Lateral diffusion of membrane lipids and proteins is increased specifically in neurites of differentiating neuroblastoma cells

Lateral diffusion of membrane lipids and proteins was determined in differentiating C1300 mouse neuroblastoma cells by fluorescence photo-bleaching recovery measurements. It is demonstrated that upon differentiation the lateral diffusion of membrane lipids and proteins is increased specifically in the extending neurites. This indicates the appearance of a topographical heterogeneity in the cell membrane, whereby more fluid domains become located in the membrane of the neurites.     

Relationship among methylation,isoprenylation, and GTP binding in 21-to 23-kDa proteins of neuroblastoma

1. Dimethylsulfoxide-induced differentiated neuroblastoma express high levels of membrane 21 to 23-kDa carboxyl methylated proteins. Relationships among methylation, isoprenylation, and GTP binding in these proteins were investigated. Protein carboxyl methylation, protein isoprenylation, and [alpha-32P]GTP binding were determined in the electrophoretically separated proteins of cells labeled with the methylation precursor [methyl-3H]methionine or with an isoprenoid precursor [3H]mevalonate. 2. A broad band of GTP-binding proteins, which overlaps with the methylated 21 to 23-kDa proteins, was detected in [alpha-32P]GTP blot overlay assays. This band of proteins was separated in two-dimensional gels into nine methylated proteins, of which four bound GTP. 3. The carboxyl-methylated 21 to 23-kDa proteins incorporated [3H]mevalonate metabolites with characteristics of protein isoprenylation. The label was not removed by organic solvents or destroyed by hydroxylamine. Incorporation of radioactivity from [3H]mevalonate was enhanced when endogenous levels of mevalonate were reduced by lovastatin, an inhibitor of mevalonate synthesis. Lovastatin blocked methylation of the 21 to 23-kDa proteins as well (greater than 70%). 4. Methylthioadenosine, a methylation inhibitor, inhibited methylation of these proteins (greater than 80%) but did not affect their labeling by [3H]mevalonate. The results suggest that methylation of the 21 to 23-kDa proteins depends on, and is subsequent to, isoprenylation. The sequence of events may be similar to that known in ras proteins, i.e., carboxyl methylation of a C-terminal cysteine that is isoprenylated. 5. Lovastatin reduced the level of small GTP-binding proteins in the membranes and increased GTP binding in the cytosol. Methylthioadensoine blocked methylation without affecting GTP binding. 6. Thus, isoprenylation appears to precede methylation and to be important for membrane association, while methylation is not required for GTP binding or membrane association. The role of methylation remains to be determined but might be related to specific interactions of the small GTP-binding proteins with other proteins.     

Detection of 23-27 kDa GTP-binding proteins in platelets and other cells.

Membrane proteins from rabbit and human platelets were separated by SDS/polyacrylamide-gel electrophoresis and the resolved polypeptides blotted on nitrocellulose. A family of GTP-binding proteins, termed Gn proteins, was detected by incubation of these blots with [alpha-32P]GTP in the presence of Mg2+. A major Gn protein with a molecular mass of 27 kDa (Gn27) and lesser amounts of 23, 24 and 25 kDa Gn proteins were observed in platelet membranes; much smaller amounts were in the platelet soluble fraction. Binding of [alpha-32P]GTP by platelet Gn proteins was blocked by GDP, GTP or guanosine 5'-[gamma-thio]triphosphate, but not by GMP or adenosine 5'-[beta gamma-imido]triphosphate. Rabbit and human red-cell membranes contained only Gn27. When rat tissues were analysed for Gn proteins, the largest amounts were found in brain, which contained two membrane-bound forms (Gn27 and Gn26) and a soluble form (Gn26).     

Carboxyl methylation of cytosolic proteins in intact human erythrocytes. Identification of numerous methyl-accepting proteins including hemoglobin and carbonic anhydrase

Intact human erythrocytes incubated with L-[methyl-3H]methionine incorporated radioactivity into base-labile linkages with membrane and cytosolic proteins which are characteristic of protein methyl esters. Kinetic analysis of the methylation reactions in intact cells shows that individual erythrocytes contain approximately 38,000 and 115,000 protein methyl esters with biological half-lives of 150 min or less in the membrane and cytosolic protein fractions, respectively. Fractionation of the methylated cytosolic species by gel filtration chromatography at pH 6.5 followed by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 reveals that many different cytosolic proteins serve as methyl acceptors and that the degree of modification varies widely for individual proteins. For example, hemoglobin is modified to the extent of 3 methyl groups/10(6) polypeptide chains, while carbonic anhydrase contains 1 methyl group/approximately 16,500 polypeptide chains at steady state. Aspartic acid beta-[3H]methyl ester (Asp beta-[3H]Me) can be isolated from carboxypeptidase Y digests of cytosol proteins. By synthesizing and separating diastereomeric L-Leu-L-Asp beta Me and L-Leu-D-Asp beta Me dipeptides, we show that all of the Asp beta-[3H]Me recovered from cytosolic proteins is in the D-stereoconfiguration. Based on these data and on previous observations that erythrocytes contain a single methyltransferase which also methylates red cell membrane proteins at D-aspartyl residues both in vivo (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464) and in vitro (O'Connor, C. M., and Clarke, S. (1983) J. Biol. Chem. 258, 8485-8492), we propose that protein carboxyl methylation is part of a generalized mechanism for metabolizing damaged proteins. The infrequent and spontaneous occurrence of D-aspartyl residues in proteins adequately explains the broad substrate specificity and limited stoichiometries of protein carboxyl methylation reactions.     

