Regulation of Sialyltransferase Activities by Phosphorylation and Dephosphorylation

Abstract: The composition of tissue gangliosides is thought to result mainly from the active regulation and selective expression of specific enzymes responsible for their metabolism. In the last few years, we have purified several rat brain sialyltransferases to homogeneity; the availability of these highly purified enzymes enabled us to investigate their regulation and expression at the molecular level. Thus, we studied the regulation of sialyltransferase activities, in particular, CMP-NeuAc:GM1 and CMP-NeuAc:LacCer sialyltransferases by a phosphorylation/dephosphorylation mechanism. Protein kinase C was added to a standard enzyme assay mixture containing [γ-³²P]ATP, and the activity of the enzyme was measured after various incubation times. We found that treatment of several sialyltransferases by protein kinase C decreased their activities in a time-dependent manner. Analyses of ³²P-labeled amino acids revealed that the major phosphorylation site of CMP-NeuAc:GM1 α2→3 sialyltransferase (ST-IV) was serine and that for CMP-NeuAc:LacCer α2→3 sialyltransferase (ST-I) was primarily threonine. Partial recovery of the enzyme activity could be achieved by treatment of the phosphorylated sialyltransferases with rat brain protein phosphatase. We conclude that the activities of sialyltransferases can be modulated by protein kinase C and protein phosphatase and this may represent a potential regulatory mechanism for ganglioside biosynthesis.     

Coordinate Regulation of Ganglioside Glycosyltransferases in Differentiating NG108-15 Neuroblastoma X Glioma Cells

The enzymatic basis for ganglioside regulation during differentiation of NG108-15 mouse neuroblastoma x rat glioma hybrid cells was studied. This cell line contains four gangliosides that lie along the same biosynthetic pathway: GM3, GM2, GM1, and GD1a. Chemically induced neuronal differentiation of NG108-15 cells led to an 80% drop in the steady-state level of their major ganglioside, GM3, a sixfold increase in the level of a minor ganglioside, GM2 (which became the predominant ganglioside of differentiated cells); and relatively little change in the levels of GM1 and GD1a, which lie further along the same biosynthetic pathway. The enzymatic basis for this selective change in ganglioside expression was investigated by measuring the activity of two glycosyltransferases involved in ganglioside biosynthesis. UDP-N-acetylgalactosamine: GM3 N-acetylgalactosaminyltransferase (GM2-synthetase) activity increased fivefold during butyrate-induced differentiation, whereas UDP-galactose: GM2 galactosyltransferase (GM1-synthetase) activity decreased to 10% of its control level. Coordinate regulation of these two glycosyltransferases appears to be primarily responsible for the selective increase of GM2 expression during NG108-15 differentiation.     

Enhancement of ganglioside GM3 synthesis in okadaic-acid-treated granulosa cells.

Okadaic acid is a potent inhibitor of type-2A (PP2A) and type-1 (PP1) protein phosphatases and has been proved to be a valuable tool for studies on the protein phosphorylation. We have investigated the effects of okadaic acid on rat granulosa cells in order to determine whether the regulation of ganglioside synthesis involves protein phosphorylation via inhibition of PP2A and PP1. Granulosa cells expressed luteinizing hormone (LH) receptors, measured as the binding of 125I-deglycosylated human chorionic gonadotropin (hCG) to intact cells, and synthesized the gangliosides NeuAc alpha 2-->3Gal beta 1-->4Glc beta 1-->1Cer (GM3) and Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer (GM1), demonstrated by metabolic labeling of glycosphingolipids with [3H]galactose, in response to follicle-stimulating hormone (FSH). When FSH-stimulated granulosa cells were treated with 10 nM okadaic acid for 15 h, down-regulation of LH receptors, dissociation of LH receptor-effector coupling and significant decreases of intracellular and extracellular 3',5'-cyclic adenosine monophosphate (cAMP) levels were observed. The okadaic acid-induced desensitization to gonadotropin in granulosa cells was accompanied by increased ganglioside synthesis. The amount of 3H-labeled ganglioside GM3, the major ganglioside (about 95% of the total) synthesized by mature granulosa cells, was enhanced in okadaic acid-desensitized cells (to 215% of the control value) and in those desensitized by hCG (by 354%), forskolin (by 190%) and 12-O-tetradecanoylphorbol 13-acetate (by 143%). The results of this study suggest that an increase in the phosphorylation state of cells is accompanied by enhancement of ganglioside synthesis.     

