FcgammaRII tyrosine phosphorylation differs between FcgammaRII cross-linking and platelet-activating anti-platelet monoclonal antibodies.

Using glutathione S-transferase Syk fusion proteins, we evaluated the mode of platelet FcgammaRII tyrosine phosphorylation induced by FcgammaRII cross-linking or anti-CD9 monoclonal antibodies (mAb). The N-terminal SH2 domain of Syk (Syk-N-SH2), the C-terminal SH2 domain of Syk (Syk-C-SH2), and the domain having both the N- and C-terminal SH2 of Syk (Syk-NC-SH2) all bound to tyrosine-phosphorylated FcgammaRII with FcgammaRII cross-linking. In the case of anti-CD9 mAb-induced platelet activation, only Syk-C-SH2 and Syk-NC-SH2 bound to tyrosine-phosphorylated FcgammaRII. Since the SH2 domain is specific for a particular structure containing phosphotyrosine, these findings suggest that only one tyrosine residue in the immunoreceptor tyrosine-based activation motif (ITAM) is phosphorylated with anti-CD9 mAb, and that both are phosphorylated with FcgammaRII cross-linking. Synthetic peptides corresponding to the ITAM of human platelet FcgammaRII with the N-terminal tyrosine residue phosphorylated (N-P) or the C-terminal tyrosine residue phosphorylated (C-P), were used. N-P more potently dissociated Syk-C-SH2 from tyrosine-phosphorylated FcgammaRII than C-P, suggesting that the N-terminal tyrosine residue is phosphorylated upon anti-CD9 mAb-induced activation. Furthermore, these findings imply that Syk-N-SH2 binds to the phosphorylated C-terminal tyrosine residue of ITAM, and Syk-C-SH2 to the N-terminal tyrosine. Taken together, our findings suggest that FcgammaRII-dependent platelet activation without FcgammaRII dimerization, such as with anti-CD9 mAb, is distinct from that induced by FcgammaRII cross-linking.     

Sphingomyelinase generation of ceramide promotes clustering of nanoscale domains in supported bilayer membranes

The effects of ceramide incorporation in supported bilayers prepared from ternary lipid mixtures which have small nanoscale domains have been examined using atomic force and fluorescence microscopy. Both direct ceramide incorporation in vesicles used to prepare the supported bilayers and enzymatic hydrolysis of SM by sphingomyelinase were compared for membranes prepared from 5:5:1 DOPC/sphingomyelin/cholesterol mixtures. Both methods of ceramide incorporation resulted in enlargement of the initial small ordered domains. However, enzymatic ceramide generation led to a much more pronounced restructuring of the bilayer to give large clusters of domains with adjacent areas of a lower phase. The individual domains were heterogeneous with two distinct heights, the highest of which is assigned to a ceramide-rich phase which is hypothesized to occur via ceramide flip-flop to the lower leaflet with formation of a raised domain due to negative membrane curvature. A combination of AFM and fluorescence showed that the bilayer restructuring starts rapidly after enzyme addition, with formation of large clusters of domains at sites of high enzyme activity. The clustering of domains is accompanied by redistribution of fluid phase to the periphery of the domain clusters and there is a continued slow evolution of the bilayer over a period of an hour or more after the enzyme is removed. The relevance of the observed clustering of small nanoscale domains to the postulated coalescence of raft domains to form large signaling platforms is discussed.     

Sphingomyelinase generation of ceramide promotes clustering of nanoscale domains in supported bilayer membranes

The effects of ceramide incorporation in supported bilayers prepared from ternary lipid mixtures which have small nanoscale domains have been examined using atomic force and fluorescence microscopy. Both direct ceramide incorporation in vesicles used to prepare the supported bilayers and enzymatic hydrolysis of SM by sphingomyelinase were compared for membranes prepared from 5:5:1 DOPC/sphingomyelin/cholesterol mixtures. Both methods of ceramide incorporation resulted in enlargement of the initial small ordered domains. However, enzymatic ceramide generation led to a much more pronounced restructuring of the bilayer to give large clusters of domains with adjacent areas of a lower phase. The individual domains were heterogeneous with two distinct heights, the highest of which is assigned to a ceramide-rich phase which is hypothesized to occur via ceramide flip-flop to the lower leaflet with formation of a raised domain due to negative membrane curvature. A combination of AFM and fluorescence showed that the bilayer restructuring starts rapidly after enzyme addition, with formation of large clusters of domains at sites of high enzyme activity. The clustering of domains is accompanied by redistribution of fluid phase to the periphery of the domain clusters and there is a continued slow evolution of the bilayer over a period of an hour or more after the enzyme is removed. The relevance of the observed clustering of small nanoscale domains to the postulated coalescence of raft domains to form large signaling platforms is discussed.     