Bovine lens 23, 21 and 19 kDa intrinsic membrane proteins have an identical amino-terminal amino acid sequence

We have isolated bovine lens intrinsic membrane proteins (MP) having molecular masses of 19, 21 and 23 kDa. Limited amino acid sequence analysis of the amino-terminal portion of each of these polypeptides revealed a 100% homology in sequence for the number of residues determined (20 amino acids). Northern blot analysis of bovine lens mRNA using a labeled antisense oligonucleotide probe common to the amino acid sequence of these three peptides revealed a single band having an apparent molecular size of 0.8 kb. Taken together, these findings suggest a genetic commonality between these polypeptides.     

Carboxyl methylation of human erythrocyte band 3 in intact cells. Relation to anion transport activity.

The anion transport protein of the human erythrocyte membrane, band 3, is reversibly methylated by an endogenous protein carboxyl methyltransferase. The physiological consequence of this modification was studied by measuring the rate of phosphate transport by intact erythrocytes incubated under conditions where protein methylation reactions are inhibited. No change in phosphate transport was detected when cells were treated with either methionine-free media or cycloleucine, whereas cells incubated with adenosine and homocysteine thiolactone displayed a marginally slower rate of transport, which was not reversed by subsequent remethylation of the membrane proteins. These results suggest that erythrocyte protein carboxyl methylation does not directly regulate this activity of band 3.     

A change in membrane microviscosity of mouse neuroblastoma cells in association with morphological differentiation

Changes in membrane microviscosity as well as in membrane constituents of mouse neuroblastoma clone N-18 were studied in association with neurite formation. The membrane microviscosity studied by fluorescence technique increased with the formation of neurites. The concomitant increase increase in the ratio of cholesterol to phospholipids was also observed.     

Inhibition of mir-21, which is up-regulated during MYCN knockdown-mediated differentiation, does not prevent differentiation of neuroblastoma cells

Background

Neuroblastoma is a malignant childhood tumour arising from precursor cells of the sympathetic nervous system. Genomic amplification of the MYCN oncogene is associated with dismal prognosis. For this group of high-risk tumours, the induction of tumour cell differentiation is part of current treatment protocols. MicroRNAs (miRNAs) are small non-coding RNA molecules that effectively reduce the translation of target mRNAs. MiRNAs play an important role in cell proliferation, apoptosis, differentiation and cancer. In this study, we investigated the role of N-myc on miRNA expression in MYCN-amplified neuroblastoma. We performed a miRNA profiling study on SK-N-BE (2) cells, and determined differentially expressed miRNAs during differentiation initiated by MYCN knockdown, using anti-MYCN short-hairpin RNA (shRNA) technology.

Results

Microarray analyses revealed 23 miRNAs differentially expressed during the MYCN knockdown-mediated neuronal differentiation of MNA neuroblastoma cells. The expression changes were bidirectional, with 11 and 12 miRNAs being up- and down-regulated, respectively. Among the down-regulated miRNAs, we found several members of the mir-17 family of miRNAs. Mir-21, an established oncomir in a variety of cancer types, became strongly up-regulated upon MYCN knockdown and the subsequent differentiation.Neither overexpression of mir-21 in the high-MYCN neuroblastoma cells, nor repression of increased mir-21 levels during MYCN knockdown-mediated differentiation had any significant effects on cell differentiation or proliferation.

Conclusions

We describe a subset of miRNAs that were altered during the N-myc deprived differentiation of MYCN-amplified neuroblastoma cells. In this context, N-myc acts as both an activator and suppressor of miRNA expression. Mir-21 was up-regulated during cell differentiation, but inhibition of mir-21 did not prevent this process. We were unable to establish a role for this miRNA during differentiation and proliferation of the two neuroblastoma cell lines used in this study.     

