Frederick B. St. C. Palmer

Dissociation of phosphorylation and translocation of a myristoylated protein kinase C substrate (MARCKS protein) in C6 glioma and N1E-115 neuroblastoma cells.

Abstract: An 80‐kDa protein labeled with [3H]myristic acid in C6 glioma and N1E‐115 neuroblastoma cells has been identified as the myristoylated alanine‐rich C kinase substrate (MARCKS protein) on the basis of its calmodulin‐binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [3H]myristate were treated with 200 nM 4β‐12‐O‐tetradecanoylphorbol 13‐acetate (β‐TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X‐100–soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of β‐TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, β‐TPA–stimulated phosphorylation of MARCKS in N1E‐115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to α‐TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell‐specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.

Differential Expression of Marcks and Other Calmodulin-Binding Protein-Kinase-C Substrates in Cultured Neuroblastoma and Glioma-Cells

Abstract: Expression of the protein kinase C substrate MARCKS and other heat‐stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [3H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB‐11 (SK‐N‐SH) neuroblastoma cells than in HTB‐10 (SK‐N‐MC) or mouse N1E‐115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional 3H‐myristoylated proteins of 50 and 40–45 kDa were observed in cell extracts heated to >80°C or treated with perchloric acid. The 50‐kDa protein, which bound to calmodulin in the presence of Ca2+, was more prominent in cells (N1E‐115 and HTB‐10) with less MARCKS, whereas neuromodulin (GAP‐43) was detected in N1E‐115 and HTB‐11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [3H]myristate‐labeled MARCKS and may be due to a conformational change affecting the C‐terminal epitope or enhanced retention of the protein on nitrocellulose. Addition of β‐12‐O‐tetradecanoylphorbol 13‐acetate resulted in three‐ to fourfold increased phosphorylation of MARCKS in HTB‐11 cells, with little increase noted in HTB‐10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin‐binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.

Phosphoinositide metabolism in cultured glioma and neuroblastoma cells: subcellular distribution of enzymes indicate incomplete turnover at the plasma membrane.

The hypothesis that the small portion of cellular phosphoinositide participating in signal transduction might be preferentially recycled within the plasma membrane was tested in rat glioma (C6) and murine neuroblastoma (N1E-115) cells. Percoll density gradient centrifugation was used to isolate a purified plasma membrane fraction and the subcellular distribution of all enzymes mediating phosphoinositide turnover was assessed. A small but significant proportion of PtdInsP2-specific phosphodiesterase was located in the plasma membrane but only two of the five enzymes required to replace PtdInsP2 (diacylglycerol kinase and PtdInsP kinase) also were present. CTP:phosphatidate cytidylyltransferase and CMP-phosphatidate:inositol phosphatidyltransferase were located exclusively in a microsomal fraction containing enriched levels of endoplasmic reticulum markers. Thus, diacylglycerol from agonist-stimulated cleavage of PtdInsP2, or phosphatidic acid formed from it, must be transferred to the endoplasmic reticulum for conversion to PtdIns. Plasma membrane also lacked PtdIns kinase. If the soluble PtdIns kinase has access to membrane-bound substrate, PtdIns may be phosphorylated to PtdInsP before or during transport to the plasma membrane. Phosphorylation by the predominantly plasma membrane PtdInsP kinase to form PtdInsP2 completes the cycle. PtdInsP phosphatase was present in all membrane fractions suggesting that PtdInsP can be returned to the PtdIns pool in plasma membrane and elsewhere. PtdInsP2 phosphatase was almost exclusively in the cytosol suggesting that reversible interchange between PtdInsP and PtdInsP2 in the plasma membrane may be modulated by the ability of this phosphatase to act on PtdInsP2 in the membrane. Thus, PtdIns resynthesis in the plasma membrane of these cells does not occur and is not required for phosphoinositide-mediated signal transduction.

Niemann-Pick Type II fibroblasts exhibit impaired cholesterol esterification in response to shingomyelin hydrolysis

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (greater than 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [3H]oleic acid into cholesterol-[3H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimulated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquine abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.

Isolation of plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover

A rapid and reliable method for the isolation of plasma membranes and microsomes of high purity and yield from cultured glioma cells is described. The procedure involves disruption by N2 cavitation, preliminary separation by centrifugation in Tricine buffer, and final separation on a gradient formed from 40% Percoll at pH 9.3. Enzyme and chemical markers indicated greater than 60% yield with six- to eightfold enrichment for plasma membranes and greater than 25% yield with three- to fourfold enrichment for a microsomal fraction consisting mainly of endoplasmic reticulum. The final fractions were obtained with high reproducibility in less than 1 h from the time of cell harvesting. Application of this procedure to human fibroblasts in culture is assessed. The isolation procedure was applied to investigations of synthesis and turnover of sphingomyelin and phosphatidylcholine in plasma membranes of glioma cells following incubation for 4-24 h with [methyl-3H]choline. These studies indicated that radioactivity from phosphatidylcholine synthesized in microsomes from exogenous choline may serve as a precursor of the head-group of sphingomyelin accumulating in the plasma membrane.