Activated FcgammaRII and signalling molecules revealed in rafts by ultra-structural observations of plasma-membrane sheets

To reveal topography of FcgammaRII components of the receptor-signalling complex, large plasma-membrane sheets were obtained by cell cleavage and analysed by immuno-electron microscopy. Non-activated FcgammaRII was dispersed in the plane of the plasma membrane and only rarely was localized in the proximity of Lyn, an Src family tyrosine kinase, and CD55, a glycosylphosphatidylinositol-anchored protein. After FcgammaRII activation by cross-linking with antibodies, clusters of an electron-dense material acquiring about 86% of FcgammaRII and reaching up to 300 nm in diameter were formed within 5 min. These structures also accommodated about 85% of Lyn and 63% of CD55 labels that were located in close vicinity of gold particles attributed to the cross-linked FcgammaRII . The electron-dense structures were also abundant in tyrosine phosphorylated proteins. At their margins PIP2 was preferentially located. Based on a concentration of Lyn, CD55 and activated FcgammaRII , the electron-dense structures seem to reflect coalescent membrane rafts.     

Association of FcgammaRII with low-density detergent-resistant membranes is important for cross-linking-dependent initiation of the tyrosine phosphorylation pathway and superoxide generation.

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcgammaRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcgammaR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcgammaRs resulted in a marked increase in the tyrosine phosphorylation of FcgammaRIIa, p58(lyn), and p120(c-cbl), which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcgammaRs and tyrosine-phosphorylated proteins including p120(c-cbl) were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcgammaRs as well as p120(c-cbl) with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-beta-cyclodextrin, the cross-linking did not induce the association of FcgammaRs as well as p120(c-cbl) with DRMs. In addition, although the physical association between FcgammaRIIa and p58(lyn) was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-beta-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcgammaRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcgammaRII to lipid rafts is important for the activation of FcgammaRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.     

Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.The sphingomyelin pathway is initiated by the hydrolysis of sphingomyelin to generate the second messenger ceramide.¹ Sphingomyelin hydrolysis is a major pathway for stress-induced ceramide generation. Neutral sphingomyelinase (nSMase) is activated by a variety of environmental stress conditions, such as heat shock,^{1, 2, 3} oxidative stress (hydrogen peroxide (H₂O₂), oxidized lipoproteins),¹ ultraviolet (UV) radiation,¹ chemotherapeutic agents,⁴ and β-amyloid peptides.^{5, 6} Cytokines, including tumor necrosis factor (TNF)-α,^{7, 8, 9} interleukin (IL)-1β,¹⁰ Fas ligand,¹¹ and their associated proteins, also trigger the activation of nSMase.¹² Membrane-bound Mg²⁺-dependent nSMase is considered to be a strong candidate for mediating the effects of stress and inflammatory cytokines on ceramide.³Among the four vertebrate nSMases, nSMase1 (SMPD2) was the first to be cloned and is localized in the endoplasmic reticulum (ER) and Golgi apparatus.¹³ Several studies have focused on the potential signaling roles of nSMase1, and some reports have suggested that nSMase1 is important for ceramide generation in response to stress.^{5, 6, 14, 15} In addition, nSMase1 is responsible for heat-induced apoptosis in zebrafish embryonic cultured (ZE) cells, and a loss-of-function study showed a reduction in ceramide generation, caspase-3 activation, and apoptosis in zebrafish embryos.¹⁶ However, nSMase1-knockout mice showed no lipid storage diseases or abnormalities in sphingomyelin metabolism.¹⁷ Therefore, the molecular mechanisms by which nSMase1 is activated have yet to be elucidated.Environmental stress and inflammatory cytokines^{1, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27} stimulate stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) signaling, which involves the sequential activation of members of the mitogen-activated protein kinase (MAPK) family, including MAPK/ERK kinase kinase (MEKK)1/MAPK kinase (MKK)4, and/or SAPK/ERK kinase (SEK)1/MKK7, JNK, and c-jun. Both the JNK and sphingomyelin signaling pathways coordinately mediate the induction of apoptosis.¹ However, possible crosstalk between the JNK and sphingomyelin signaling pathways has not yet been characterized. Previously, we used SDS-PAGE to determine that nSMase1 polypeptides migrated at higher molecular masses,¹⁶ suggesting that the sphingomyelin signaling pathway might cause the production of a chemically modified phosphorylated nSMase1, which is stimulated under stressed conditions in ZE cells.¹⁶ Here, we demonstrate that JNK signaling results in the phosphorylation of Ser-270 of nSMase1, which initiates ceramide generation and apoptosis. We also provide evidence for a signaling mechanism that integrates cytokine- and stress-activated apoptosis in vertebrate cells. We studied stress-induced ceramide generation in two cell types: ZE cells and human leukemia Jurkat T-lymphoid cells. Stress-induced apoptosis has been investigated in these systems previously.^{16, 28}     