In vivo modulation of epidermal growth factor receptor phosphorylation in mice expressing different gangliosides

We studied in this work the in vivo phosphorylation of the epidermal growth factor receptor (EGFr) in skin from knockout mice lacking different ganglioside glycosyltransferases. Results show an enhancement of EGFr phosphorylation, after EGF stimulation, in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice as compared with wild-type and Sial-T1 knockout mice. Qualitative analysis of ganglioside composition in mice skin suggest that the increase of EGFr phosphorylation observed in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice in response to EGF might not be primary attributed to the expression of GD3 or a-series gangliosides in mice skin. These studies provide, for the first time, an approach for studying the molecular mechanisms involved in the in vivo regulation of EGFr function by gangliosides.     

Sialidase occurs in both membranes of the nuclear envelope and hydrolyzes endogenous GD1a

Previous reports indicated the presence of both gangliosides and sialidase in the nuclear envelope (NE) of primary neurons and the NG108-15 neural cell line. GM1, one of the major gangliosides of this membrane, was shown to be tightly associated with a sodium-calcium exchanger in the inner membrane of the NE and to potentiate exchanger activity. GD1a was the other major ganglioside detected in the NE and, like GM1, occurs in both inner and outer membranes. A subsequent report indicated the presence of sialidase activity in the NE without specification as to which of the two membranes express it. The present study was undertaken to determine the nature and locus of this activity within the NE of two cell lines: NG108-15 and SH-SY5Y. Western blot analysis of the separated membranes revealed occurrence of Neu3 in the inner membrane and Neu1 in the outer membrane of the NE. Moreover, sialidase activity at both sites was shown capable of catalyzing conversion of endogenous GD1a to GM1.     

Relationship between the regulation of membrane enzyme activities by gangliosides and a possible ganglioside segregation in membrane microdomains

Laser and neutron scattering experiments showed that in mixed micelles of ganglioside GM2 and GT1b, a membrane mimicking system, the segregation of gangliosides may occur spontaneously. Photolabeling experiments using nitrophenylazide containing ganglioside GM1 proved that gangliosides added to cells in culture enter the cell and bind to its membrane as components of microdomains, which specifically interact with a protein of about 30 kDa. This suggests that ganglioside segregation may be a natural phenomenon. Gangliosides when added to granule cells in culture led to increase in protein phosphorylation, the effect exerted being related to the amount of ganglioside molecules inserted stably into the cell lipid layer and an increase of 0.7% of the cell original ganglioside content promoted an increase of 57% in the incorporation of 32P into cell membrane proteins. From the above results a possible relationship between ganglioside segregation and involvement of ganglioside in enzyme activity control is suggested.     

Gene-linked shift in ganglioside distribution influences growth and vascularity in a mouse astrocytoma

Brain tumor growth and progression is dependent upon vascularity, and is associated with altered ganglioside composition and distribution. In this study, we examined the influence of gangliosides on growth and vascularity in a malignant mouse astrocytoma, CT-2A. Ganglioside distribution was altered in CT-2A tumor cells using an antisense construct to beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T), a key enzyme that uses the simple ganglioside GM3 as a substrate for the synthesis of the more complex gangliosides, GM2, GM1 and GD1a. GalNAc-T gene expression was significantly lower in CT-2A cells stably transfected with the antisense GalNAc-T plasmid, pcDNA3.1/TNG (CT-2A/TNG) than in either non-transfected CT-2A or mock-transfected (CT-2A/V) control tumor cells. GM3 was elevated from 16% to 58% of the total ganglioside distribution, whereas GM1 and GD1a were reduced from 17% and 49% to 10% and 17%, respectively, in CT-2A/TNG tumor cells. Growth, vascularity (blood vessel density and Matrigel assay) and vascular endothelial growth factor (VEGF) expression was significantly less in CT-2A/TNG tumors than in control CT-2A brain tumors. In addition, the expression of VEGF, hypoxia-inducible factor 1alpha (HIF-1alpha) and neuropilin-1 (NP-1) was significantly lower in CT-2A/TNG tumor cells than in control CT-2A tumor cells. These data suggest that gene-linked changes in ganglioside composition influence the growth and angiogenic properties of the CT-2A astrocytoma.     

