhibits the catalytic subunit of bovine brain protein kinase. The rate of inactivation exhibits a hyperbolic dependence on the dye concentration. This suggests that an enzyme-dye complex forms prior to inactivation. Protein and peptide substrates or MgATP protects the enzyme against dye inactivation. Kinetic measurements show that the dissociation constant is approximately 100 micro M and the maximal rate of inactivation is 0.13 min-1 at 22 degree C. Inactivation is temperature and time dependent. Exhaustive dialysis, gel filtration, or the addition of substrate fails to reactivate inhibited enzyme. The failure to reverse the inhibition suggests that the dye forms a covalent complex with the enzyme. Denaturation by sodium dodecyl sulfate also fails to dissociate the dye from enzyme. The hyperbolic kinetics, moreover, suggest that the dye functions as an active site directed reagent. The holoenzyme is resistant to Cibacron Blue inactivation. Addition of cAMP converts the enzyme to a form susceptible to inhibition. In agreement with our previous studies, these results also suggest that the regulatory subunit shields, either physically or functionally, the active site of the catalytic subunit.

Immunocytochemical localization, in synapses, of protein I, an endogenous substrate for protein kinases in mammalian brain.

Protein I is a principal endogenous substrate for cyclic AMP- and Ca2+-dependent protein kinases of mammalian brain. Antibodies raised against purified protein I have been used to localize this protein in the rat central nervous system. At the light microscope level, immunoreactivity was detected in punctate deposits in selected zones of synaptic termination. These deposits varied in density among brain regions. At the electron microscope level, immunoreactivity was observed at some but not all synaptic regions and was restricted to the perimeter of synaptic vesicles and to submembranous material in the postsynaptic neuron.

Determination of binding parameters of cyclic AMP and its analogs to cyclic AMP-dependent protein kinase by the fluorescent probe method

The method for determination of dissociation constants for cyclic AMP and its analogs bound to cyclic AMP-dependent protein kinase from pig brain is described. The technique for measuring the binding parameters of the ligands is based on the changes in the fluorescent spectrum of etheno cyclic AMP once it is bound to protein kinase. The dissociation constants for a number of non-fluorescent cyclic AMP analogs were determined in the competitive displacement of etheno cyclic AMP by these analogs. The number of cyclic AMP-binding sites in the pig brain protein kinase was found to be 2.2; no cooperativity was observed upon binding. The holoenzyme complex ( M r = 180 000 ) of the protein kinase under study was established to have the stoichiometry of R 2C 2 type under native conditions.

Soluble and Membrane-Bound Protein Kinases in Newborn and Mature Rat Brain

We studied the developmental changes of soluble and membrane-bound protein kinases (EC 2.7.1.37) in rat brain and found that the elution profiles from a DEAE-cellulose column by NaCl of adenosine-3':5'-monophosphate (cyclic AMP) dependent protein kinase from cytosol were not significantly different in newborn (0.19 M NaCl) vs. mature (0.21 M NaCl). In addition, there was little change in Kd for cyclic AMP binding of newborn and mature soluble enzyme. In contrast, using endogenous protein as the phosphate acceptor, cyclic AMP dependent and calcium-dependent protein kinases from brain synaptic membranes of mature rats were significantly higher than that of newborn rats.

Isolation and properties of the bovine brain protein inhibitor of adenosine 3′:5′-monophosphate-dependent protein kinases

While the heat-stable protein inhibitor (PKI) of the CAMP-dependent protein kinase was first described in skeletal muscle [I] from which it was recently isolated in pure form [2] , its greatest concentration occurs in the brain [3] . This is not surprising in view of the very high concentration of protein kinase found in this tissue [4] . It has recently been proposed that CAMP-dependent phosphorylations might be involved in neuronal functions, such as tissue growth, membrane permeability and synaptic transmission (reviewed [S-7] ). Another protein kinease inhibitor has also been described in nervous tissue that can block phosphorylations catalyzed by both cyclic nucleotidedependent and -independent enzymes [8]. It thus appeared desirable to reinvestigate the protein kinase system of the brain. As a first step towards this goal, beef brain protein kinase inhibitor was purified by the procedure established for isolation of the skeletal muscle protein [2] ; some of its properties were also investigated.

Ethanol-induced changes in activities of adenylate cyclase, guanylate cyclase and cyclic adenosine 3′,5′-monophosphate dependent protein kinase in the brain and liver

