Phosphorylation of Tyrosine Hydroxylase by Cyclic GMP-Dependent Protein Kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.     

Regulation of Sensory Neuron Precursor Proliferation by Cyclic GMP-Dependent Protein Kinase

Abstract: Cyclic GMP (cGMP) is a molecular messenger involved in diverse cellular processes. Recently, cGMP-dependent protein kinase (cGK) type II was determined to be a regulator of endochondral ossification and bone growth, identifying a role for cGMP in the regulation of cellular proliferation. Here, we demonstrate the presence of cGK type I (cGKI) in cells of the developing trigeminal ganglia. cGKI occurs in some proliferating precursors as evidenced by double labeling with an antibody to cGKI and 5-bromo-2'-deoxyuridine(BrdU) incorporation. Inhibition of cGKI with KT5823 or Rp -8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate ( Rp -8-pCPT-cGMPS) in chick embryos results in a 30–40% decrease in trigeminal ganglia cell number, and this effect is independent of nitric oxide synthase (NOS). In addition, inhibition of cGKI with Rp -8-pCPT-cGMPS results in a 60% decrease in BrdU incorporation in the trigeminal ganglia of embryonic day 5 chicks. We find that PC12 cells expressing cGKI proliferate more rapidly and incorporate more BrdU than do control cells. The cGKI inhibitor Rp -8-pCPT-cGMPS decreases proliferation and BrdU incorporation in transfected PC12 cells but has no effect on control cells. The PC12 cells do not express NOS, indicating that this effect is also independent of NOS. Thus, cGKI regulates the proliferation of sensory neurons as a result of activation of a NOS-independent pathway, representing a novel pathway by which the number of sensory neurons is regulated.     

Kinetics of Protein Phosphorylation in Microvessels Isolated from Rat Brain: Modulation by Second Messengers

The role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the kinetics of 32P incorporation into specific protein substrates were evaluated by computer-aided x-ray film densitometry. With the use of this method, Ca2+-calmodulin (CAM)-, Ca2+/phospholipid (PK C)-, cyclic GMP (cGMP)-, and cyclic AMP (cAMP)-dependent protein kinases were detected. CAM-dependent protein kinase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP-dependent protein kinase was much higher than that of the cAMP-dependent one. Autophosphorylation of both the alpha- and beta-subunits of CAM-dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothelial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood-brain barrier.     

Chronic Antidepressant Administration Alters the Subcellular Distribution of Cyclic AMP-Dependent Protein Kinase in Rat Frontal Cortex

The influence of chronic administration of antidepressants on cyclic AMP-dependent protein kinase activity was examined in rat frontal cortex. Chronic administration of imipramine, tranylcypromine, or electroconvulsive seizures decreased cyclic AMP-dependent protein kinase activity in soluble fractions by approximately 25%, whereas enzyme activity was increased in the particulate fractions by approximately 20%. In contrast, enzyme activity in crude homogenates was not altered. This effect appears to be specific to antidepressant drugs, because representatives of several other classes of psychotropic drugs-namely, haloperidol, morphine, and diazepam--failed to alter either soluble or particulate levels of cyclic AMP-dependent protein kinase activity in this brain region following chronic administration. When the total particulate fraction was subfractionated, it was found that chronic imipramine treatment significantly increased the activity of cyclic AMP-dependent protein kinase in crude nuclear fractions but not in crude synaptosomal or microsomal fractions. Taken together, the data raise the possibility that chronic antidepressant treatments may stimulate the translocation of cyclic AMP-dependent protein kinase from the cytosol to the nucleus. This effect would represent a novel action of antidepressants that could contribute to the long-term adaptive changes in brain thought to be essential for the clinical actions of these treatments.     

Intracellular pH on Protein Kinase C and Ionomycin Potentiation of Isoproterenol-Stimulated Cyclic AMP and Cyclic GMP Production in Rat Pinealocytes

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.     

Cyclic AMP-Dependent Protein Kinase Activity in Human Brain Across Age

Abstract: Cyclic AMP-dependent protein kinase activity was measured in the cerebral cortex of humans 2 days to 83 years of age and in the cortex of F344 rats 3, 22, or 30 months of age. Protein kinase activity was detected in the human brain, but no age-related differences in activity were observed in the presence or absence of cyclic AMP. Age differences were also not seen in protein kinase in the rat cerebral cortex. Enzyme activities in rat and human brain were similar.     

Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

Cyclic GMP-Dependent Pathways Protect Differentiated Oligodendrocytes from Multiple Types of Injury

The cyclic GMP analog 8-bromo-cyclic GMP (8-Br-cGMP) protects differentiated murine oligodendrocytes (OLs) from caspase-mediated death initiated by staurosporine, thapsigargin or kainate. Caspase-independent death caused by high levels of NO is also partially prevented by 8-Br-cGMP. Inhibitors of protein kinase G (cGMP-dependent protein kinase, cGK) reversed protection, supporting involvement of cGK. Since NO stimulates soluble guanylate cyclase, increasing cGMP, we treated OLs with low levels of NO and observed partial protection against thapsigargin, staurosporine and kainate. Two inhibitors of mitochondrial pore transition (MPT), cyclosporin A and bongkrekic acid, were poorly protective, indicating that cGMP is not acting primarily by blocking MPT. 8Br-cGMP was more effective than 8Br-cAMP in protecting against staurosporine or release of intracellular Ca++ by thapsigargin. The cAMP analog exhibited little or no protection against kainate or high levels of NO. Thus cGK signaling is more effective than protein kinase A or phosphodiesterase 3 signaling in preventing OL death. Special issue dedicated to Anthony Campagnoni.     

Polyamine Regulation of the Microtubule-Associated Protein Kinase

Microtubule protein prepared by cycles of assembly-disassembly contains a cyclic AMP-dependent protein kinase that phosphorylates the high-molecular-weight microtubule-associated protein MAP-2. The polyamine spermine at 2mM affected the phosphorylation of MAP-2 in a manner that depended on the cyclic AMP concentration. At cyclic AMP concentrations below 10(-6) M, spermine increased the rate of phosphorylation, while at cyclic AMP concentrations above 10(-6) M, spermine decreased the rate of phosphorylation. Spermine also decreased the final extent of cyclic AMP-dependent phosphorylation but did not affect the protein substrate specificity of the microtubule-associated protein kinase. MAP-2 was the principal substrate both in the presence and in the absence of spermine. Because of these results, we propose that microtubule protein phosphorylation may be regulated in vivo by spermine as well as by cyclic AMP levels.     

Calcium/Diacylglycerol-Dependent Protein Kinase and Its Major 87-Kilodalton Protein Substrate Are Differentially Distributed in Rat Basal Ganglia

When brain tissue is subjected to subcellular fractionation, both calcium/diacylglycerol-dependent protein kinase (protein kinase C) and an 87-kilodalton (kDa) protein substrate for this enzyme are enriched in the crude nerve terminal fraction. The present study, using chemical and surgical lesions of neurons in the rat neostriatum and substantia nigra, has examined whether the 87-kDa protein is colocalized with protein kinase C in identified neurons and nerve terminals. Our results show that, in the basal ganglia, protein kinase C is highly enriched in local striatal neurons and the striatonigral fibers and terminals. In contrast, the 87-kDa protein appears to be widely and evenly distributed in both neuronal and nonneuronal cells. The 87-kDa protein may therefore mediate functions of protein kinase C not restricted to nerve terminals.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

High-Pressure Extraction of Membrane-Associated Protein Kinase C from Rat Brain

Extraction of rat brain membrane-associated protein kinase C with high specific activity was obtained by applying benzyl alcohol (a membrane fluidizer), EDTA, and high hydrostatic pressures. Approximately 50% of total brain-associated activity was extracted from membranes. The pressure-extracted activity had an eightfold enrichment in the lipid/protein ratio when compared with the cytosolic fraction. This may explain the inability of exogenous diacylglycerol to stimulate endogenous phosphorylation in pressure-extracted activity. The enzyme is extracted at greater than 1,300 atm, a result indicating it most likely has a portion inserted into the hydrophobic portion of the membrane bilayer. Perturbation of the native membrane induces a change in the membrane-associated protein kinase C-lipid interaction that permits extraction under conditions used for the cytosolic species. This is the first report of conversion of the endogenous membrane species to a cytosolic one and may be important in determining the role of protein kinase C in neuronal regulation.     

Phosphorylation of P400 Protein by Cyclic AMP-Dependent Protein Kinase and Ca2+/Calmodulin-Dependent Protein Kinase II

Purified P400 protein was phosphorylated by both purified Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Because P400 protein was suggested to function as an integral membrane protein, we investigated the phosphorylation of P400 protein using crude mitochondrial and microsomal fractions (P2/P3 fraction). Incubation of the P2/P3 fraction from mouse cerebellum with cyclic AMP or the catalytic subunit of A-kinase stimulated the phosphorylation of P400 protein. The phosphorylation of P400 protein was not observed in the P2/P3 fraction from mouse forebrain. Cyclic AMP and A-kinase enhanced the phosphorylation of several proteins, including P400 protein, suggesting that P400 protein is one of the best substrates for A-kinase in the P2/P3 fraction. Although endogenous and exogenous CaM kinase II stimulated the phosphorylation of some proteins in the P2/P3 fraction, the phosphorylation of P400 protein was weak. Immunoprecipitation with the monoclonal antibody to P400 protein confirmed that the P400 protein itself was definitely phosphorylated by the catalytic subunit of A-kinase and CaM kinase II. A-kinase phosphorylated only the seryl residue in P400 protein. Immunoblot analysis of the cells in primary culture of mouse cerebellum confirmed the expression of P400 protein, which migrated at the same position on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that in the P2/P3 fraction. Incubation of the cultured cerebellar cells with [32P]orthophosphate resulted in the labeling of P400 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycolipids Isolated from Aplysia kurodaiCan Activate Cyclic Adenosine 3', 5'-Monophosphate-Dependent Protein Kinase from Rat Brain