PLC gamma 1, a possible mediator of T cell receptor function

Stimulation of T cell antigen receptor (TCR/CD3) following the recognition of peptide-major histocompatibility antigen complex induces phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, the phospholipase C (PLC) enzyme mediating this process has not been identified. We report that PLC gamma 1 protein is expressed in human T cells. It is a phosphoprotein, and the activation of cyclic AMP-dependent protein kinase (PKA) or of protein kinase C (PKC) with forskolin or phorbol ester, respectively, increases the level of phosphorylation. CD3 stimulation of T cells induces tyrosine phosphorylation of PLC gamma 1 and causes 8-10-fold higher yield of PLC activity with anti-phosphotyrosine antibody (APTyr Ab) from activated cells than from non-activated cells. Genistein, an inhibitor of protein tyrosine kinase, decreases this yield of AP-Tyr Ab-bound PLC activity from activated cells and lowers the level of Ca2+ mobilization. Furthermore, phorbol ester and forskolin treatment of cells before CD3 stimulation reduces the level of tyrosine phosphorylation of PLC gamma 1 and the PLC activity associated with APTyr Ab. These results suggest that CD3 stimulation activates PIP2 hydrolysis by inducing tyrosine phosphorylation of PLC gamma 1, which is regulated negatively by PKC and PKA.     

Tyrosine and threonine phosphorylation of an immunoaffinity-purified 44-kDa MAP kinase.

We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester-associated changes in ganglioside metabolism

The effect of phorbol esters on ganglioside metabolism in contact-inhibited Chinese hamster V79 cells was examined. Three phorbol esters of varying structure and tumor-promoting activity were used. Treatment of cells with tumor-promoting phorbol esters resulted in accumulation of gangliosides and increased incorporation of [1-¹⁴C]palmitate and [9-³H]sialic acid into gangliosides. Moreover, the phorbol esters were found to increase the activity of CMP-sialic acid: lactosylceramide sialyltransferase, the enzyme catalysing the first step in ganglioside biosynthesis. The magnitude of phorbol ester effects on V79 cell ganglioside metabolism correlated with the in vivo phorbol ester tumor-promoting activity.     

Interaction of the extracellular domain of the epidermal growth factor receptor with gangliosides.

Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.     

Ganglioside GM1 Activates the Mitogen-Activated Protein Kinase Erk2 and p70 S6 Kinase in U-1242 MG Human Glioma Cells

Abstract: Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [³H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70^s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70^s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.     

Modulation of phospholipase A2 activity by neutral and anionic glycosphingolipids in monolayers.

The effect of neutral (galactocerebroside and asialo-ganglioside GM1) or anionic (sulphatide and gangliosides GM1, GD1a and GT1b) glycosphingolipids on the activity of phospholipase A2 from pig pancreas was studied in mixed monolayers of dilauroyl phosphatidylcholine with the glycosphingolipids in different molar fractions at various constant surface pressures. The activity of the enzyme depends on the proportion and type of glycosphingolipid in the interface. Sulphatide activates the enzyme at all proportions, whereas galactocerebroside shows inhibition or activation depending on its proportion in the film. Asialo-ganglioside GM1 and gangliosides GM1, GD1a and GT1b can strongly inhibit the enzyme at relatively low molar fractions in the film in the following order: asialo-ganglioside GM1 less than ganglioside GM1 less than ganglioside GT1b less than ganglioside GD1a. The changes of activity are not due to a direct action of the lipids on the active centre or interfacial recognition region of the enzyme.     

Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

GM1 ganglioside induces phosphorylation and activation of Trk and Erk in brain

We investigated the ability of GM1 to induce phosphorylation of the tyrosine kinase receptor for neurotrophins, Trk, in rat brain, and activation of possible down-stream signaling cascades. GM1 increased phosphorylated Trk (pTrk) in slices of striatum, hippocampus and frontal cortex in a concentration- and time-dependent manner, and enhanced the activity of Trk kinase resulting in receptor autophosphorylation. The ability of GM1 to induce pTrk was shared by other gangliosides, and was blocked by the selective Trk kinase inhibitors K252a and AG879. GM1 induced phosphorylation of TrkA > TrkC > TrkB in a region-specific distribution. Adding GM1 to brain slices activated extracellular-regulated protein kinases (Erks) in all three brain regions studied. In striatum, GM1 elicited activation of Erk2 > Erk1 in a time-and concentration-dependent manner. The GM1 effect on Erk2 was mimicked by other gangliosides, and was blocked by the Trk kinase inhibitors K252a and AG879. Pertussis toxin, as well as Src protein tyrosine kinase and protein kinase C inhibitors, did not prevent the GM1-induced activation of Erk2, apparently excluding the participation of Gi and Gq/11 protein-coupled receptors. Intracerebroventricular administration of GM1 induced a transient phosphorylation of TrkA and Erk1/2 in the striatum and hippocampus complementing the in situ studies. These observations support a role for GM1 in modulating Trk and Erk phosphorylation and activity in brain.     