Effects of acute and chronic administrations of ethanol on the activities of adenylate cyclase, guanylate cyclase and cyclic adenosine 3′,5′-monophosphate (cyclic AMP) dependent protein kinase in the mouse brain and liver were investigated. The activities in the cerebral cortex and liver of adenylate cyclase and guanylate cyclase, the enzymes responsible for the formation of cyclic AMP and cyclic GMP (cyclic guanosine 3′,5′-monophosphate), respectively, were not significantly affected by acute ethanol administration. On the other hand, adenylate cyclase activities in the cerebral cortex and liver were activated significantly following continuous administrations of ethanol. In the case of guanylate cyclase, no significant changes in the basal and NaN 3-activated activities were found in the brain, whereas activities in the liver, measured in the presence of NaN 3, were inhibited significantly following continuous administrations of ethanol. The cerebral content of cyclic AMP was also increased significantly following continuous ethanol treatments, while no such change was found in acutely ethanol-treated groups. The stimulatory effect of norepinephrine on the formation of cyclic AMP in slices of the cerebral cortex was significantly attenuated, whereas that of glucagon on hepatic adenylate cyclase activity was significantly increased following continuous administrations of ethanol. The activity of cyclic AMP dependent protein kinase in synaptosomal fractions from the brain was also significantly increased by continuous ethanol treatments. Cerebral and hepatic phosphodiesterase activities, measured in the presence of cyclic AMP or cyclic GMP, were not affected by both acute and continuous administrations of ethanol. Present results indicate that continuous administrations of ethanol induce the activation of cerebral and hepatic adenylate cyclases and the inhibition of NaN 3-activated guanylate cyclase activity in the liver. The possible significance of these findings in the occurrence of ethanol intoxication in the brain and liver is briefly discussed.

Ionic inhibition of catalytic phosphorylation of histone by bovine brain protein kinase.

The effects of various ions commonly found in protein kinase assays upon the rate of histone phosphorylation catalyzed by the highly purified bovine brain enzyme, protein kinase I, have been investigated. Sodium, potassium, and magnesium were found to inhibit histone phosphorylation by protein kinase I in a similar manner. The degree of inhibition by any of these cations was demonstrated to be directly proportional to the square root of the ionic strength of the assay medium. The relationship between the ionic strength of the assay medium and the rate of histone phosphorylation catalyzed by protein kinase I was employed to correct the rate of histone phosphorylation at various magnesium acetate concentrations to a standard ionic strength. When this was done an analysis of the previously postulated rate law for histone phosphorylation c atalyzed by protein kinase I gave a binding constant for the magnesium-ATP complex which was in agreement with that expected for this complex on the basis of various binding constants available in the literature. These results demonstrate that it is unnecessary to postulate a specific ion inhibition process for protein kinase I by the ions employed in this study. They also support the reasonable assumption that magnesium ion binds to ATP at or prior to the rate-determining step in histone phosphorylation catalyzed by protein kinase I. The expression developed in this paper for the effect of ionic strength upon protein kinase I activity can now be used to correct activity measurements made under various assay conditions to a standard assay state, allowing facile comparisons of kinetic data. It should be possible to develop similar expressions for other protein kinases and substrates to permit useful interpretation of kinetic data.

Effect of Rubidium, Lithium and Cesium on Brain ATPase and Protein Kinases

The authors studied the effect of rubidium, lithium and cesium on the ATPase system and c-AMP protein kinase in brain. They demonstrated that rubidium could replace potassium in the Na+K-ATPase system, whereas lithium and cesium had no effect on this enzyme activity in the absence of potassium. K+-dependent ATPase was activated by even low rubidium concentrations; lithium and cesium inhibited it. None of three (rubidium, lithium and cesium) affected c-AMP protein kinase.

Age related changes of cyclic AMP-dependent protein kinase in bovine brain.

Journal of NeurochemistryVolume 27, Issue 5 p. 1249-1251 Age related changes of cyclic AMP-dependent protein kinase in bovine brain K. D. Reichlmeier, K. D. Reichlmeier Basic Medical Research Department, Sandoz Ltd., Basel. SwitzerlandSearch for more papers by this author K. D. Reichlmeier, K. D. Reichlmeier Basic Medical Research Department, Sandoz Ltd., Basel. SwitzerlandSearch for more papers by this author First published: November 1976 https://doi.org/10.1111/j.1471-4159.1976.tb00336.xCitations: 12AboutPDF 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 onFacebookTwitterLinked InRedditWechat Citing Literature Volume27, Issue5November 1976Pages 1249-1251 RelatedInformation

Phosphorylation of histone catalyzed by a bovine brain protein kinase.

The use of polyethyleneimine-cellulose thin layer sheets to follow the phosphorylation of histone and decomposition of ATP catalyzed by an adenosine 3':5'-monophosphate (cyclic AMP)-stimulated protein kinase, protein kinase I, has made possible a more detailed analysis of the time course of these reactions than has been achieved previously be observing only recovered phosphorylated protein. When [gamma-32P] ATP was employed, significant error was introduced by the presence of 32Pi at the solvent front on these sheets, and this limited the accuracy of the available information. However, the analysis of assays performed with [U-14C] ATP was straightforward and appeared to have an accuracy comparable to that of the present standard assay. This appears to be the first use of [U-14C] ATP to assay protein kinases. Our physical characterization of protein kinase I showed it to be a homogeneous protein species by polyacrylamide gel electrophoresis, sodium dodecyl sulfate gel electrophoresis and analytical ultracentrifugation. Kinetic studies with protein kinase I indicated the absence of histone phosphatase and cyclic AMP phosphodiesterase activity. Furthermore, the ATPase activity seen is believed to be intimately associated with the protein kinase action, particularly in view of the observed dependence of the rate of Pi production on the presence of cyclic AMP. The kinetic data for the phosphorylation of histone catalyzed by protein kinase I under full stimulation by cyclic AMP are consistent with a double displacement mechanism.