Abstract: Cyclic AMP (cAMP)-dependent protein kinase (cAMP-kinase) partially purified from the membrane fractions of rat brains was stimulated by novel phosphonogly-cosphingolipids (glycolipids) derived from the skin and nerve fibers of Aplysia kurodai. Among various glycolipids tested, a major glycolipid from the skin, 3-O-MeGalβ 1→3GalNAcα 1→3 [6'- O -(2-aminoethylphosphonyl) Galα1→2] (2-aminoethylphosphonyl→6) Glcβ 1→4GICβ1→1ceramide (SGL-II), was most potent, giving half-maximal activation at 32.2 μ M. Activation of cAMP-kinase was maximal with 250 μ M SGL-II using kemptide as substrate. The effect of SGL-II was additive on kinase activity at submaximal concentrations of cAMP. The kinase activity activated with SGL-II was inhibited by the addition of protein kinase inhibitor peptide, a specific peptide inhibitor for cAMP-kinase. Its inhibitory pattern was similar to that for the catalytic subunit. Of the various substrates tested, the glycolipid-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I, and myelin basic protein but not histone H1 and casein. The regulatory subunit strongly inhibited the activity of purified catalytic subunit of cAMP-kinase. This inhibition was reversed by addition of SGL-II, as observed for cAMP. SGL-II was capable of partially dissociating cAMP-kinase, which was observed by gel filtration column chromatography. However, the binding activity of cAMP to the holoenzyme was not inhibited with SGL-II. These results demonstrate that the glycolipids can directly activate cAMP-kinase in a manner similar, but not identical, to that of cAMP.     

Increased Cyclic AMP-Dependent Protein Kinase Activity in Postmortem Brain from Patients with Bipolar Affective Disorder

Previous observations of reduced [3H]cyclic AMP binding in postmortem brain regions from bipolar affective disorder subjects imply cyclic AMP-dependent protein kinase function may be altered in this illness. To test this hypothesis, basal and stimulated cyclic AMP-dependent protein kinase activity was determined in cytosolic and particulate fractions of postmortem brain from bipolar disorder patients and matched controls. Maximal enzyme activity was significantly higher (104%) in temporal cortex cytosolic fractions from bipolar disorder brain compared with matched controls. In temporal cortex particulate fractions and in the cytosolic and particulate fractions of other brain regions, smaller but statistically nonsignificant increments in maximal enzyme activity were detected. Basal cyclic AMP-dependent protein kinase activity was also significantly higher (40%) in temporal cortex cytosolic fractions of bipolar disorder brain compared with controls. Estimated EC50 values for cyclic AMP activation of this kinase were significantly lower (70 and 58%, respectively) in both cytosolic and particulate fractions of temporal cortex from bipolar disorder subjects compared with controls. These findings suggest that higher cyclic AMP-dependent protein kinase activity in bipolar disorder brain may be associated with a reduction of regulatory subunits of this enzyme, reflecting a possible adaptive response of this transducing enzyme to increased cyclic AMP signaling in this disorder.     

Tryptophan Hydroxylase Is Phosphorylated by Protein Kinase A

Abstract: The effect of protein kinase A on the catalytic activity and phosphorylation of brain tryptophan hydroxylase was examined. Stimulation of endogenous protein kinase A by cyclic AMP or its analogues, dibutyryl-cyclic AMP and 8-thiomethyl-cyclic AMP, failed to activate tryptophan hydroxylase. The activation of tryptophan hydroxylase by calcium/calmodulin-phosphorylating conditions was not modified by cyclic AMP. Endogenous protein kinase A phosphorylated a large number of proteins and tryptophan hydroxylase could be identified as one substrate by sucrose gradient centrifugation, immunoprecipitation, and immunoblotting. These results indicate that tryptophan hydroxylase is phosphorylated by protein kinase A in brain and question whether this protein kinase exerts direct regulatory influence over tryptophan hydroxylase activity via phosphorylation.     

Protein Kinase C Activity in Rat Brain Cortex

The procedure used to obtain cerebral tissue for analysis of protein kinase C (PKC) activity may affect the subcellular distribution of the enzyme. We compared different methods of tissue preparation and found that the proportion of PKC activity associated with the particulate fraction of the cerebral cortex was only 30% when the brain was frozen in situ while the animal was on life support or after decapitation followed by delayed freezing. Other methods of obtaining cerebral tissue resulted in 49-56% of the PKC activity in the particulate fraction. Freezing per se had no apparent effect on the activity or subcellular distribution of PKC. In addition, whenever the particulate PKC activity was high (greater than 48%), there was also a significant increase in the proportion of particulate protein (from 51 to approximately 63%, p less than 0.05).