GANGLIOSIDE1−COMPOSITION OF BRAIN IN TAY-SACHS DISEASE: INCREASED AMOUNTS OF GD2 AND N-ACETYL-β-D-GALACTOSAMINYL GD1a GANGLIOSIDE

Abstract— The ganglioside composition of the brain of a patient with Tay-Sachs disease (TS-brain) was determined by a newly developed ganglioside-mapping procedure and compared with that of an age-matched control brain. GM2 ganglioside was the predominant component in TS-brain and the following gangliosides were also found, GM1, GD1a, GD1b and GT1 (major gangliosides in normal brain), and GM3, GD3, GD2 and GD1a-GAN (minor or undetectable components of normal brain). Individual gangliosides were isolated by column chromatography using a combination of DEAE-Sepharose, Iatrobeads and Silica Gel 60 and their structures were confirmed by comparing them with authentic standards using TLC, analysing their carbohydrate compositions by gas-liquid chromatography and cleaving them sequentially with glycosidases. The amounts of individual components were measured by quantitative densitometric scanning of the thin-layer plates. As a reflection of myelin breakdown, no sialosylgalactosyl ceramide was detectable in TS-brain. Although the total amounts of all gangliosides except GM2 in TS-brain were low, there were normal molar ratios of the main gangliosides in normal brain, that is, GM1, GD1a, GD1b and GT1. In comparison with the amount of GDla ganglioside, the amounts of GM2, GD2 and GD1a-GAN, which contain N-acetylgalactosamine as a terminal carbohydrate residue, were all elevated in TS-brain. The long chain bases of individual gangliosides contained both C-18 and C-20 sphingosine in different ratios and the ratio of C-20 to C-18 increased in the gangliosides in the order: GM2 < GM1 < GD1a < GD1a-GAN < GD1b < GT1 in both normal brain and TS-brain. In contrast, GD2 and GD3 gangliosides consisted mainly of C-18 sphingosine. The C-20 to C-18 ratios of individual gangliosides in the TS-brain were lower than those of age-matched control brain. Hexosaminidase from Turbo cornutus showed the same specific activity and K_m value in catalysing the cleavage of terminal N-acetylgalactosaminyl residues from GM2, GD2 and GD1a-GAN, suggesting that the brain gangliosides that increase in Tay-Sachs disease may be cleaved by the same enzyme.     

Na+,K(+)-ATPase phosphorylation in the choroid plexus: synergistic regulation by serotonin/protein kinase C and isoproterenol/cAMP-PK/PP-1 pathways.

BACKGROUND: The ion pump Na+,K(+)-ATPase is responsible for the secretion of cerebrospinal fluid from the choroid plexus. In this tissue, the activity of Na+,K(+)-ATPase is inhibited by serotonin via stimulation of protein kinase C-catalyzed phosphorylation. The choroid plexus is highly enriched in two phosphoproteins which act as regulators of protein phosphatase-1 activity, DARPP-32 and inhibitor-1. Phosphorylation catalyzed by cAMP-dependent protein kinase on a single threonyl residue converts DARPP-32 and inhibitor-1 into potent inhibitors of protein phosphatase-1. Previous work has shown that in the choroid plexus, phosphorylation of DARPP-32 and I-1 is enhanced by isoproterenol and other agents that activate cAMP-PK. We have now examined the possible involvement of the cAMP-PK/protein phosphatase-1 pathway in the regulation of Na+,K(+)-ATPase. MATERIALS AND METHODS: The state of phosphorylation of Na+,K(+)-ATPase was measured by determining the amount of radioactivity incorporated into the ion pump following immunoprecipitation from 32P-prelabeled choroid plexuses incubated with various drugs (see below). Two-dimensional phosphopeptide mapping was employed to identify the protein kinase involved in the phosphorylation of Na+,K(+)-ATPase. RESULTS: The serotonin-mediated increase in Na+,K(+)-ATPase phosphorylation is potentiated by okadaic acid, an inhibitor of protein phosphatases-1 and -2A, as well as by forskolin or the beta-adrenergic agonist, isoproterenol, activators of cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps suggest that this potentiating action occurs at the level of a protein kinase C phosphorylation site. Forskolin and isoproterenol also stimulate the phosphorylation of DARPP-32 and protein phosphatase inhibitor-1, which in their phosphorylated form are potent inhibitors of protein phosphatase-1. CONCLUSIONS: The results presented here support a model in which okadaic acid, forskolin, and isoproterenol achieve their synergistic effects with serotonin through phosphorylation of DARPP-32 and inhibitor-1, inhibition of protein phosphatase-1, and a reduction of dephosphorylation of Na+,K(+)-ATPase at a protein kinase C phosphorylation site.     