Phorbol ester stimulation of phosphatidylcholine synthesis in four cultured neural cell lines: correlations with expression of protein kinase C isoforms.

Phosphatidylcholine (PtdCho) can provide lipid second messengers involved in signal transduction pathways. As a measure of phospholipid turnover in response to extracellular stimulation, we investigated differential enhancement of [3H]choline incorporation into PtdCho by phorbol esters. In C6 rat glioma and SK-N-SH human neuroblastoma cells, [3H]PtdCho synthesis was 2–4 fold stimulated by β-12-O-tetradecanoylphorbol-13-acetate (β-TPA) when [3H]choline was incubated simultaneously with, or 15 min prior to, β-TPA treatment. By contrast, in N1E-115 mouse and SK-N-MC human neuroblastoma cells, phorbol esters had no appreciable effect on [3H]choline incorporation; however, in all cells, 200 μM oleic acid enhanced PtdCho synthesis, indicating a stimulable process. Alterations by thymeleatoxin (TMT), an activator of conventional PKC isoforms (α, β and γ), were similar to β-TPA. We investigated whether expression of specific PKC isoforms might correlate with these effects of phorbol esters on PtdCho synthesis. All cell lines bound phorbol esters, had PKC activity that was translocated by phorbol esters and differentially expressed isoforms of PKC. Northern and western blot analyses, using specific cDNA and antibodies for PKC-α,-β,-γ,-δ,-ε, and-ζ, revealed that expression of α-isoform predominated in C6 and SK-N-SH cells. In contrast, TPA-responsive β-isoform predominated in SK-N-MC cells. γ-PKC was not detected in any cells and only in C6 cells was PKC-δ present and translocated by β-TPA treatment. PKC-ε was not detected in SK-N-MC cell lines but translocated with TPA treatment in the other three cell lines. PKC-ζ was present in all cells but was unaltered by TPA treatment. Accordingly, stimulation of PtdCho turnover by phorbol esters correlated only with expression of PKC-α; presence of PKC-β alone was insufficient for a TPA response.

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis by cytochalasin B in cultured glioma cells: Potential regulation of biosynthesis by Ca2+-dependent mechanisms

The major route of phosphatidylcholine (PtdCho) biosynthesis in mammalian cells is the sequence: choline (Cho)----phosphocholine (PCho)----cytidinediphosphate choline (CDP-Cho)----PtdCho. Recently, we have found that intermediates of this pathway are not freely diffusible in cultured rat glioma (C6) cells but are channeled towards PtdCho biosynthesis (George et al. (1989). Biochim. Biophys. Acta. 1004, 283-291). Channeling of intermediates in other mammalian systems is thought to be mediated through adsorption of enzymes to membranes and cytoskeletal elements to form multienzyme complexes. In this study, agents which perturb the structure and function of cytoskeletal elements were tested for effects on phospholipid metabolism in glioma cells. The filament-disrupting agent cytochalasin B (CB), but not other cytochalasins or the microtubule depolymerizer colchicine inhibited PtdCho and phosphatidylethanolamine (PtdEtn) biosynthesis as judged by dose-dependent reduction of labeling from [3H]Cho and [14C]ethanolamine (Etn). 32Pi pulse-labeling indicated that CB selectively decreased PtdCho and PtdEtn biosynthesis without affecting synthesis of other phospholipids. Synthesis of water-soluble intermediates of PtdCho metabolism was unaffected but the conversion of phosphoethanolamine to CDP-ethanolamine was reduced by CB. Effects of CB on phospholipid biosynthesis were not due to inhibition of glucose uptake as shown by experiments with 2-deoxyglucose, glucose-starved cells and other cytochalasins. Experiments with Ca(2+)-EGTA buffers and digitonin-permeabilized cells, and the Ca(2+)-channel blocker verapamil suggest that effects of CB on PtdCho and PtdEtn biosynthesis are due to alteration of intracellular Ca2+. Taken together, these results suggest that CB acts at sites distinct from glucose transport and cellular microfilaments to specifically inhibit PtdCho and PtdEtn biosynthesis by mechanisms dependent on intracellular Ca2+.