Tyrosine phosphorylation and association of FcgammaRII and p72(Syk) are not limited to the FcgammaRII signalling pathway

The tyrosine kinase p72(Syk) plays a critical role in platelet signal transduction. It associates with the platelet receptor for the Fc domain of IgGs, FcgammaRII, following stimulation by FcgammaRII cross-linking. Here, we show that p72(Syk) and FcgammaRII tyrosine phosphorylation and association occured following platelet stimulation by: (1) two monoclonal antibodies, which form a bridge between a target antigen and FcgammaRII, and (2) the G-protein-coupled receptor agonist thrombin. The kinetics of the p72(Syk)/FcgammaRII association depended on the signalling pathway (i.e., the antigen targeted or the thrombin receptor). We established a direct relationship between the level of FcgammaRII phosphorylation and the detection of its association with p72(Syk). Inhibition of p72(Syk) by piceatannol resulted in partial or total inhibition of FcgammaRII phosphorylation, after immunological activation or addition of thrombin, respectively, suggesting that p72(Syk) participates in FcgammaRII phosphorylation. The results provide evidence that p72(Syk)/FcgammaRII association is not restricted to immunological activation.     

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.     

Sphingosine-phosphate lyase enhances stress-induced ceramide generation and apoptosis

Sphingosine-1-phosphate lyase is a widely expressed enzyme that catalyzes the essentially irreversible cleavage of the signaling molecule sphingosine 1-phosphate. To investigate whether sphingosine-1-phosphate lyase influences mammalian cell fate decisions, a recombinant human sphingosine-1-phosphate lyase fused to green fluorescent protein was expressed in HEK293 cells. The recombinant enzyme was active, localized to the endoplasmic reticulum, and reduced baseline sphingosine and sphingosine 1-phosphate levels. Stable overexpression led to diminished viability under stress, which was attributed to an increase in apoptosis and was reversible in a dose-dependent manner by exogenous sphingosine 1-phosphate. In contrast to sphingosine 1-phosphate, the products of the lyase reaction had no effect on apoptosis. Lyase enzymatic activity was required to potentiate apoptosis, because cells expressing a catalytically inactive enzyme behaved like controls. Stress increased the amounts of long- and very long-chain ceramides in HEK293 cells, and this was enhanced in cells overexpressing wild type but not catalytically inactive lyase. The ceramide increases appeared to be required for apoptosis, because inhibition of ceramide synthase with fumonisin B1 decreased apoptosis in lyase-overexpressing cells. Thus, sphingosine-1-phosphate lyase overexpression in HEK293 cells decreases sphingosine and sphingosine 1-phosphate amounts but elevates stress-induced ceramide generation and apoptosis. This identifies sphingosine-1-phosphate lyase as a dual modulator of sphingosine 1-phosphate and ceramide metabolism as well as a regulator of cell fate decisions and, hence, a potential target for diseases with an imbalance in these biomodulators, such as cancer.     

Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER

Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and transported by ceramide transfer protein to the Golgi for conversion to sphingomyelin (SM) by SM synthase SMS1. In this study, we report the identification of an SMS1-related (SMSr) enzyme, which catalyses the synthesis of the SM analogue ceramide phosphoethanolamine (CPE) in the ER lumen. Strikingly, SMSr produces only trace amounts of CPE, i.e., 300-fold less than SMS1-derived SM. Nevertheless, blocking its catalytic activity causes a substantial rise in ER ceramide levels and a structural collapse of the early secretory pathway. We find that the latter phenotype is not caused by depletion of CPE but rather a consequence of ceramide accumulation in the ER. Our results establish SMSr as a key regulator of ceramide homeostasis that seems to operate as a sensor rather than a converter of ceramides in the ER.     

Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase.

We have recently shown that changes in tyrosine phosphorylation of a 130-kDa protein(s) (pp130) may be involved in integrin signaling (Kornberg, L., Earp, H.S., Turner, C., Prokop, and Juliano, R. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8392-8396). One component of the pp130 protein complex reacts with an antibody generated against p125fak, which is a focal contact-associated tyrosine kinase (Schaller, M.D., Borgman, C. A., Cobb, B. S., Vines, R. R., Reynolds, A. B., and Parsons, J. T. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5192-5196). Both antibody-mediated integrin clustering and adhesion of KB cells to fibronectin leads to increased tyrosine phosphorylation of p125fak. The phosphorylation of p125fak is coincident with adhesion of cells to fibronectin and is maximal prior to cell spreading. Tyrosine phosphorylation of p125fak is induced when KB cells are allowed to adhere to fibronectin, collagen type IV, or laminin, but is not induced on polylysine. When KB cells are subjected to indirect immunofluorescence microscopy, p125fak colocalizes with talin in focal contacts. These data provide additional evidence that tyrosine kinases are involved in integrin signaling.     

IGF-I induces caveolin 1 tyrosine phosphorylation and translocation in the lipid rafts

Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IR(WT) cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R(-) cells overexpressing IRs.     

Involvement of protein kinase C-regulated ceramide generation in inostamycin-induced apoptosis

Activation of caspases is commonly involved in the apoptosis induced by various anticancer drugs. However, the upstream events leading to the activation of caspases seem to be specific to each anticancer drug. In the present study, we examined the possible involvement of protein kinase C (PKC) and ceramide generation in caspase-3(-like) protease activation induced by inostamycin, a phosphatidylinositol synthesis inhibitor. Treatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, suppressed the release of cytochrome c from mitochondria and the activation of caspase-3(-like) proteases in inostamycin-treated cells, but not in other anticancer drug-treated cells. Inostamycin induced the elevation of intracellular ceramide levels, and fumonisin B1, an inhibitor of ceramide synthase, inhibited inostamycin-induced cytochrome c release, caspase-3(-like) protease activation, and apoptosis. Moreover, TPA also inhibited inostamycin-induced ceramide synthesis. Taken together, our results suggest that inostamycin-induced apoptosis is mediated by PKC-regulated ceramide generation, leading to the activation of a caspase cascade.     

In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.     

Cell nonhomologous end joining capacity controls SAF-A phosphorylation by DNA-PK in response to DNA double-strand breaks inducers

Aiming to identify novel phosphorylation sites in response to DNA double-strand breaks (DSB) inducers, we have isolated a phosphorylation site on KU70. Unexpectedly, a rabbit antiserum raised against this site cross-reacted with a 120 kDa protein in cells treated by DNA DSB inducers. We identified this protein as SAF-A/hnRNP U, an abundant and essential nuclear protein containing regions binding DNA or RNA. The phosphorylation site was mapped at S59 position in a sequence context favoring a "S-hydrophobic" consensus model for DNA-PK phosphorylation site in vivo. This site was exclusively phosphorylated by DNA-PK in response to DNA DSB inducers. In addition, the extent and duration of this phosphorylation was in inverse correlation with the capacity of the cells to repair DSB by nonhomologous end joining. These results bring a new link between the hnRNP family and the DNA damage response. Additionaly, the mapped phospho-site on SAF-A might serve as a potential bio-marker for DNA-PK activity in academic studies and clinical analyses of DNA-PK activators or inhibitors.     

Free radical generation in the brain precedes hyperbaric oxygen-induced convulsions.