Stimulation of Transglutaminase Activity by GM1-Ganglioside and α-Sialylcholesterol in Superior Cervical and Nodose Ganglia Excised from Adult Rat

Changes in transglutaminase (TG) activity in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined following application of selected membrane transport-altering agents, including GM1-ganglioside (GM1) and alpha-sialylcholesterol (alpha-SC). Although TG activity of freshly dissected SCG and NG was relatively low, it increased gradually during 30 min of incubation, and it stayed at this elevated level for 2 h. Addition of alpha-SC at its maximal effective concentration, 20 microM, stimulated TG activity more than eightfold in SCG and more than twofold in NG by 30 min. Addition of GM1 at its most effective concentration, 5 nM, had similar effects, but of lesser magnitude. Cycloheximide, a potent inhibitor of protein biosynthesis, did not affect the GM1- or alpha-SC-evoked increases in ganglionic TG activity, suggesting that enzyme activation rather than synthesis of new enzyme was occurring. The stimulation of TG activity in both ganglia caused by either GM1 or alpha-SC was associated with a decrease in Km and an increase in Vmax values. Addition of cholera toxin B, which specifically masks the oligosaccharide chain of GM1, reduced the GM1-induced increase in TG activity by approximately 60% in SCG and 88% in NG. The alpha-SC-induced increase in TG activity was only partially mimicked by free cholesterol. Although application of either dibutyryl cyclic AMP or dibutyryl cyclic GMP produced little change in TG activity of either ganglion, phorbol ester clearly inhibited the enzymic activity. Because TG is a calcium-dependent enzyme, we measured 45Ca2+ influx into either ganglion, and found that it was reduced by GM1 and alpha-SC in SCG and by alpha-SC in NG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium/Ganglioside-Dependent Protein Kinase Activity in Rat Brain Membrane

The effects of gangliosides on phosphorylation were studied in rat brain membrane. Gangliosides stimulated phosphorylation only in the presence of Ca2+ with major phosphoproteins of 45,000, 50,000, 60,000, and 80,000 daltons and high-molecular-weight species. In addition, gangliosides inhibited the phosphorylation of three proteins with molecular weights of 15,000, 20,000, and 78,000 daltons. The two low-molecular-weight proteins comigrated with rat myelin basic proteins. Ganglioside stimulation was dependent on the formation of a Ca2+-ganglioside complex since the calcium salt of gangliosides stimulated phosphorylation maximally. Disialo and trisialo gangliosides were more potent stimulators of kinase activity than the monosialo GM1 X GD1a was the most potent activator tested. Asialo-GM1, cerebroside, sialic acid, neuraminyllactose, sulfatide, and the acidic phospholipids phosphatidylserine and phosphatidylinositol did not stimulate kinase activity. The Ca2+-dependent, ganglioside-stimulated phosphorylation was qualitatively similar to the pattern for calmodulin-dependent phosphorylation. However, while calmodulin-dependent kinase activity was inhibited with an IC50 of 10 microM trifluoperazine, ganglioside-stimulated kinase was inhibited with an IC50 of 200 microM trifluoperazine. These results indicate that gangliosides have complex effects on membrane-associated kinase activities and suggest that Ca2+-ganglioside complexes are potent stimulators of membrane kinase activity.     

Neurochemical and morphological studies on differentiation of NG108-15 cells by phorbol ester and forskolin

12-O-tetradecanoylphorbol 13-acetate (TPA), forskolin or dibutyryl cAMP induced neurite outgrowth and inhibition of cell growth in NG108-15 cells. TPA, forskolin and dibutyryl cAMP significantly increased specific activity of choline acetyltransferase. Forskolin markedly stimulated cAMP accumulation, but not TPA, suggesting that forskolin could induce differentiation by increasing the cAMP content via adenylate cyclase activation, but TPA-induced differentiation seems not to be due to the raise of the cAMP level. Incubation of the cells with TPA, forskolin or dibutyryl cAMP for 24 h resulted in enhancement of 50 mM K⁺-evoked Ca²⁺ influx and neurite elongation, although incubation with these agents for 1 h didn't affect these events. From these results, it is suggested that TPA and forskolin induce differentiation of NG108-15 cells to acetylcholine neurons via different mechanisms: protein kinase C activation by TPA and cAMP-dependent protein kinase activation by forskolin. In addition, it is likely that Ca²⁺ channels in cells differentiated by TPA, forskolin or dibutyryl cAMP become sensitive to depolarization.