An enzymatically coupled assay for rat brain polyphosphoinositide phosphodiesterase in an optimized reaction mixture

Bovine intestinal alkaline phosphatase was found to hydrolyze inositol phosphates many times faster than the monoester phosphate groups of the polyphosphoinositides. A convenient and sensitive in vitro assay for the Ca2+-dependent polyphosphoinositide phosphodiesterase was devised in which inositol trisphosphate released from exogenous phosphatidylinositol 4,5-bisphosphate was hydrolyzed by alkaline phosphatase. The resulting inorganic phosphate was measured by an automated method after solubilization of the reaction mixture with sodium dodecyl sulfate. The phosphodiesterase was maximally stimulated by combining the known positive effects of cetyltrimethylammonium bromide (at the optimum detergent-to-substrate ratio of 2.3), monovalent cations (0.1 M KCl), and Ca2+ (0.5 mM) with the additional enhancement by Triton X-100 (0.2% w/v). Activities obtained for rat brain homogenates and microsomal and cytosol fractions were 126 +/- 3.8 (17), 110 +/- 5.7 (10), and 252 +/- 15.5 (8) nmol X min-1 X mg protein-1 (mean +/- SE for n determinations), respectively.

Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann-Pick type C and type D patients

Regulation of intracellular cholesterol metabolism has been studied in Epstein-Barr virus-transformed lymphoblasts from patients with Niemann-Pick type C (NPC) and the Nova Scotia type D (NPD) disease. Addition of LDL to normal lymphoblasts cultured in lipoprotein-deficient medium increased cholesterol esterification 10-fold (to a maximum of 1.0 nmol/h/mg protein at 15 h), while little stimulation was seen in NPC cells. The response by NPD lymphoblasts was intermediate, reaching approximately half of normal values by 14-24 h. Lymphoblasts from both NPC and NPD obligate heterozygotes exhibited 50% of normal LDL-stimulated cholesterol esterification at 6 h, when activity was < 10% of normal values in patient cells. Fluorescence staining with filipin indicated excessive intracellular accumulation of LDL-derived cholesterol in both NPC and NPD lymphoblasts. Downregulation of LDL receptor mRNA levels by LDL, measured by S1 nuclease protection assay, was also impaired in NP lymphoblasts and fibroblasts (NPC > NPD), although a similar rate of receptor protein down-regulation by LDL (t1/2 = 10-15 h) was observed in normal and NP lymphoblasts. In contrast, LDL down-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA did not appear to be affected in NP cells: LDL produced a 3-fold (lymphoblasts) or > 10-fold (fibroblasts) decrease by 12 h in both normal and affected cells. Thus, NPC and NPD lymphoblasts exhibit distinct defects in cholesterol esterification and storage, similar to those observed in mutant fibroblasts. Other regulatory responses are also impaired in NPC lymphoblasts but appear to be less affected in NPD cells. Lymphoblasts should provide a valuable immortalized cell line model for study of defective regulation of cholesterol esterification and transport in Niemann-Pick type II disease, and may also be suitable for diagnosis and carrier detection.

Cultured Fibroblasts from Patients with Niemann-Pick Disease Type-C and Type-D Exhibit Distinct Defects in Cholesterol Esterification

The Niemann-Pick group of diseases can be broadly classified into two types based on clinical and biochemical characteristics. Type I is characterized by a primary deficiency of lysosomal sphingomyelinase while Type II may have a defect in the regulation of intracellular cholesterol metabolism. We have studied cholesterol esterification in cultured fibroblasts from patients with two phenotypes of Type II disease: an Acadian population of southwestern Nova Scotia (Canada) with a form of the disease known as Niemann-Pick type D (NPD) and a group of panethnic origin with Niemann-Pick type C (NPC). Addition of whole serum to normal fibroblasts grown initially in lipoprotein-deficient serum caused a rapid (within 6 h) increase in cholesterol esterification, reaching maximum values at around 24 h, while NPC fibroblasts showed little increase (less than 10% of normal). In contrast, cholesterol esterification in NPD fibroblasts increased slowly during the first 6-12 h and reached 50% of normal values by 24 h. 25-Hydroxycholesterol, a non-lipoprotein stimulator of cholesterol esterification, caused a similar stimulation of cholesterol esterification in NPC, NPD and normal cells. This was inhibited by addition of serum in mutant but not in normal cells. Within 24 h of serum addition, free cholesterol accumulated in all cell types with NPC greater than NPD greater than normal. These observations indicate that (a) regulation of cholesterol esterification in response to serum lipoproteins (but not 25-hydroxycholesterol) is abnormal in both NPC and NPD fibroblasts, and (b) the biochemical phenotypes of fibroblasts from NPC and NPD patients are distinct.