We tested the hypothesis that hyperbaric oxygenation (HBO) generates free radicals in the brain before the onset of neurological manifestations of central nervous system (CNS) oxygen poisoning. Chronically cannulated, conscious rats were individually placed in a transparent pressure chamber and exposed to (1) 5 atmospheres absolute (ATA) oxygen for 15 min (n = 4); (2) 5 ATA oxygen for 30 min (n = 5), during which no visible convulsions occurred; (3) 5 ATA oxygen for 30 min with recurrent convulsions (n = 6); (4) 5 ATA oxygen until the appearance of the first visible convulsions (n = 5); (5) 4 ATA oxygen for 60 min during which no convulsions occurred (n = 5); and (6) 5 ATA air for 30 min (n = 5, controls). Immediately before compression, 1 mL of 0.1 M of alpha-phenyl-N-tert-butyl nitrone (PBN) was administered intravenously (iv) for spin trapping. At the termination of each experiment, rats were euthanized by pentobarbital iv and decompressed within 1 min. Brains were rapidly removed for preparation of lipid extracts (Folch). The presence of PBN spin adducts in the lipid extracts was examined by electron spin resonance (ESR) spectroscopy. ESR spectra from unconvulsed rats exposed to 5 ATA oxygen for 30 min revealed both oxygen-centered and carbon-centered PBN spin adducts in three of the five brains. One of the five rats in this group showed an ascorbyl signal in the ESR spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)     

O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein

Secretory proteins in yeast are N- and O-glycosylated while they enter the endoplasmic reticulum. N-glycosylation is initiated by the oligosaccharyl transferase complex and O-mannosylation is initiated by distinct O-mannosyltransferase complexes of the protein mannosyl transferase Pmt1/Pmt2 and Pmt4 families. Using covalently linked cell-wall protein 5 (Ccw5) as a model, we show that the Pmt4 and Pmt1/Pmt2 mannosyltransferases glycosylate different domains of the Ccw5 protein, thereby mannosylating several consecutive serine and threonine residues. In addition, it is shown that O-mannosylation by Pmt4 prevents N-glycosylation by blocking the hydroxy amino acid of the single N-glycosylation site present in Ccw5. These data prove that the O- and N-glycosylation machineries compete for Ccw5; therefore O-mannosylation by Pmt4 precedes N-glycosylation.     

Cell adhesion-dependent differences in endogenous protein phosphorylation on the surface of various cell lines

Endogenous phosphorylation of intact cells was studied with four mouse, hamster and human cell lines using [gamma-32P]ATP and [gamma-32P]GTP as exogenous substrates. With all four cell lines distinct differences in the phosphoprotein patterns could be demonstrated for cells grown in suspension culture compared to cells grown in monolayers. Two major, apparently ubiquitous phosphoproteins with molecular weights of 135 000 (128 000 in HeLa cells) and 105 000, representing up to 60% of total phosphorylation, were phosphorylated only in cells grown in suspension. These phosphoproteins and the kinase(s) were located on the surface of the suspension cells. Evidence showed that phosphorylation was apparently not a true endogenous reaction, that rather it occurred by cell-cell collision, showing exponentially increasing 32P incorporation with increasing cell population density. Phosphorylation of pp135 and pp105 was established with ATP as well as with GTP and was not dependent on cyclic nucleotides cyclic AMP, cyclic GMP and cyclic CMP. The substrate-attached cells of all four cell lines have protein kinases on the cell surface. The lack of pp135 and pp105 phosphorylation may be due to the fact that these phosphoproteins are not expressed at all on the surface of substrate-attached cells or that these phosphoproteins are already fully phosphorylated.     

Compartmentalization of the exocyst complex in lipid rafts controls Glut4 vesicle tethering

Lipid raft microdomains act as organizing centers for signal transduction. We report here that the exocyst complex, consisting of Exo70, Sec6, and Sec8, regulates the compartmentalization of Glut4-containing vesicles at lipid raft domains in adipocytes. Exo70 is recruited by the G protein TC10 after activation by insulin and brings with it Sec6 and Sec8. Knockdowns of these proteins block insulin-stimulated glucose uptake. Moreover, their targeting to lipid rafts is required for glucose uptake and Glut4 docking at the plasma membrane. The assembly of this complex also requires the PDZ domain protein SAP97, a member of the MAGUKs family, which binds to Sec8 upon its translocation to the lipid raft. Exocyst assembly at lipid rafts sets up targeting sites for Glut4 vesicles, which transiently associate with these microdomains upon stimulation of cells with insulin. These results suggest that the TC10/exocyst complex/SAP97 axis plays an important role in the tethering of Glut4 vesicles to the plasma membrane in adipocytes.