Identification of a 170 kDa membrane kinase with increased activity in KB-V1 multidrug resistant cells

Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of ³²P incorporation in proteins that were renatured after being subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723–20729, 1989; Mol Cell Biol 10:3020–3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB-V1 cells as compared to the sensitive parental KB-3-1 cells. The KB-V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca⁺⁺, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P-glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF-7/Adr^R and murine melanoma B16/Adr^R. cells, but the activity was not present in murine leukemia P-388 sensitive or multidrug resistant cells.     

Surfactant-free purification of membrane proteins with intact native membrane environment

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10?nm disc-like structure ideally suited to purification and further biochemical study.     

Phosphorylation of zymogen granule membrane proteins in intact rat pancreatic acinar cells

The comparative effects of insulin and ethanolamine on 14CO2 production and lipid synthesis from [U-14C]-D-glucose in isolated rat adipocytes were studied. Ethanolamine (10 mM) increased 14CO2 production (glucose oxidation) about 5-fold and lipogenesis about 3-fold as compared to the control. Ethanolamine was more efficient than 25 microU/ml insulin regarding both parameters, but it was less efficient than 200 microU/ml insulin in glucose oxidation, and equally potent in lipogenesis. The combination of ethanolamine and insulin was more active than insulin alone. The mechanisms of ethanolamine action include facilitation of glucose transport and increase of pyruvate dehydrogenase activity.     

A 165 kDa membrane antigen mediating fibronectin-vinculin interaction is involved in murine odontoblast differentiation

Membrane-mediated matrix-microfilament interactions are involved in odontoblast differentiation. In this study, we analyzed the interactions of vinculin and fibronectin with plasma membrane proteins separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene-difluoride (PVDF) paper. Vinculin was found to interact with 58, 63 and 165 kDa plasma membrane proteins. Fibronectin interacted with three high molecular weight (145, 165, and 185 kDa) membrane proteins. Attempts were made to characterize the 165 kDa protein which interacted with vinculin and with fibronectin. The interaction of the 165 kDa protein with fibronectin was not competitively inhibited by synthetic peptides such as GRGDS or GRGDSP, suggesting that the protein was not related to integrins. Antibodies directed against the 165 kDa protein allowed the identification of the precise localization and biological role of this membrane antigen. The data presented in this paper and previous observations indicate that the 165 kDa protein, involved in odontoblast elongation and polarization, mediates a fibronectin-vinculin transmembrane interaction.     

Carboxyl methylation of platelet rap1 proteins is stimulated by guanosine 5'-(3-O-thio)triphosphate

Carboxyl methylation of platelet ras-related proteins, known as rap proteins, was investigated in this study. Platelet membrane proteins of Mr 23,000 incorporated radioactivity in the presence of S-[methyl-3H]adenosylmethionine and platelet cytosol. About 97% of the radioactivity present in the Mr 23,000 proteins was liberated as volatile methanol under basic (1 M sodium hydroxide) conditions. Cycloheximide, an inhibitor of protein synthesis, inhibited incorporation of S-[methyl-3H]adenosylmethionine by 25%. These results suggest that at least 75% of the radioactivity present in the Mr 23,000 proteins is due to carboxyl methylation and not due to the incorporation of S-[methyl-3H]adenosylmethionine into proteins or due to the incorporation of base-stable methyl groups into side chains of arginine, histidine, or lysine residues. Protein methylation did not occur if membranes or cytosol alone was incubated with S-[methyl-3H]adenosylmethionine. Guanosine 5'(3-O-thio)triphosphate increased methylation of the Mr 23,000 proteins in a time- and concentration-dependent manner. Acetyl-farnesylcysteine, a synthetic substrate for carboxyl methyltransferases, completely blocked methylation of the Mr 23,000 membrane proteins. On the basis of one- and two-dimensional Western blots using rap-specific antisera, the Mr 23,000 methylated proteins were identified as rap1 proteins. The existence of the carboxyl-terminal CAAX motif in rap1 proteins, similar to the CAAX motif present in p21ras as well as in the yeast mating factors, leads us to suggest that methylation of rap1 proteins possibly occurs at the alpha-carboxyl-terminal cysteine.     

IgE-dependent activation of human lung mast cells is not associated with increased phospholipid methylation

Enhanced phospholipid methylation has been suggested to be an obligatory process in IgE-dependent stimulus-secretion coupling in human lung mast cells. Our studies with mast cell-enriched lung preparations do not support this hypothesis, demonstrating no increased 3H-methyl radiolabeling of chloroform/methanol-extracted lipids or chromatographically separated phospholipids accompanying anti-IgE-dependent histamine secretion. Inhibitors of transmethylation, 3-deazaadenosine, and homocysteine thiolactone inhibited histamine secretion by both anti-IgE and calcium ionophore A23187, reflecting a requirement of secretion for overall integrity of cellular transmethylation. These agents induced small increases in cAMP concentration which are considered to make at most a minor contribution to this inhibition. The inability of methylation inhibitors to diminish anti-IgE-dependent increases in lung mast cell cAMP levels would suggest that not only does phospholipid methylation have no role in histamine secretion but also it does not participate in the activation of adenylate cyclase by this stimulus.     

Expression of external-surface membrane proteins in differentiated and undifferentiated mouse neuroblastoma cells.

Murine neuroblastoma cultures were labeled externally with the cationic reagent N,N,N-[3H]-trimethylamino-beta-alanyl-N-hydroxy-succinimide ester ([3H]Me3N-beta Ala-NSuc) or with 125I/lactoperoxidase. The cells were labeled in the logarithmic and confluent growth phases as well as in a highly differentiated state following treatment with 2% dimethylsulfoxide. The labeled exterior membrane proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Major changes in the exterior membrane proteins were observed during maturation and differentiation of the cells. Most of these changes were clonal-specific, while others were common to several clones. Two proteins of Mr 55,000 and 65,000 were labeled by both 125I/lactoperoxidase and Me3N-[3H]-beta Ala-NSuc. The level of labeling was dependent on the clonal lines used and the state of the cell maturation. A group of proteins displaying a molecular weight between 150,000 and 200,000 was found to be related to the transition of a culture from logarithmic to confluent growth phases. An additional protein, with an apparent molecular weight of 95,000, was common to differentiated cells of the two inducible clones used. In general the maturation of logarithmic phase cells into confluent cells resulted in a less complex electrophoretic distribution of the pattern of labeling. After dimethyl-sulfoxide treatment, further reduction in the complexity of the externally labeled proteins was observed.     

Differences in metabolite levels upon differentiation of intact neuroblastoma X glioma cells observed by proton NMR spectroscopy

Proton NMR spectroscopy was used to study the effect of differentiation with prostaglandin E1 and theophylline on intact hybrid neuroblastoma X glioma cells. The standard proton NMR method showed more resolvable signals than the spin echo NMR spectra. The differentiated cells were found to contain significantly higher levels of glutamine than the undifferentiated precursors. Observations on cell extracts confirmed these results.     

The phosphorylation of coated membrane proteins in intact neurons

To complement studies that have demonstrated the prominent phosphorylation of a 50-kD coated vesicle polypeptide in vitro, we have evaluated the phosphorylation of coated membrane proteins in intact cells. A co-assembly assay has been devised in which extracts of cultured rat sympathetic neurons labeled with [32P]-Pi were combined with unlabeled carrier bovine brain coat proteins and reassembled coat structures were isolated by gradient centrifugation. Two groups of phosphorylated polypeptides, of 100-110 kD (pp100-110) and 155 kD (pp155) apparent molecular mass, were incorporated into reassembled coats. The neuronal pp100-110 are structurally and functionally related to the 100-110-kD component of the bovine brain assembly protein (AP), a protein complex that also contains 50-kD and 16.5-kD components and is characterized by its ability to promote the reassembly of clathrin coat structures under physiological conditions of pH and ionic strength (Zaremba, S. and J. H. Keen, 1983, J. Cell Biol., 97:1337-1348). The neuronal pp155 detected in reassembled coat structures was readily observable in total extracts of [32P]-Pi-labeled neurons dissolved in SDS-containing buffer. A bovine brain counterpart to the neuronal pp155 was also observed when brain coated vesicles were subjected to two-dimensional gel electrophoresis. Phosphoserine was the predominant phosphoaminoacid found in both the pp100 and pp155. A structural and functional counterpart to the 50-kD brain assembly polypeptide (AP50) was also identified in these neurons. Although the brain AP50 is prominently phosphorylated by an endogenous protein kinase in isolated coated vesicle preparations, the neuronal AP50 was not detectably phosphorylated in intact cells as assessed by two-dimensional non-equilibrium pH gradient gel electrophoresis of labeled cells dissolved directly in SDS-containing buffers. These results demonstrate that the bovine brain assembly polypeptides of 50 kD and 100-110 kD that we have previously described, as well as a novel 155-kD polypeptide reported here, have structural and functional counterparts in cultured neurons. They also indicate that phosphorylation of the 100-110-kD AP may be involved in the regulation of coated membrane structure and function. The extent of phosphorylation of the AP50 in intact cells and in isolated coated vesicles is strikingly different: it has been suggested that the latter process reflects an autophosphorylation reaction (Campbell C., J. Squicciarini, M. Shia, P. F. Pilch, and R. E. Fine, 1984, Biochemistry, 23:4420-4426).(ABSTRACT TRUNCATED AT 400 WORDS)